Biological Monitoring for Pesticide Exposure: Measurement, Estimation, and Risk Reduction (ACS Symposium Series)

ACS

SYMPOSIUM

SERIES

382

Biological Monitoring for Pesticide Exposure Measurement, Estimation, and Risk Reduction Rhoda G. M. Wang, EDITOR State of California, Department of Food and Agriculture

Claire A. Franklin, EDITOR Department of National Health and Welfare, Ottawa

Richard C. Honeycutt, EDITOR Ciba-Geigy

Joseph C. Reinert, EDITOR U.S. Environmental Protection Agency

Developed from a symposium sponsored by the Division of Agrochemicals at the 194th Meeting of the A m e r i c a n C h e m i c a l Society, N e w Orleans, Louisiana, August 30-September 4, 1987

American Chemical Society, Washington, DC 1989

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Library of Congress Cataloging-in-Publication Data Biological monitoring for pesticide exposure : measurement, estimation, andriskreduction Rhoda G. M. Wang, editor ... [et al.]. p. cm.—(ACS Symposium Series; 382) Includes bibliographies and index. ISBN 0-8412-1559-6 1. Pesticides—Toxicology—Congresses. 2. Biological monitoring—Congresses. 3. Agricultural workers—Health risk assessment—Congresses. I. Wang, Rhoda G. M., date. II [DNLM: 1. Environmental Exposure-congresses. 2. Environmental Monitoring-congresses. 3. Pesticides—toxicity—congresses. 4. Risk—congresses. WA 465 B61615 1988] RA1270.P4B53 1988 632'.95042—dc19 DNLM/DLC for Library of Congress

88-39189 CIP

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Copyright 1989 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprograpnic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of anyrightor permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

ACS Symposium Series M. Joan Comstock, Series Editor 1988 ACS Books Advisory Board Paul S. Anderson

Vincent D. McGinniss

Merck Sharp & Dohme Research Laboratories

Battelle Columbus Laboratories

Harvey W. Blanch University of California—Berkeley

Malcolm H. Chisholm

James C. Randall

Indiana University

Exxon Chemical Company

Alan Elzerman

E. Reichmanis

Clemson University

AT&T Bell Laboratories

John W. Finley Nabisco Brands, Inc.

Natalie Foster Lehigh University

C. M. Roland U.S. Naval Research Laboratory

W. D. Shults Oak Ridge National Laboratory

Marye Anne Fox The University of Texas—Austin

Geoffrey K. Smith Rohm & Haas Co.

Roland F. Hirsch U.S. Department of Energy

G. Wayne Ivie

Douglas B. Walters National Institute of Environmental Health

USDA, Agricultural Research Service

Wendy A. Warr Michael R. Ladisch

Imperial Chemical Industries

Purdue University

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Foreword The ACS SYMPOSIUM SERIES was founded in 1974 to provide a

medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, lished papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Preface SESSING WORKER EXPOSURE TO PESTICIDES through biological mon­

itoring is the main thrust of this book. Historically, in the absence of dermal absorption data in humans and animals, regulatory agencies often assumed that pesticides were 100% absorbed by dermally exposed work­ ers. However, 100% absorption may not actually occur. Accurate meas­ urement of the dose absorbed is necessary in order to estimate the health risk to exposed workers In recent years, pesticid effectively communicating the need to develop dermal pesticide absorp tion data. However, despite the improved quality and availability of exposure and dermal absorption data generated in rodents and other species, current methods are not accurate for estimating total pesticide uptake during exposure. Poor estimates result from attempting to meas­ ure the amount of pesticide found on limited areas of dermal pads and to extrapolate this information to a much greater body surface area (which magnifies or reduces any inherent errors) and from assuming that percutaneous absorption in animals approximates or equals that in humans. Interspecies differences affect individual dermal absorption rates, and humans tend to have a lower dermal absorption rate than many common test animals. The kinetics of absorption, biotransformation, and excretion cannot be addressed by passive dosimetry, even when the percent of absorption has been factored in. The critical dose of a toxicant must be transported dermally, reach plasma concentration, and be distributed to the target organs and tissues before it can evoke a toxicological effect. Therefore, without this vital information, the assessment of exposure health risk is at best a presumptuous estimation. Nevertheless, the combined use of dermal pad and absorption data remains a valid choice. In conjunction with sensitive biomarkers, biological monitoring can offer a distinct advantage over conventional methods. However, biologi­ cal monitoring methods have intrinsic limitations and barriers. For instance, unless the pharmacokinetic behavior of a pesticide in humans is well understood, it could be difficult to relate the urinary excretion of a pesticide to the internal dosage. Obtaining full cooperation of field workers to provide 24-hour urine samples during and after exposure can be difficult. Isolation and identification of unlabeled metabolites in bio-

ix In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

logical fluids is a complex procedure, even with modern analytical chemi­ cal techniques. I hope this book marks the beginning of a long journey devoted to improved estimation of pesticide or other chemical exposure. RHODA G . M . W A N G

California Department of Food and Agriculture Sacramento, C A 95814 April 19, 1988

x In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 1

Biological Monitoring for Pesticide Dose Determination Historical Perspectives, Current Practices, and New Approaches H. N. Nigg and J. H. Stamper Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850 The rationales governin biological monitorin experiments are past studies an Purpose shortcoming are covered. Deficiencies i n blood and urine monitoring are reviewed. Sampling procedures and s t a t i s t i c a l considerations are discussed. New approaches are outlined, including vagal tone monitoring, saliva and perspiration assays and erythrocyte adducts. There are various ways of estimating pesticide exposure. Environmental monitoring includes measurement of ambient levels of pesticide i n the worker's environment and passive dosimetry to estimate the quantity which comes into contact with the worker. Dermal patches and personal a i r samplers have been used to estimate dermal and inhalation exposure i n pesticide workers. Biological monitoring includes the measurement of parent compounds, their metabolites, or an indicator of response (e.g. acetylcholinesterase inhibition) i n a biological sample such as urine, blood, sweat, s a l i v a , exhaled breath, hair or n a i l s . The use of biological samples as indicators of pesticide dose i s a new research area and there i s not yet agreement on the meaning of even the most commonly used terms. By "exposure" to pesticide, we mean here the deposit of compound onto the clothing or skin of the worker, the l a t t e r being "dermal exposure". "Dose" i s that amount which enters the body, including skin sequestration. The majority of the dose to an agricultural worker arises through transdermal absorption, but other routes such as respiratory or oral ingestion also play a r o l e . A "biological indicator" i s any biological sample taken from the worker which contains r e l i a b l e evidence that a pesticide dose has been received. General Rationale Our purposes here are to describe current and prospective biological monitoring methods and to raise questions. The kinds of questions we ask the reader to consider throughout this a r t i c l e are 0097-6156/89/0382-0006$06.50/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

NIGG AND STAMPER

7

Pesticide Dose Determination

the f o l l o w i n g . I f human b i o l o g i c a l samples a r e n e g a t i v e f o r a g i v e n p e s t i c i d e , what can be concluded about dose? On the o t h e r hand, i f they a r e p o s i t i v e , can dose be r e l i a b l y q u a n t i f i e d ? Many of the b i o l o g i c a l methods we r e v i e w here have been combined w i t h n o n - b i o l o g i c a l methods f o r measuring e x p o s u r e . R e f e r e n c e s 1-5 p r o v i d e r e v i e w s o f n o n - b i o l o g i c a l methods. F a c t o r s A f f e c t i n g Exposure o f A g r i c u l t u r a l Workers For a p p l i c a t o r / m i x e r - l o a d e r g r o u p s , the type of equipment used, the number of tanks a p p l i e d per u n i t t i m e , the c o n c e n t r a t i o n of the tank mix, the l o a d i n g method, and the p r o t e c t i v e c l o t h i n g worn a f f e c t the dermal exposure p r o c e s s . E s p e c i a l l y i m p o r t a n t are p e r s o n a l work h a b i t s . T h i s has been known f o r years and i s d e s c r i b e d i n many p u b l i s h e d r e p o r t s 6). R e g a r d l e s s of crop t y p e the p r o d u c t i o n r a t e of h a r v e s t e r s appears to be r e l a t e d t r a t e i s r e l a t e d t o the which has been s t u d i e d u s i n g movies and time a n a l y s i s (7) and e s t i m a t e d w i t h surveys ( & ) • The p r o d u c t i o n r a t e can be q u a n t i f i e d as the number o f boxes p i c k e d , c r a t e s l o a d e d , t a s s e l s removed, e t c . , but i s confounded w i t h r e s i d u e l e v e l s i n a f f e c t i n g exposure. E s t i m a t i o n of Dose One of the reasons f o r measuring worker p e s t i c i d e exposure i s t o a i d i n d e t e r m i n i n g the s a f e t y o f the p r o d u c t . To do t h i s , i t must be known whether the amount of p e s t i c i d e t o which the worker i s exposed may l e a d to a t o x i c e f f e c t . The t o x i c e f f e c t o f a p e s t i c i d e i s e v a l u a t e d t h r o u g h t e s t i n g i n animals by e s t a b l i s h i n g i t s r e l a t i o n s h i p w i t h dose ( g e n e r a l l y g a s t r o i n t e s t i n a l ) . In o r d e r to e x t r a p o l a t e the animal d a t a to humans, t h e r e must be an e q u i v a l e n t e s t i m a t e of dose. Two approaches have been used to do t h i s i n p e s t i c i d e w o r k e r s . One i n v o l v e s the measurement of dermal exposure (most p r e v a l e n t r o u t e ) w i t h a c o r r e c t i o n f o r the e s t i m a t e d percentage of percutaneous p e n e t r a t i o n . The o t h e r approach r e l a t e s the u r i n a r y m e t a b o l i t e l e v e l ( s ) t o dose. Most b i o l o g i c a l s t u d i e s i n the l i t e r a t u r e were never designed t o q u a n t i t a t i v e l y e s t i m a t e exposure and c a r e must be e x e r c i s e d i n u s i n g such s t u d i e s to draw c o n c l u s i o n s about the u t i l i t y o f b i o l o g i c a l m o n i t o r i n g . B i o l o g i c a l I n d i c a t o r s of P e s t i c i d e E x p o s u r e — P r e v a l e n t l y Methods

Used

B l o o d . One of the e a r l i e r b i o l o g i c a l m o n i t o r i n g s t u d i e s was undertaken by Quinby e t a l . ( 9 ) . C h o l i n e s t e r a s e a c t i v i t y was measured i n a e r i a l a p p l i c a t o r s , t o g e t h e r w i t h r e s i d u e s on worker c l o t h i n g and i n r e s p i r a t o r f i l t e r s . In s p i t e of p h y s i c a l c o m p l a i n t s by the p i l o t s , e i t h e r normal or o n l y s l i g h t l y depressed c h o l i n e s t e r a s e v a l u e s were r e p o r t e d . C h o l i n e s t e r a s e v a l u e s were compared w i t h the 'normal' range f o r the U.S. p o p u l a t i o n r a t h e r than the p i l o t s ' own i n d i v i d u a l 'normal' v a l u e s . In 1952, Kay e t a l . (10) measured c h o l i n e s t e r a s e l e v e l s i n o r c h a r d p a r a t h i o n a p p l i c a t o r s . These were compared w i t h c h o l i n e s t e r a s e l e v e l s t a k e n from the same workers d u r i n g non-spray p e r i o d s . Plasma

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

c h o l i n e s t e r a s e f o r workers r e p o r t i n g p h y s i c a l symptoms was 16% lower d u r i n g the spray p e r i o d . The c o r r e s p o n d i n g r e d u c t i o n f o r symptomless workers was about 13%. Red b l o o d c e l l (RBC) c h o l i n e s t e r a s e was d e p r e s s e d 27% f o r the symptom group v s . 17% f o r the non-symptom group, but t h e s e means were not s t a t i s t i c a l l y d i f f e r e n t . Roan e t a l . (11) measured plasma and e r y t h r o c y t e c h o l i n e s t e r a s e and serum l e v e l s o f e t h y l and m e t h y l p a r a t h i o n i n a e r i a l a p p l i c a t o r s . Serum l e v e l s of the p a r a t h i o n s c o u l d not be c o r r e l a t e d w i t h c h o l i n e s t e r a s e l e v e l s . However, serum l e v e l s d i d c o r r e l a t e w i t h the u r i n e c o n c e n t r a t i o n of p - n i t r o p h e n o l . Drevenkar e t a l . (12) measured plasma and e r y t h r o c y t e c h o l i n e s t e r a s e l e v e l s and u r i n e c o n c e n t r a t i o n s o f organophosphorus and carbamate p e s t i c i d e s i n p l a n t workers who f o r m u l a t e these m a t e r i a l s . No c o r r e l a t i o n e x i s t e d between u r i n a r y m e t a b o l i t e s and c h o l i n e s t e r a s e depression. Bradway e t a l . (13) s t u d i e d c h o l i n e s t e r a s e , b l o o d r e s i d u e s , and u r i n a r y m e t a b o l i t e s i n r a t s E i g h t organophosphorus compounds were i n c l u d e c o n d i t i o n s and known d o s e s w i t h b l o o d r e s i d u e s and u r i n e m e t a b o l i t e l e v e l s were poor. The o v e r r i d i n g g e n e r a l c o n c l u s i o n from t h e s e d a t a i s t h a t c h o l i n e s t e r a s e i n h i b i t i o n c o n t a i n s too many v a r i a b l e s , known and unknown, t o be u s e f u l f o r e s t i m a t i n g dose. Whole b l o o d has r a r e l y been used t o m o n i t o r dose i n humans d e s p i t e p l e a s from p e s t i c i d e c h e m i s t s , t o x i c o l o g i s t s , and p h a r m a c o l o g i s t s ( 1 4 ) . V e r y n i c e s t u d i e s o f 2,4,5-T and 2,4-D i n human b l o o d and u r i n e are r e f e r e n c e s JJ5, 16^ and J7. An o r a l dose of 2,4,5-T was e x c r e t e d i n the u r i n e of f i v e human v o l u n t e e r s w i t h a h a l f - l i f e o f 23 h w h e r e i n 88.5 + 5.1% of the a d m i n i s t e r e d dose was r e c o v e r e d . I t s h a l f - l i f e i n plasma was a l s o 23 h ( 1 5 ) . 2,4^D was t a k e n o r a l l y and a l s o e x c r e t e d unchanged i n u r i n e (75% recovered i n 96 h) ( 1 6 ) . There was marked i n d i v i d u a l v a r i a t i o n i n when the peak o f 2,4-D o c c u r r e d i n plasma. Sauerhoff e t a l . (17) a d m i n i s t e r e d a s i n g l e 5 mg/kg o r a l dose t o f i v e male human v o l u n t e e r s . 2,4-D d i s a p p e a r e d from plasma w i t h an average h a l f - l i f e o f 11.6 h and from u r i n e w i t h an average h a l f - l i f e of 17.7 h. From 88-106% o f the dose was recovered i n u r i n e over 144 h; o v e r 50% was r e c o v e r e d i n 24 h. 2-(2, 4 , 5 - t r i c h l o r o p h e n o x y ) p r o p i o n i c a c i d ( s i l v e x ) was a d m i n i s t e r e d to seven men and one woman at 1 mg/kg. B i p h a s i c d i s a p p e a r a n c e from plasma and u r i n e w i t h h a l f - l i v e s o f 4.0 + 1.9/16.5 + 7.3 h and 5.0 + 1.8/25.9 + 6.3 h, r e s p e c t i v e l y were determined (18) and 65% o f the dose was e x c r e t e d i n u r i n e i n 2 4 h. Human exposure to phenoxy h e r b i c i d e s has been reviewed by Lavy ( 1 9 ) . There are s e v e r a l problems w i t h b l o o d m o n i t o r i n g . It i s d i f f i c u l t ( p r o b a b l y even i m p o s s i b l e w i t h the p r e s e n t human s u b j e c t committee o v e r s i g h t ) to conduct human experiments as i n L5, J£ and 17. A l s o , workers are a v e r s e t o blood t e s t i n g ; n o n i n v a s i v e methods are much p r e f e r r e d . Blood has been used i n e p i d e m i o l o g i c a l s t u d i e s 20» 21) , but an o r i g i n a l dose can p r o b a b l y n e v e r be i n f e r r e d . For the f u t u r e , the r a p i d c l e a r a n c e of newer, b i o d e g r a d a b l e p e s t i c i d e s from b l o o d may e l i m i n a t e b l o o d as an exposure m o n i t o r i n g t o o l f o r p e s t i c i d e dose and may a l s o e l i m i n a t e b l o o d as an e p i d e m i o l o g i c a l m o n i t o r i n g t o o l . However, L e w a l t e r and K o r a l l u s (22) reviewed e r y t h r o c y t e p r o t e i n c o n j u g a t e s as a m o n i t o r i n g t e c h n i q u e f o r p e s t i c i d e e x p o s u r e . Though not v a l i d a t e d , t h i s b l o o d

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

NIGG AND

STAMPER

Pesticide Dose Determination

m o n i t o r i n g t e c h n i q u e appears p r o m i s i n g f o r e s t i m a t i n g dose f o r some p e s t i c i d e s which form e r y t h r o c y t e a d d u c t s . The e r y t h r o c y t e i s e x t r a c t e d f o r adducts of p e s t i c i d e p a r e n t and/or m e t a b o l i t e s . Dose-concentration-effect r e l a t i o n s h i p s may be p o s s i b l e w i t h t h i s t e c h n i q u e . Readers are encouraged to c o n s u l t r e f e r e n c e (22) and references t h e r e i n . U r i n e . The f i r s t problem i n m o n i t o r i n g u r i n e i s the v a r i a b i l i t y i n u r i n a r y o u t p u t . Newborn i n f a n t s may e x c r e t e 17-34 mL/d, 6-10 y e a r o l d boys 459 + 175 mL/d, 10-14 y e a r o l d boys 605 + 2 9 4 mL/d, 6-10 y e a r o l d g i r l s 274 + 125 mL/d, 10-14 year o l d g i r l s 441 + 305 mL/d, men 1360 + 443 mL/d, and women 1000 + 430 mL/d ( 2 3 ) . Men working i n heat e x c r e t e about 900 mL/d (Nigg amd Stamper, u n p u b l i s h e d ) . In one s t u d y , l o n g d i s t a n c e r u n n e r s e x c r e t e d o n l y 600 mL/d whereas c o n t r o l s e x c r e t e d 1300 mL/d (2 4 ) . Some a u t h o r s propose the use of c r e a t i n i n e to n o r m a l i z e d i f f e r e n c e s i n the u r i n a r d i f f e r e n c e s can a r i s e fro p h y s i o l o g i c a l changes such as s w e a t i n g . In o r d e r to be u s e f u l , c r e a t i n i n e i n u r i n e s h o u l d show a c o n s t a n t day-to-day e x c r e t i o n r a t e r e g a r d l e s s o f the volume of u r i n e o u t p u t , food i n t a k e , o r p h y s i c a l a c t i v i t y , and s h o u l d r e f l e c t o n l y s m a l l i n t e r i n d i v i d u a l v a r i a t i o n . In a l a r g e s t u d y o f c r e a t i n i n e e x c r e t i o n , A l e s s i o e t a l . (25) found t h a t men e x c r e t e d more c r e a t i n i n e than women, younger s u b j e c t s more than o l d , and i n d i v i d u a l s v a r i e d i n t h e i r 2 4 h c r e a t i n i n e o u t p u t from 9.2 t o 79.4%, perhaps due to f l u i d i n t a k e . I t appears t h a t g r e a t c a r e must be t a k e n i f c r e a t i n i n e i s to be used to n o r m a l i z e u r i n e samples. Perhaps the b e s t s t u d y o f a d j u s t i n g u r i n e c o n c e n t r a t i o n s of e x c r e t e d s u b s t a n c e s i s by A r a k i e t a l . ( 2 6 ) . These a u t h o r s s t u d i e d the e x c r e t i o n of l e a d , i n o r g a n i c mercury, z i n c , and copper i n u r i n e . They c o n c l u d e d t h a t u r i n a r y f l o w - a d j u s t e d c o n c e n t r a t i o n i s a p p l i c a b l e t o m o n i t o r i n g a l l substances i n u r i n e , c r e a t i n i n e included. C r e a t i n i n e , u r i n a r y s p e c i f i c g r a v i t y , and timed e x c r e t i o n p l u s u r i n a r y f l o w had l i m i t e d u s e s . C o n s e q u e n t l y , i t i s n e c e s s a r y to use 2 4 h, t o t a l u r i n e c o l l e c t i o n when u r i n e w i l l be used to e s t i m a t e the p e s t i c i d e dose. U r i n e m o n i t o r i n g to determine dose n e c e s s a r i l y depends on c e r t a i n assumptions and d i s p a r a t e p i e c e s of a v a i l a b l e d a t a . The e x c r e t i o n k i n e t i c s o f the p e s t i c i d e employed must be known f o r b o t h dermal and o r a l a d m i n i s t r a t i o n . I f the t o t a l dose i s e x c r e t e d by s m a l l a n i m a l s i n 2 4-48 h, the same may a l s o be t r u e of humans. I f the dose i s e x c r e t e d over a p e r i o d of a week, a s i m p l e t e s t c o r r e l a t i n g dermal exposure w i t h an immediate e f f e c t on u r i n e p e s t i c i d e l e v e l s w i l l not work. However, the use of prolonged sampling p e r i o d s over which mean v a l u e s of u r i n a r y p e s t i c i d e e x c r e t i o n r a t e s are c a l c u l a t e d p r e s e n t s problems a l s o . I t i s common to see a 100% c o e f f i c i e n t of v a r i a t i o n among u r i n a r y e x c r e t i o n samples. As a consequence, a major change i n u r i n a r y p e s t i c i d e l e v e l s would have to o c c u r b e f o r e a s i g n i f i c a n t d i f f e r e n c e c o u l d be c o n f i r m e d s t a t i s t i c a l l y . P a r t of t h i s u n f o r t u n a t e v a r i a t i o n no doubt r e s u l t s from r e c k l e s s work p r a c t i c e s . Workers s p i l l c o n c e n t r a t e d m a t e r i a l s . They s e r v i c e contaminated machinery w i t h b a r e hands. We have observed one worker remove h i s g l o v e s , r o l l up h i s p r o t e c t i v e s u i t s l e e v e , and

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

r e t r i e v e a c r e s c e n t wrench from a mixed tank of p e s t i c i d e . However, i f the dermal dose e x c r e t i o n k i n e t i c s are known, i f u r i n e i s r e f l e c t i v e o f d o s e , i f 24 h u r i n e s are c o l l e c t e d and i f sample numbers a r e based on expected v a r i a t i o n , then u r i n e c o u l d be used to m o n i t o r dose and a l s o the e f f i c a c y of p r o t e c t i v e d e v i c e s . Many p e s t i c i d e s i n common use are not e x c r e t e d i n u r i n e i n p r o p o r t i o n t o dose. O r g a n o c h l o r i n e s a r e u s u a l l y e x c r e t e d more i n f e c e s than i n u r i n e . S i g n i f i c a n t s t o r a g e i n body t i s s u e may o c c u r . An example of c u r r e n t i n t e r e s t i s d i c o f o l . W i t h m u l t i p l e o r a l d o s e s , t h i s o r g a n o c h l o r i n e r a p i d l y reached a p l a t e a u i n u r i n e , w h i l e e x c r e t i o n l e v e l s s t e a d i l y i n c r e a s e d i n the f e c e s ( 2 7 ) • The e x c r e t i o n k i n e t i c s a v a i l a b l e f o r most compounds have r e s u l t e d from s t u d i e s u s i n g o n l y one, o r a v e r y few o r a l d o s e s . Workers, however, r e c e i v e d a i l y dermal and r e s p i r a t o r y doses. There are no animal d a t a r e f l e c t i n g t h e s e c o n t i n u o u s and m u l t i p l e r o u t e s of e x p o s u r e . There are k i n e t i c models which c o u l d r e p r e s e n t pesticides recycled int account f o r the d i f f e r e n c e frequency o f dose, o r the subsequent e x c r e t i o n d i f f e r e n c e s i n workers compared to s m a l l a n i m a l s . U r i n a r y m e t a b o l i t e s o f p e s t i c i d e s have been used f o r i n d i c a t i n g a b s o r p t i o n and e x c r e t i o n . Swan (29) measured paraquat i n the u r i n e o f spraymen, G o l l o p and G l a s s (30) and Wagner and Weswig (31) measured a r s e n i c i n timber a p p l i c a t o r s , and Lieben e t a l . (32) measured p a r a n i t r o p h e n o l i n u r i n e a f t e r p a r a t h i o n exposure as d i d Durham e t a l . ( 3 3 ) . C h l o r o b e n z i l a t e m e t a b o l i t e ( d i c h l o r o b e n z i l i c a c i d ) was d e t e c t e d i n c i t r u s workers ( 3 4 ) , phenoxy a c i d h e r b i c i d e s i n farmers (35) , and organophosphorus m e t a b o l i t e s i n the u r i n e o f mosquito t r e a t m e n t a p p l i c a t o r s ( 3 6 ) . Davies e t a l . (37) used u r i n a r y m e t a b o l i t e s of organophosphorus compounds and carbamates t o c o n f i r m p o i s o n i n g c a s e s . These s t u d i e s document e x p o s u r e , but no q u a n t i t a t i v e e s t i m a t e o f dose can be made from u r i n a r y m e t a b o l i t e l e v e l s a l o n e . Other s t u d i e s have used a i r sampling and m o n i t o r e d hand exposure i n c o m b i n a t i o n w i t h u r i n e l e v e l s (38) , and a i r sampling plus cholinesterase i n h i b i t i o n plus urine l e v e l s (39). N o n - b i o l o g i c a l methods, combined w i t h measurement a F u r i n a r y m e t a b o l i t e s , have a l s o been used to compare worker exposure f o r d i f f e r e n t p e s t i c i d e a p p l i c a t i o n methods (^0, _41) as w e l l as t o m o n i t o r f o r m u l a t i n g p l a n t worker exposure (42) and homeowner exposure ( 4 3 ) • S e v e r a l s t u d i e s have used n o n - b i o l o g i c a l methods i n an attempt to c o r r e l a t e u r i n e l e v e l s w i t h an exposure e s t i m a t e (40, 43-48)• Lavy e t a l . (_47 , 48) found no such c o r r e l a t i o n w i t h 2,4-D or 2,4,5-T. Wojeck e t a l . (40) found no paraquat i n u r i n e and c o n s e q u e n t l y no r e l a t i o n s h i p between dose and u r i n e l e v e l s . However, the group d a i l y mean c o n c e n t r a t i o n o f u r i n a r y m e t a b o l i t e s of e t h i o n and the group mean t o t a l e s t i m a t e d dermal dose to e t h i o n on t h a t day c o r r e l a t e d p o s i t i v e l y , w i t h s i g n i f i c a n c e a t the 97% c o n f i d e n c e l e v e l ( 4 6 ) . For a r s e n i c , the c u m u l a t i v e t o t a l e s t i m a t e d dose and d a i l y u r i n a r y a r s e n i c c o n c e n t r a t i o n c o r r e l a t e d p o s i t i v e l y , w i t h s i g n i f i c a n c e a t the 99% c o n f i d e n c e l e v e l ( 4 5 ) . F r a n k l i n e t a l . (43) found a p o s i t i v e c o r r e l a t i o n between 48 h e x c r e t i o n o f a z i n p h o s m e t h y l m e t a b o l i t e s and the amount o f a c t i v e i n g r e d i e n t s p r a y e d . A s i g n i f i c a n t c o r r e l a t i o n c o u l d not be made, however,

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

NIGG AND STAMPER

Pesticide Dose Determination

11

between 48 h e x c r e t i o n and an exposure e s t i m a t e u s i n g d o s i m e t e r s . In the F r a n k l i n e t a l . (43) e x p e r i m e n t , a f l u o r e s c e n t t r a c e r had been added t o the s p r a y m i x t u r e . Q u a l i t a t i v e l y , unmonitored areas ( f a c e , hands, neck) a l s o r e c e i v e d s i g n i f i c a n t e x p o s u r e , perhaps l e a d i n g t o a weak c o r r e l a t i o n between the exposure e s t i m a t e and u r i n a r y m e t a b o l i t e s . F r a n k l i n et a l . (49) a l s o studied e x c r e t i o n of DMTP i n o r c h a r d workers i n two o t h e r areas i n Canada and a g a i n found t h a t u r i n e d a t a more c l o s e l y r e f l e c t e d the amount o f p e s t i c i d e a p p l i e d . Grover e t a l . ( 5 0 ) a l s o o b t a i n e d a c o r r e l a t i o n (R = 0 . 8 2 ) between the amount o f 2,4-D a p p l i e d and u r i n a r y e x c r e t i o n . T o t a l d e p o s i t i o n o f 2,4-D e s t i m a t e d w i t h d o s i m e t e r s and urinary e x c r e t i o n c o r r e l a t e d at only R = 0.47. W i n t e r l i n et a l . ( 5 1 ) monitored the dermal exposure o f a p p l i c a t o r s , m i x e r - l o a d e r s , and s t r a w b e r r y h a r v e s t e r s to captan u s i n g exposure pads. Although the a p p l i c a t o r , m i x e r - l o a d e r group showed h i g h e r e s t i m a t e d dermal e x p o s u r e , no m e t a b o l i t e was d e t e c t e d i n t h e i r u r i n e ; h a r v e s t e r u r i n e had d e t e c t a b l e l e v e l s p r o p e r l y c o l l e c t e d . Contaminatio e t c . , c o u l d have l e d to an o v e r e s t i m a t e d exposure i n the a p p l i c a t o r , m i x e r - l o a d e r group. Leng e t a l . ( 5 2 ) reviewed the e x c r e t i o n o f 2 , 4 , 5 - T i n humans exposed i n the f i e l d . E x c r e t i o n was s l o w e r w i t h dermal f i e l d exposure than w i t h o r a l doses o f 2 , 4 , 5 - T i n humans. K i n e t i c e x c r e t i o n models o f 2 , 4 , 5 - T i n d i c a t e d t h a t the absorbed d a i l y dose d i d not exceed 0 . 1 mg/kg/day. T h i s dose e s t i m a t i o n d i d not agree w i t h exposure e s t i m a t e s made from dermal c o l l e c t i o n patches. P a r t o f the problem appears t o be the e x c r e t i o n k i n e t i c s and e x c r e t i o n r o u t e s o f p e s t i c i d e s , the c o m p l e x i t y o f which may r e n d e r u s e l e s s any s e a r c h f o r a s i m p l e l i n e a r c o r r e l a t i o n between exposure and u r i n a r y m e t a b o l i t e s . Funckes e t a l . ( 5 3 ) exposed the hand and forearm o f human v o l u n t e e r s to 2% p a r a t h i o n d u s t . During e x p o s u r e , the v o l u n t e e r s breathed pure a i r and p l a c e d t h e i r forearm and hand i n t o a p l a s t i c bag which c o n t a i n e d the p a r a t h i o n . T h i s exposure l a s t e d 2 h and was conducted a t v a r i o u s t e m p e r a t u r e s . There was an i n c r e a s e d e x c r e t i o n o f p a r a n i t r o p h e n o l w i t h i n c r e a s i n g exposure temperature. I m p o r t a n t l y , p a r a n i t r o p h e n o l c o u l d s t i l l be d e t e c t e d i n the u r i n e 40 h post exposure a f t e r t h i s dermal exposure. JeJLdmann and Maibach (5 4) dosed human v o l u n t e e r s w i t h r a d i o l a b e l e d C - p e s t i c i d e s i n t r a v e n o u s l y and f o l l o w e d u r i n a r y e x c r e t i o n f o r 5 days u s i n g t o t a l u r i n e c o l l e c t i o n . The same v o l u n t e e r s were s u b s e q u e n t l y exposed d e r m a l l y on the forearm w i t h the same X - p e s t i c i d e s , and u r i n a r y m e t a b o l i t e s were a g a i n f o l l o w e d f o r 5 days. The % p e s t i c i d e e x c r e t e d a f t e r forearm ( d e r m a l ) d o s i n g was then c o r r e c t e d f o r i n c o m p l e t e i . v . e x c r e t i o n . For i n s t a n c e , the dermal e x c r e t i o n ( a b s o r p t i o n ) o f a p e s t i c i d e was doubled i f the i . v . dose was o n l y 50% e x c r e t e d i n u r i n e . ^ C - a z o d r i n , e t h i o n , g u t h i o n , m a l a t h i o n , p a r a t h i o n , baygon, c a r b a r y l , a l d r i n , d i e l d r i n , l i n d a n e , 2,4-D and^diquat were the compounds a p p l i e d . Wester e t a l . (55J) a p p l i e d C-paraquat d i c h l o r i d e to the l e g s , hands and forearms i n a s i m i l a r e x p e r i m e n t . U r i n a r y e x c r e t i o n o f C was f o l l o w e d over 5 days. The h i g h e s t c o r r e c t e d u r i n a r y r e c o v e r y i n these two s t u d i e s was f o r c a r b a r y l a t a p p r o x i m a t e l y 7 4% o f the d e r m a l l y a p p l i e d compound ( f o r e a r m ) ; the l o w e s t was f o r paraquat a t 0.23% (hand). The r e s t o f t h e s e d e r m a l l y - a p p l i e d C-pesticides were r e c o v e r e d i n u r i n e from 0 . 3 % ( d i q u a t ) t o 19.6% (baygon) o f the 1

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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d e r m a l l y a p p l i e d dose. I n d i v i d u a l s v a r y i n t h e i r a b s o r p t i o n o f c h e m i c a l s ; a l s o , d i f f e r e n t anatomic s i t e s absorb c h e m i c a l s a t d i f f e r e n t r a t e s i n b o t h humans and a n i m a l s (56^, 5 7 ) . O c c l u s i o n o f the anatomic s i t e may a l s o i n c r e a s e a b s o r p t i o n o f c h e m i c a l s (5 4, 58, 5 9 ) . I n another human e x p e r i m e n t , Kolmodin-Hedman e t al.~C60) a p p l i e d m e t h y l c h l o r o p h e n o x y a c e t i c a c i d (MCPA) t o t h e t h i g h . Plasma MCPA reached a maximum i n 12 h and MCPA appeared i n t h e u r i n e f o r 5 days w i t h a maximum i n about 48 h. Given o r a l l y , u r i n a r y MCPA peaked i n u r i n e i n 1 h w i t h about 40% o f t h e dose e x c r e t e d i n 2 4 h. U s i n g t h r e e male and two female v o l u n t e e r s , Akerblom e t a l . (61) performed t h r e e experiments w i t h MCPA. I n one e x p e r i m e n t , t h e hands and forearms were d i p p e d i n t o a 2% s o l u t i o n . The d r i e d s o l u t i o n was washed o f f a f t e r 30 min. I n a second e x p e r i m e n t , o r a l MCPA (15 yg/kg) was g i v e n . I n a t h i r d e x p e r i m e n t , 10 mL o f a 10% aqueous s o l u t i o n o f commercial MCPA f o r m u l a t i o n was p l a c e d on a 10 cm^ pad which was a f f i x e d t o t h e t h i g h f o r 2 h U r i n e and b l o o d were m o n i t o r e d u r i n e w i t h t h e hand an c o n c e n t r a t i o n o c c u r r e d a t about 6 h i n u r i n e and 1 h i n plasma. I n the MCPA t h i g h a b s o r p t i o n s t u d y , t h e u r i n e peak o c c u r r e d a t 2 4-48 h. Nolan e t a l . (62) dosed s i x male v o l u n t e e r s o r a l l y w i t h 4 ^ - a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c a c i d ( p i c l o r a m ) . They were exposed d e r m a l l y i n a s e p a r a t e e x p e r i m e n t . I n 72 h , o v e r 90% o f the o r a l dose was e x c r e t e d i n t h e u r i n e . The dermal a p p l i c a t i o n was p o o r l y absorbed (0.2%) and was s t i l l b e i n g e x c r e t e d i n u r i n e a t 72 h. Nolan e t a l . (63) s t u d i e d the e x c r e t i o n o f c h l o r p y r i f o s i n human u r i n e a f t e r an o r a l o r a dermal a p p l i c a t i o n . T o t a l u r i n e was c o l l e c t e d from 24-48 h p r i o r t o a d m i n i s t r a t i o n t h r o u g h 120 h a f t e r a d m i n i s t r a t i o n . 70 +_ 11% ( n - 6) o f t h e o r a l dose was r e c o v e r e d i n urine. 1.28 + 0.83% ( n = 6) o f t h e dermal dose was r e c o v e r e d i n u r i n e . B l o o d l e v e l s peaked i n 6 h a f t e r t h e o r a l dose and i n 24 h a f t e r t h e 5 mg/kg dermal dose. The u r i n e e l i m i n a t i o n h a l f - l i f e was about 27 h f o r b o t h o r a l and dermal doses. In a r a t e x p e r i m e n t , seven d i f f e r e n t organophosphorus coupounds a t two d i f f e r e n t doses were f e d t o r a t s ( 1 3 ) . Rats were removed from exposure a f t e r t h e t h i r d day and b l o o d and u r i n e c o l l e c t e d f o r t h e n e x t 10 d a y s . The average p e r c e n t s o f t h e t o t a l dose e x c r e t e d i n u r i n e over t h e 10 days were: d i m e t h o a t e , 12%; d i c h l o r v o s , 10%; r o n n e l , 11%; d i c h l o f e n t h i o n , 57%; c a r b o p h e n o t h i o n , 66%; p a r a t h i o n , 40%; and l e p t o p h o s , 50%. Very l i t t l e o f t h i s e x c r e t i o n o c c u r r e d beyond t h e t h i r d day post exposure. Intact r e s i d u e s o f r o n n e l , d i c h l o f e n t h i o n , c a r b o p h e n o t h i o n , and l e p t o p h o s were found i n f a t on day 3 and day 8 post exposure. Morgan e t a l . (64) dosed humans o r a l l y w i t h methyl and e t h y l p a r a t h i o n . P a r a n i t r o p h e n o l , w h i c h appears i n u r i n e a f t e r exposure to e i t h e r p e s t i c i d e , was e x c r e t e d a t 37% o f t h e o r e t i c a l w h i l e t h e a l k y l phosphates were e x c r e t e d a t 50%+ o f t h e o r e t i c a l i n 2 4 h . Thus, about 50% o f t h e o r a l dose a d m i n i s t e r e d t o humans was n o t r e c o v e r e d i n u r i n e i n 2 4 h . I n another r a t e x p e r i m e n t , a n i m a l s were a d m i n i s t e r e d a z i n p h o s r a e t h y l d e r m a l l y and i n t r a m u s c u l a r l y ( 6 5 ) . The purpose o f t h e experiment was t o determine whether a l i n e a r response e x i s t e d between t h e amount o f a z i n p h o s m e t h y l a p p l i e d d e r m a l l y and t h e e x c r e t i o n o f DMTP. I f i t d i d , i t i s p o s s i b l e t h a t the DMTP l e v e l s might be u s e f u l t o p r e d i c t dermal exposure. A s i m i l a r l i n e a r r e l a t i o n s h i p appears t o e x i s t i n humans. T h i s

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suggests t h a t DMTP l e v e l s can be m u l t i p l i e d by the r a t i o ( a z i n p h o s m e t h y l exposure)/(DMTP l e v e l i n u r i n e ) . T h i s i s a 10 to 1 r a t i o . U s i n g d a t a from s e v e r a l f i e l d a p p l i c a t i o n s , i t appears t h a t the DMTP-times-10 r e s u l t i s more c l o s e l y r e l a t e d to the amount sprayed than the dermal exposure e s t i m a t e d from patches ( 4 9 ) . F r a n k l i n e t a l . (49) concluded t h a t w i t h o u t adequate p h a r m a c o k i n e t i c i n f o r m a t i o n f o r the parent compound, u r i n e i s u s e f u l o n l y as a s c r e e n i n g t o o l f o r i n d i c a t i n g exposure or as a p r e d i c t o r of overexposure. These experiments i l l u s t r a t e the e x c r e t i o n d i f f e r e n c e s between d e r m a l , i n t r a m u s c u l a r , and o r a l a d m i n i s t r a t i o n , the e x c r e t i o n d i f f e r e n c e s between compounds, and a l s o problems about which u r i n a r y m e t a b o l i t e to monitor ( 6 5 ) . Consequently, a v e r y d i s c r i m i n a t i n g e x p e r i m e n t a l d e s i g n would be n e c e s s a r y t o model dermal exposure and a b s o r p t i o n v s . u r i n a r y m e t a b o l i t e l e v e l s . S t a t i s t i c a l problems, c e n t e r e d on r e p l i c a t e v a r i a t i o n and the resulting necessity fo c o s t o f e i t h e r a human A s m a l l a n i m a l e x c r e t i o n study w i t h m u l t i p l e a p p l i c a t i o n s c o u l d be done. However, w h i c h a d m i n i s t r a t i o n method s h o u l d be used? Should i t be d e r m a l , s i n c e the human worker r e c e i v e s m o s t l y dermal exposure, o r s h o u l d i t be a m i x t u r e o f o r a l , d e r m a l , and r e s p i r a t o r y ? What i f the compound has a s s o c i a t e d d a t a s u g g e s t i n g s k i n p e n e t r a t i o n i s low? Wester and Noonan (66) have reviewed the r e l e v a n c e o f a n i m a l models f o r percutaneous a b s o r p t i o n . The p i g and the monkey were the most n e a r l y l i k e humans i n t h i s r e s p e c t . These a u t h o r s warn t h a t a c l e a r u n d e r s t a n d i n g o f the s p e c i e s and methods used i s a p r e r e q u i s i t e to e x t r a p o l a t i o n to humans. Dedek (67) p r e s e n t s an i n t e r e s t i n g case f o r the e f f e c t o f s o l u b i l i t y o f p e s t i c i d e s i n s o l v e n t s and s k i n p e n e t r a t i o n . P o l a r compounds p e n e t r a t e d b e t t e r when a p p l i e d i n n o n p o l a r s o l v e n t s and nonpolar compounds p e n e t r a t e d b e t t e r i n p o l a r s o l v e n t s . The e f f e c t o f s o l v e n t and c o n c e n t r a t i o n s h o u l d be i n c l u d e d i n dermal p e n e t r a t i o n e x p e r i m e n t s . S i n c e r e g u l a t i o n s a r e designed to p r o t e c t humans, r e a l w o r l d e s t i m a t e s , r e l e v a n c e t o work exposure, and a c c u r a c y i n the p r e l i m i n a r y s m a l l a n i m a l s t u d i e s a r e e x t r e m e l y i m p o r t a n t . Another p o s s i b i l i t y i s t o perform an a c t u a l human e x c r e t i o n s t u d y . At the end o f the s p r a y season, w i t h the workers removed from f u r t h e r ex posure , a s e r i e s o f 24 h u r i n e samples might y i e l d the n e c e s s a r y k i n e t i c d a t a . We suggest s p l i t t i n g the d a i l y sampling p e r i o d s i n t o two 12 h o r f o u r 6 h segments. For compounds which a r e v e r y r a p i d l y e x c r e t e d , t h i s d i v i s i o n i n t o timed segments w i l l h e l p to o b t a i n the n e c e s s a r y d a t a p o i n t s . Removal o f workers from exposure i s paramount, even i f the e x p e r i m e n t e r must c l o s e l y m o n i t o r t h e i r work a c t i v i t i e s and wash the workers' c l o t h i n g over t h i s p e r i o d . We have d i s c o v e r e d , t o our s u r p r i s e , t h a t workers have i n t e r p r e t e d 'removal from exposure' as 'stop s p r a y i n g , ' and then proceeded to c l e a n and m a i n t a i n h e a v i l y contaminated equipment over our sampling p e r i o d . S p i t t l e r e t a l . (68) d i s c u s s e d a v a r i e t y of 'extraneous' p e s t i c i d e exposure p o s s i b i l i t i e s . Even i f a l l of t h e s e f a c t o r s are c o n t r o l l e d , the c h e m i c a l t y p e , i t s metabolism and v a r i a b i l i t y among s u b j e c t s , may p r o h i b i t the drawing o f s t a t i s t i c a l l y valid inferences.

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B i o l o g i c a l I n d i c a t o r s o f P e s t i c i d e E x p o s u r e — N e w Methods Vagal Tone. V a g a l tone m o n i t o r i n g r e f e r s t o the r e s p i r a t o r y - c a r d i a c n e u r a l r e f l e x and i s measured by q u a n t i f y i n g the a m p l i t u d e o f the r e s p i r a t o r y s i n u s a r r h y t h m i a . R e s p i r a t o r y s i n u s a r r h y t h m i a i s d e s c r i b e d as a decrease i n h e a r t p e r i o d w i t h i n s p i r a t i o n and an i n c r e a s e w i t h e x p i r a t i o n (6£, 7 0 ) . T h i s i s a complex p h y s i o l o g i c a l f u n c t i o n , but i t can be measured. I t s measurement i l l u s t r a t e s a d e l a y e d e f f e c t from organophosphorus compounds ( 6 9 , 70, 7 1 ) . The exposure of m i l i t a r y p e r s o n n e l to n e u r o t o x i c agents has g e n e r a t e d i n t e r e s t i n t h i s method. I n d i c h l o v o s t r e a t e d dogs, v a g a l tone was not a l t e r e d 90 min a f t e r treatment. Plasma CHE was 47% of normal; RBC c h o l i n e s t e r a s e 56% of normal. N i n e t y min a f t e r 2-PAM and a t r o p i n e a d m i n i s t r a t i o n , v a g a l tone was l e s s d i s r u p t e d i n treatment dogs v s . c o n t r o l dogs. Plasma CHE was i n h i b i t e d a t 47% i . e . not r e a c t i v a t e d ; RBC CHE was 83% o f normal. Three weeks l a t e r a c t i v i t i e s were i n h i b i t e a t r o p i n e , the v a g a l tone response of e x p e r i m e n t a l dogs was a t t e n u a t e d compared to c o n t r o l dogs. The d e l a y e d e f f e c t on v a g a l tone by OP compounds may s i g n a l d e l a y e d human performance changes. A s i m i l a r experiment was conducted w i t h Rhesus monkeys t r e a t e d w i t h p y r i d o s t i g m i n e . A l t h o u g h a d e l a y e d e f f e c t was not i n v e s t i g a t e d , the same a t t e n u a t e d v a g a l tone response to a t r o p i n e was demonstrated i n the s h o r t term ( 7 2 ) . The a t r o p i n e r e s p o n s e , i t s e l f , i s also important. Since a t r o p i n e i m p a i r s the performance of a v i a t o r s (71) and monkeys ( 6 9 ) , the i m p l i c a t i o n s f o r s e l f - d o s i n g m i l i t a r y p e r s o n n e l are o b v i o u s . I t may be p o s s i b l e t o measure v a g a l tone response to dose and t h i s might be p o s s i b l e over a l o n g p e r i o d of t i m e . I f s o , a ( q u a l i t a t i v e ) dose of an organophosphorus compound might be i n f e r r e d from a n o n i n v a s i v e p h y s i o l o g i c a l measurement l o n g a f t e r the event. These experiments i l l u s t r a t e an i n n o v a t i v e and a t t r a c t i v e approach to b i o l o g i c a l m o n i t o r i n g . Perhaps we have o v e r l o o k e d o t h e r p h y s i o l o g i c a l responses f o r m o n i t o r i n g exposure to pesticides. S a l i v a . S a l i v a has never been popular as a m o n i t o r i n g medium f o r p e s t i c i d e s . Perhaps s u s p i c i o n t h a t o r a l c o n t a m i n a t i o n i s too l i k e l y o r t h a t d a i l y t o t a l s a l i v a p r o d u c t i o n i s too d i f f i c u l t to q u a n t i f y has l e d to t h i s s i t u a t i o n . I t a l s o may be t h a t a l l s a l i v a r y p e s t i c i d e , once swallowed, w i l l u l t i m a t e l y appear as u r i n a r y p e s t i c i d e , but t h i s has not been e s t a b l i s h e d . N o n e t h e l e s s , s a l i v a has been used to i n d i c a t e exposure f o r a wide v a r i e t y o f other environmental contaminants. S a l i v a would seem t o o f f e r advantages f o r m o n i t o r i n g p e s t i c i d e s . We produce about 1000 mL of s a l i v a per day ( 2 3 ) . S a l i v a c o l l e c t i o n i s r e l a t i v e l y easy and procedures such as p r e - r i n s e s may be used to a v o i d c o n t a m i n a t i o n . Saliva secretion i s v a r i a b l e w i t h t i m e ; t h e r e f o r e , samples may have t o be taken a t the same time each day ( 7 3 ) . I t s use f o r m o n i t o r i n g d r u g s , m e t a l s , hormones, immunoglobulins, t e s t o s t e r o n e , s t r e s s , phosphate, p r o g e s t e r o n e s , e s t r i o l s and p e s t i c i d e s has been shown. Stowe and P l a a (7_4) i n t h e i r r e v i e w o f the l i t e r a t u r e through June 1967 on e x t r a r e n a l s e c r e t i o n c i t e d r e f e r e n c e s which demonstrated the

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excretion i n s a l i v a of s u l f a drugs, b a r b i t u r a t e s , p e n i c i l l i n , d i h y d r o s t r e p t o m y c i n and t e t r a c y c l i n e s . Drugs such as d i g i t a l i s (75, 7 6 ) , nefopam ( 7 7 ) , c a f f e i n e , t h e o p h y l l i n e and theobromine ( 7 8 ) , c a n n a b i s ( 7 9 ) , and p h e n y l c y c l i d i n e (80) have been i s o l a t e d i n s a l i v a and p a i r e d w i t h d e t e r m i n a t i o n s i n u r i n e o r plasma. S a l i v a has been used w i t h p s y c h i a t r i c p a t i e n t s undergoing l i t h i u m t h e r a p y as an i n d i c a t o r f o r l i t h i u m (81) and as a s y s t e m i c i n d i c a t o r f o r z i n c d u r i n g pregnancy (82) as w e l l as f o r p o t a s s i u m and c a l c i u m (83, 8 4, 85) and magnesium ( 8 6 ) . M e t a l s i n the environment, e.g., l e a d T87) , cadmium (88) , c o p p e r , s i l v e r , t i n , mercury, and z i n c (89) have been found i n the a n a l y s i s o f s a l i v a r y samples. S a l i v a r y phosphate has been used as an i n d i c a t o r o f o v u l a t i o n ( 8 3 ) . IgA l e v e l s i n s a l i v a i n c r e a s e d u r i n g pregnancy ( 9 0 ) and d e c r e a s e i n cases o f academic s t r e s s ( 9 1 ) . IgA and IgM changes a r e r e f l e c t e d i n rheumatoid a r t h r i t i s ( 9 2 ) . S a l i v a r y hormones may be used i n the d i a g n o s i s o f some forms o f c a n c e r ( 9 3 94 9 5 ) Testosterone i n s a l i v a has been used a and C o r t i s o l as a d e t e r m i n a n S a l i v a r y p r o g e s t e r o n e s and e s t r i o l s have been used i n the assessment o f o v a r i a n f u n c t i o n ( 9 7 , 98, 99) and d u r i n g pregnancy as i n d i c a t o r s o f f e t a l v i a b i l i t y (100-105). S e c r e t i o n o f p e s t i c i d e s i n s a l i v a has been demonstrated ( 8 3 , 106, 107, 1 0 8 ) . S k a l s k y e t a l . (106) s t u d i e d the e f f e c t s o f p a r a t h i o n and c a r b o f u r a n on c h o l i n e s t e r a s e a c t i v i t y i n plasma, e r y t h r o c y t e s and s a l i v a . Rats were dosed o r a l l y by d i r e c t i n t u b a t i o n . A c o r r e l a t i o n between c h o l i n e s t e r a s e a c t i v i t i e s was o b t a i n e d . S k a l s k y e t a l . (107) a l s o s t u d i e d the e f f e c t o f c a r b a r y l on s a l i v a r y and serum c h o l i n e s t e r a s e and c a r b a r y l d i s t r i b u t i o n i n t e a r s , u r i n e , s a l i v a , and b l o o d o f r a t s . The abdominal w a l l was opened and C - c a r b a r y l was i n j e c t e d d i r e c t l y i n t o t h e stomach a t doses o f 0, 50, 100 o r 200 mg/kg. At a 200 mg/kg dose, c a r b a r y l s e c r e t i o n i n s a l i v a peaked i n about 60 m i n . The plasma peak o c c u r r e d i n about 75 min. Maximum plasma c o n c e n t r a t i o n was 35 ppm, s a l i v a about 28 ppm. By t h i n l a y e r chromatography ( a t t h e 200 mg/kg dose) t h e same t h r e e compounds, i n c l u d i n g c a r b a r y l , appeared i n s a l i v a , plasma, and RBC. Tears c o n t a i n e d c a r b a r y l and cx-naphthol. U r i n e c o n t a i n e d 9 compounds, t h e major b e i n g a - n a p h t h o l . At t h e 100 and 50 mg/kg d o s e s , s a l i v a showed o n l y two compounds: c a r b a r y l and a n o t h e r m e t a b o l i t e w h i c h d i d n o t appear a t the 200 mg/kg dose. S a l i v a , plasma, and RBC c h o l i n e s t e r a s e s were i n h i b i t e d a t 50 mg/kg. B o r z e l l e c a and S k a l s k y (108) s t u d i e d s a l i v a r y s e c r e t i o n o f c h l o r d e c o n e i n r a t s . Dosing was by stomach i n j e c t i o n as i n 107 a t 50 mg/kg. S a l i v a r y f l o w was s t i m u l a t e d by p i l o c a r p i n e . Chlordecone peaked i n s a l i v a i n 6-2 4 h. C-chlordecone appeared i n s a l i v a a t l e s s than 1/2 t h e plasma l e v e l ; however, c h l o r d e c o n e e x c r e t i o n was m o n i t o r e d f o r o n l y 48 h. There a r e problems w i t h u s i n g s a l i v a as i n t h e s e e x p e r i m e n t s . Human s a l i v a r y e s t e r a s e s a r e c a r b o x y l e s t e r a s e s (109) and much o f t h i s "human" enzyme a c t i v i t y may be c o n t r i b u t e d by o r a l b a c t e r i a l enzymes ( 1 1 0 ) . I t i s unknown how t h e s e r e s u l t s w i t h p e s t i c i d e s r e l a t e t o humans. There was no mass b a l a n c e work done w i t h t h e s e a n i m a l s , so we have no i d e a o f t h e r e l a t i o n s h i p between t h e p e s t i c i d e i n s a l i v a and the t o t a l p e s t i c i d e e x c r e t e d o r dose r e c e i v e d . These were stomach, o r e s s e n t i a l l y o r a l , doses. What

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might the r e s u l t s have been w i t h dermal a p p l i c a t i o n s ? Mass b a l a n c e experiments i n s m a l l a n i m a l s and semi-mass b a l a n c e experiments i n humans would be r e q u i r e d f o r d e t e r m i n i n g the r e l a t i o n s h i p t o dose. S a l i v a appears to p r e s e n t l e s s a n a l y t i c a l c o m p l e x i t y compared to u r i n e . At l e a s t i n r a t s , no c a r b a r y l was d e t e c t e d i n s a l i v a a f t e r 2 4 h. I f t h i s were t r u e f o r humans, s a l i v a might be used t o p r e d i c t when an exposure had o c c u r r e d , i . e . , i f a compound were p r e s e n t , exposure might have o c c u r r e d w i t h i n 2 4 h. For workers exposed t o p e s t i c i d e s on a d a i l y b a s i s , s a l i v a might be an e s t i m a t o r o f dose. As f o r u r i n e , e x c r e t i o n k i n e t i c s would have t o be known. E x c r e t i o n p a t t e r n s o f p a r e n t and m e t a b o l i t e i n r e l a t i o n to dose would have to be known. U n f o r t u n a t e l y , we cannot c o l l e c t the t o t a l d a i l y e x c r e t i o n o f s a l i v a as we can f o r u r i n e . These problems, s p e c u l a t i o n s and s u p p o s i t i o n s a w a i t e x p e r i m e n t a l resolution. Sweat. Feldmann and Maibac sweating on dermal p e n e t r a t i o was perhaps the f i r s t s u g g e s t i o n t h a t sweat might be i m p o r t a n t to the human p e s t i c i d e a b s o r p t i o n / e x c r e t i o n p r o c e s s . L e t us assume t h a t a l l problems a s s o c i a t e d w i t h m o n i t o r i n g u r i n e f o r p e s t i c i d e s and m e t a b o l i t e s have been s o l v e d . F u r t h e r , assume t h a t the dermal exposure o f s e v e r a l workers i s e x a c t l y the same i n each of two weeks. Can we then expect the mean l e v e l o f u r i n a r y m e t a b o l i t e s d u r i n g the second week t o e q u a l t h a t d u r i n g the f i r s t week? Rosenberg e t a l . (111) used sweat patches to c o l l e c t d i c h l o r o b e n z i l i c a c i d ( a m e t a b o l i t e of c h l o r o b e n z i l a t e ) from the backs o f c h l o r o b e n z i l a t e a p p l i c a t o r s . U r i n e a n a l y s e s were a l s o performed. The p r o p o r t i o n found i n sweat and u r i n e was a p p r o x i m a t e l y 1:1. S e l l e t a l . (112) conducted a human exposure experiment w i t h 2,4-D and monitored u r i n e and sweat. Of the t o t a l 2,4-D e x c r e t e d over one week a f t e r a s i n g l e dermal a p p l i c a t i o n t o the hand, 15% was e x c r e t e d i n u r i n e and 85% was found i n sweat (average f o r t h r e e w o r k e r s ) . There a r e , however, many q u e s t i o n s about j u s t how the 2,4-D experiment was performed based on the 2,4-D human e x c r e t i o n d a t a r e f e r e n c e d here i n the u r i n e s e c t i o n , v a l i d a t i o n o f a n a l y t i c a l s e n s i t i v i t y and the c o n t i n u e d wearing of contaminated p e r s o n a l a r t i c l e s . Although the sweat m o n i t o r i n g d a t a f o r p e s t i c i d e s i s p r e l i m i n a r y , we have o v e r l o o k e d sweat e x c r e t i o n i n our d e s i g n . U n f o r t u n a t e l y , r a t s do not sweat, so r a t u r i n a r y e x c r e t i o n d a t a may not be a p p l i c a b l e to humans. I f a p e s t i c i d e and/or m e t a b o l i t e i s e x c r e t e d i n sweat, the s w e a t / u r i n e e x c r e t i o n r a t i o may change w i t h the human p h y s i o l o g i c a l response to h e a t . C o n s i d e r how l i t t l e we know about t h i s p r o c e s s . Could a dermal exposure, s u b s e q u e n t l y m a n i f e s t e d i n sweat, s i m p l y be p e s t i c i d e r e c y c l e d a t the s k i n l e v e l ? I s t h i s then an " i n t e r n a l " dose? Does a sweat c o l l e c t i o n d e v i c e a l s o c o l l e c t i n t e r s t i t i a l f l u i d ? If i t c o l l e c t e d o n l y i n t e r s t i t i a l f l u i d , we c o u l d q u i c k l y e s t i m a t e an i n t e r n a l dose. Peck e t a l . ( 1 1 3 , 114) have s t u d i e d the t r a n s d e r m a l c o l l e c t i o n o f drugs and p a r a t h i o n i n s m a l l a n i m a l s . A model f o r c h e m i c a l d i s p o s i t i o n i n s k i n i s a v a i l a b l e ( 1 1 5 ) . The experiments w i t h humans are w a i t i n g t o be r u n . There i s the problem of q u a n t i f y i n g sweat which makes i t a l e s s a t t r a c t i v e medium t o m o n i t o r . However, c y s t i c f i b r o s i s i s diagnosed w i t h a sweat t e s t which depends on q u a n t i f i c a t i o n and

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

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Pesticide Dose Determination

17

Webster (116) has reviewed the problems of sweat q u a n t i f i c a t i o n . Q u a n t i f i c a t i o n may not be a s e r i o u s problem l o c a l l y , but the r a t e of sweating and t o t a l d a i l y p r o d u c t i o n would have to be e s t i m a t e d . There a r e e s t i m a t e s o f f l u i d l o s s due to sweating a v a i l a b l e . A n a t i o n a l l y ranked 47 kg runner sweated a t 1.2-1.3 L/h ( r e d u c t i o n i n body wt = 5%+) over 10 m i l e s and 82 min ( 1 1 7 ) . Twelve, u n a c c l i m a t i z e d males were t e s t e d a t 30.0 + 0.7°C WBGT, 1.0 + 1 m/s wind v e l o c i t y on a t r e a d m i l l ( 1 1 8 ) . S u b j e c t s walked/climbed f o r 30 min and r e s t e d s i t t i n g f o r 30 min over 6 h. They sweated a t a r a t e of about 425 mL/h w h i l e w a l k i n g . T h i s r a t e decreased to 125 mL/h w h i l e s i t t i n g . T h e i r c u m u l a t i v e sweat l o s s over 6 h averaged about 3300 mL. T h i s l o s s was o f f s e t by water i n t a k e over the 6 h. Seven men e x e r c i s i n g a t 50% of maximum oxygen uptake averaged 2.19 L water l o s s through sweat i n 2 h ( 1 1 9 ) . Marathoners i n a warm environment may l o s e 2.8 L/h through sweat and i n c u r 8% l o s s o f body weight through sweat r e s u l t i n g i n a 13-14% l o s s of body water (2 4). I n s p i t e of q u a n t i f i c a t i o r o u t e of p e s t i c i d e e x c r e t i o estimates i s warranted. When the e x c r e t i o n o f p e s t i c i d e m e t a b o l i t e s and parent compounds i n human sweat i s u n d e r s t o o d , we may be a b l e to determine our p o s s i b l e u n d e r e s t i m a t i o n of exposure or dose through the use of u r i n e o n l y . For comments on the f u t u r e o f dermal m o n i t o r i n g t e c h n o l o g y , we r e f e r the reader to Peck e t a l . ( 1 2 0 ) . I f u r i n a r y e x c r e t i o n k i n e t i c s f o l l o w i n g dermal exposure a r e i g n o r e d , the i n t e r p r e t a t i o n o f the r e s u l t s o f a u r i n e experiment might be 100% wrong. I f , i n a d d i t i o n , sweat e x c r e t i o n i s i g n o r e d , a d d i t i o n a l 100% m i s t a k e s appear p o s s i b l e . Table I p r e s e n t s a h y p o t h e t i c a l case o f p e s t i c i d e e x c r e t i o n i n a worker i n a hot environment. T y p i c a l a d u l t d a i l y f l u i d e l i m i n a t i o n r a t e s were a s s i g n e d to u r i n e , sweat, and sebum. From t y p i c a l p e s t i c i d e c o n c e n t r a t i o n s w i t h i n those f l u i d s f o l l o w i n g a s i n g l e dermal exposure, d a i l y p e s t i c i d e e x c r e t i o n r a t e s were e s t i m a t e d f o r each o f the t h r e e e l i m i n a t i o n pathways. A t o t a l i n t e r n a l dose was then i n f e r r e d under the assumption t h a t 100% of the dose was executed d u r i n g the day. I f the i n t e r n a l dose were e s t i m a t e d from u r i n e d a t a a l o n e , the r e s u l t would have been r o u g h l y one-half of that c a l c u l a t e d . Do the rough c a l c u l a t i o n s o f Table I suggest a r e a l problem? I f we r e t u r n to the d a t a of Feldmann and Maibach (5 4 ) , 100% of an i . v . dose o f 2,4HD was e x c r e t e d i n u r i n e i n 120 h. In 24 h, about 70% was r e c o v e r e d . The range f o r r e c o v e r y over 120 h of i . v . doses of b i o d e g r a d a b l e ( m e t a b o l i z a b l e ) p e s t i c i d e s was 7.4% ( c a r b a r y l ) 90.2% ( m a l a t h i o n ) . I n 2 4 h, i . v . m a l a t h i o n appeared i n u r i n e a t about 88% and c a r b a r y l a t about 5% of the i . v . d o s e . An i . v . a d m i n i s t r a t i o n i s the i d e a l case s i n c e i t r e p r e s e n t s a known i n t e r n a l dose. The average t o t a l dose r e c o v e r e d i n u r i n e f o r a z o d r i n , e t h i o n , g u t h i o n , m a l a t h i o n and p a r a t h i o n , a l l c o n s i d e r e d b i o d e g r a d a b l e , was 62.3% over 5 days o f t o t a l u r i n e c o l l e c t i o n a f t e r an i . v . dose. We s h o u l d , as s c i e n t i s t s , account f o r the r e s t of the dose. Do Feldmann and M a i b a c h s d a t a suggest o t h e r r o u t e s of e x c r e t i o n ? Are some of these compounds or t h e i r m e t a b o l i t e s e x c r e t e d i n sweat? Are some e x c r e t e d i n s a l i v a e i t h e r as p a r e n t o r m e t a b o l i t e ( s ) and o n l y p a r t i a l l y reabsorbed and e x c r e t e d i n u r i n e 1

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

18

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE Table I . T y p i c a l E x c r e t i o n Rates* o f N o n i n v a s i v e B i o l o g i c a l F l u i d s and an E s t i m a t e d I n t e r n a l Dose a f t e r a S i n g l e Dermal Exposure

Fluid excretion rate

Ref. No.

Estimated pesticide cone.

Urine + swallowed saliva (excreted in urine)

900 mL/d

**

50 ng/mL

Sweat***

6,400 mL/

Sebum

3.8 mg/d

23

8 pg/mg

Fluid

Total

Pesticide excretion rate

% of total

45 yg/d

3 x 10~

47

5

yg/d

—

96 yg/d

T o t a l Dose = 96 yg, o r 0.0014 mg/kg

* L i g h t , s t e a d y work by a 70 hg a d u l t , temperatures above 32°C **Nigg and Stamper, u n p u b l i s h e d ***8h, 0.6 L/h; 16 h , 0.1 L/h, r e f e r e n c e 23, 117-119 or s t o r e d i n adipose t i s s u e o r m e t a b o l i z e d and e x c r e t e d as CO^? Only f o r 2,4-D does i t appear t h a t u r i n e m o n i t o r i n g would be adequate. The f i r s t problem o f modeling the e x c r e t i o n p r o c e s s as i n Table I l i e s w i t h t h e o r a l (stomach) d o s i n g o f t h e r a t s i n most k i n e t i c s t u d i e s . The k i n e t i c s o f e x c r e t i o n a r e d i f f e r e n t f o r o r a l v s . dermal a d m i n i s t r a t i o n s . S i n c e r a t s do n o t sweat, r a t d a t a , even f o l l o w i n g a dermal exposure, may n o t be a p p l i c a b l e . We do not p r e s e n t l y have t h e d a t a t o make an e s t i m a t e as i n Table I . We have f a i l e d t o c o l l e c t the b a s i c i n f o r m a t i o n on human e x c r e t i o n o f o r g a n i c m o l e c u l e s , i . e . , t h e r a t and mouse models r e a l l y a p p l y o n l y to r a t s and m i c e . Table I I p r e s e n t s t h e i . v . d a t a o f Feldmann and Maibach ( r e f . 54, p. 129, Table I ) i n a d i f f e r e n t form. Since the o r i g i n a l h a l f - l i v e s were e x c r e t i o n r a t e h a l f - l i v e s , we transformed t h e o r i g i n a l d a t a t o p e r c e n t - d o s e - u n a c c o u n t e d - f o r by m u l t i p l y i n g e x c r e t i o n r a t e by h o u r s , p r o d u c i n g a c u m u l a t i v e t o t a l f o r each o f the 8 time p e r i o d s monitored and s u b t r a c t i n g t h i s v a l u e from 100. These d e c r e a s i n g p e r c e n t v a l u e s were then s u b j e c t e d t o a first-order linear regression analysis. Correlation coefficients from t h a t a n a l y s i s a r e p r e s e n t e d . The l i n e a r r e g r e s s i o n l i n e s a l l o w e d us t o p r e d i c t s t a t i s t i c a l l y t h e times a t which 50% (and a l s o 90%) o f t h e i . v . dose would be e l i m i n a t e d i n u r i n e . N i n e t y - f i v e % c o n f i d e n c e i n t e r n a l s were c a l c u l a t e d f o r each o f t h e two t i m e s . Except f o r 2,4-D, t h e p r o j e c t i o n s o f Table I I had t o be

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.

NIGG AND STAMPER

19

Pesticide Dose Determination

extended beyond t h e range o f the d a t a . T h i s procedure adds t o t h e u n c e r t a i n t y i n t h e p r o j e c t i o n s , as evidenced by some o f t h e l a r g e r c o n f i d e n c e i n t e r v a l s . Because o f the n e c e s s i t y i n t h i s experiment to extend t h e model beyond e x p e r i m e n t a l d a t a , Maibach e t a l . extend u r i n a r y e x c r e t i o n experiments t o n o n - d e t e c t i o n ( H . Maibach, p e r s o n a l communication). We encourage o t h e r i n v e s t i g a t o r s i n t h i s a r e a t o conduct u r i n a r y e x c r e t i o n experiments t o n o n - d e t e c t i o n . Elimination or excretion rate h a l f - l i v e s are v i r t u a l l y useless f o r p r e d i c t i n g dose i f o n l y a s m a l l percentage o f t h e dose i s accounted f o r w i t h d a t a . The same r a t e h a l f - l i f e c o u l d r e s u l t , r e g a r d l e s s o f whether 10 o r 100% o f the dose was e x c r e t e d . M a t h e m a t i c a l r e p r e s e n t a t i o n might p r e s e n t problems w i t h u r i n a r y e x c r e t i o n o f o r a l doses and w i l l c e r t a i n l y be a problem w i t h Table I I .

P r o j e c t e d U r i n a r y E x c r e t i o n o f P e s t i c i d e s Using Data o f Feldman d Maibach (5 4)*

P r o j e c t e d p e r i o d f o r 50%and 90% e x c r e t i o n o f t o t a l i . v . dose first-order correlation 50% e x c r e t i o n 90% e x c r e t i o n Pesticide coefficient p e r i o d (h) p e r i o d (h) (n = 8)

Azodrin Ethion Guthion Malathion Parathion Baygon Carbaryl 2,4-D Diquat *95%

76 187 68 73 134 99 1670 17 204

confidence

[60, 1 0 4 ] * [129, 337] [58, 83] [37, 1284] [90, 258] [51, 1263] [1063, 389 4] [16, 19] [129 , 493]

253 620 226 2 41 445 329 55 48 58 679

[200, 3 4 5 ] * [429, 1120] [192, 276] [12 4, 4264] [300, 857] [171, 4194] [3532, 12936] [53, 64] [429, 1637]

0.966 0.913 0.984 0.727 0.901 0.735 0.868 0.997 0.863

i n t e r v a l (h)

d e r m a l l y - a p p l i e d p e s t i c i d e s w h i c h p e n e t r a t e human s k i n p o o r l y and are e x c r e t e d i n a n o n - f i r s t - o r d e r manner. Problems which have n o t been r e s o l v e d w i t h any human e x c r e t i o n o f p e s t i c i d e s i n c l u d e whether t h e e x c r e t i o n r a t e o r r o u t e i s dose dependent. We encourage, i n any e v e n t , mass-balance o r near mass-balance experiments. Combination o f N o n b i o l o g i c a l and B i o l o g i c a l E s t i m a t i o n Methods Here, we d i s c u s s i n g r e a t e r d e t a i l the s t a t i s t i c a l c o n s i d e r a t i o n s i n e x p e r i m e n t a l d e s i g n f o r t h e f i e l d e s t i m a t i o n o f dose by combining b i o l o g i c a l and n o n - b i o l o g i c a l methods. Data a n a l y s i s f o r c u r r e n t experiments u s u a l l y n e c e s s i t a t e s comparing two mean v a l u e s , each from about t h e same number o f r e p l i c a t i o n s . F o r examples, we might w i s h t o compare t h e mean u r i n a r y l e v e l o f a p e s t i c i d e m e t a b o l i t e i n a week when c o v e r a l l s were worn v s . a week when they were n o t worn o r t o compare t h e mean p e s t i c i d e c o n c e n t r a t i o n on one c o l l e c t i o n d e v i c e w i t h t h a t on another c o l l e c t i o n d e v i c e . The

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE number of r e p l i c a t i o n s per mean i s t y p i c a l l y the product o f the number o f sampling p e r i o d s ( s a m p l i n g days) and the number o f p a r t i c i p a t i n g s u b j e c t s , but t h i s i s c o n t i n g e n t upon v a l i d a t i o n , by an a n a l y s i s o f v a r i a n c e o f the o b t a i n e d d a t a , t h a t exposure l e v e l s do not v a r y s i g n i f i c a n t l y day-to-day o r s u b j e c t - t o - s u b j e c t . T h i s may be the case i f a l l s u b j e c t s performed the same t a s k s each day. The q u e s t i o n must be addressed as t o how many r e p l i c a t e s ( s u b j e c t s X days) w i l l be n e c e s s a r y to c o n f i r m s t a t i s t i c a l l y a d i f f e r e n c e between means. Two parameters must be e s t i m a t e d f i r s t . What d i f f e r e n c e i n means i s expected to a r i s e from the d a t a and what v a r i a t i o n among r e p l i c a t e s i s expected? A l a r g e e s t i m a t e f o r the f i r s t e x p e c t a t i o n or a s m a l l e s t i m a t e f o r the second e x p e c t a t i o n would reduce the number of r e p l i c a t e s n e c e s s a r y per mean. Our e x p e r i e n c e has shown t h a t p r o t e c t i v e c l o t h i n g reduces mean pad exposure by about 90% ( 1 2 1 ) . S i m i l a r l y , the use of g l o v e s may reduce handwash r e s i d u e s by about 85% a c c o r d i n g t o a r e c e n t study o f d i c o f o l exposur among r e p l i c a t e s , the c o e f f i c i e n 100% f o r exposure pad and handwash r e s i d u e s ( 6 , 121) • T h i s v a r i a t i o n c o u l d a l s o s u b s e q u e n t l y a f f e c t the v a r i a t i o n i n p a r e n t / m e t a b o l i t e q u a n t i t i e s i n b i o l o g i c a l samples. Standard s t a t i s t i c a l procedures then show (123) t h a t , on the b a s i s of the above e s t i m a t e s , two means may be judged s i g n i f i c a n t l y d i f f e r e n t a t t h e 95% c o n f i d e n c e l e v e l by t a k i n g a t l e a s t t e n r e p l i c a t i o n s per mean. The (approximate) c a l c u l a t i o n i s n > 8 (100% r 90%) , or n > 10 r e p l i c a t i o n s . For g l o v e s , the c o r r e s p o n d i n g c a l c u l a t i o n would be n > 8 (100% * 85%) , o r n > 11 r e p l i c a t i o n s . These numbers r e p r e s e n t an a b s o l u t e minimum based on the above two expected v a l u e s . An i n c r e a s e i n n o f 50% t o p r o v i d e some margin f o r e r r o r , i n the above cases to n > 15 and n > 17, i s c e r t a i n l y warranted i n v i e w o f the guesswork i n v o l v e d about sample means and v a r i a n c e s . I f more than two means a r e to be compared c o n c u r r e n t l y , as w i t h comparing r e s i d u e s a t v a r i o u s body l o c a t i o n s , the s i t u a t i o n becomes somewhat more c o m p l i c a t e d . While i t i s now harder to g e n e r a l i z e , the optimum number o f r e p l i c a t i o n s per mean can be r o u g h l y adduced by the same c a l c u l a t i o n , w i t h the f i n a l r e s u l t t h a t the means are grouped i n t o s i g n i f i c a n t l y d i f f e r e n t c a t e g o r i e s , a t some c o n f i d e n c e l e v e l . Whether to u t i l i z e , e.g., t h r e e s u b j e c t s f o r s i x sampling p e r i o d s e a c h , o r s i x s u b j e c t s f o r t h r e e sampling p e r i o d s e a c h , to o b t a i n , s a y , 18 r e p l i c a t i o n s i s u s u a l l y d i c t a t e d by f a c t o r s o t h e r than s t a t i s t i c a l ones. A good r u l e to f o l l o w i s not t o o v e r l o a d the d e s i g n too h e a v i l y i n f a v o r of e i t h e r v a r i a b l e . I f many s u b j e c t s are used f o r a v e r y few sampling d a y s , and the d a t a i n d i c a t e l a r g e d i f f e r e n c e s from s u b j e c t to s u b j e c t the a v a i l a b l e number o f r e p l i c a t e s now d e c r e a s e s to the number o f sampling days a l o n e . T h i s experiment makes a v e r y u n r e l i a b l e statement about each o f many s u b j e c t s ; no v a l i d o v e r a l l c o n c l u s i o n may emerge. Remember t h a t these s t a t i s t i c a l c a l c u l a t i o n s show s i g n i f i c a n t d i f f e r e n c e s between means, but do not e s t i m a t e the d i f f e r e n c e . Suppose one mean i s 90% l e s s than the o t h e r , as above, but t h a t one wishes to v a l i d a t e the c l a i m t h a t 50 o f t h a t 90% i s s t a t i s t i c a l l y s i g n i f i c a n t . The approximate c a l c u l a t i o n f o r the r e q u i s i t e number

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o f r e p l i c a t i o n s per mean i s now n > 8 [100% * (90% - 5 0 % ) ] - 50, or n > 75 w i t h the s a f e t y f a c t o r . From another p e r s p e c t i v e , suppose 24 h u r i n e s were c o l l e c t e d from 20 workers d u r i n g two d i f f e r e n t p e r i o d s : one where workers wore p r o t e c t i v e c l o t h i n g and one where they d i d n o t . The u r i n e e x c r e t i o n means f o r the two p e r i o d s would have t o d i f f e r by 48% t o be s t a t i s t i c a l l y d i f f e r e n t a t the 95% l e v e l , assuming a c o e f f i c i e n t of v a r i a t i o n o f 75%. For f i e l d s t u d i e s u s i n g fewer than 20 s u b j e c t s ( r e p l i c a t e s ) , d i f f e r e n c e s i n u r i n a r y e x c r e t i o n means would have to exceed 48% f o r a s t a t i s t i c a l d i f f e r e n c e to be v a l i d a t e d . Another c o n s i d e r a t i o n i s the amount o f c h e m i c a l removed by a c o l l e c t i o n d e v i c e which s u b s e q u e n t l y i s not e x c r e t e d i n u r i n e because i t never became an i n t e r n a l dose. I f twenty 103 cm £4 i n . x 4 i n . ) pads are used f o r m o n i t o r i n g a w o r k e r , about 2000 cm of body s u r f a c e a r e a would be c o v e r e d . The average human has a s u r f a c e a r e a o f c a . 20,000 cm • Since 10% o f the body s u r f a c e a r e a i s c o v e r e d , w i l l t h i s reduc H a r d l y . F i r s t , the pad u s u a l l y o n l y one o r two h o u r s , over the e i g h t hour workday. The maximum percentage r e d u c t i o n i s thus 5%. T h i s 5% d i f f e r e n c e c o u l d be reduced by i n c o m p l e t e percutaneous a b s o r p t i o n . We c h a l l e n g e any r e s e a r c h e r t o show a s t a t i s t i c a l l y v a l i d 5% d i f f e r e n c e i n u r i n a r y e x c r e t i o n i n any f i e l d experiment. The use o f a hand r i n s e o r c o t t o n g l o v e s t o m o n i t o r hand exposure may i n v a l i d a t e u r i n e r e s u l t s . Suppose workers who n o r m a l l y work ungloved and do not c u s t o m a r i l y wash t h e i r hands p a r t i c i p a t e i n an exposure s t u d y . R e q u i r i n g t h a t they wear g l o v e s i n o r d e r t o m o n i t o r hand exposure might reduce t h e i r t o t a l dermal exposure, and, hence, t h e i r u r i n a r y m e t a b o l i t e l e v e l , by 21%. This i s on the assumption t h a t hand exposure o r i g i n a l l y c o n s t i t u t e d 50% of the t o t a l dermal exposure and t h a t the g l o v e s , worn f o r 1/2 o f the workday, gave 85% p r o t e c t i o n . R e q u i r i n g handwashes might l e a d to a s i m i l a r r e d u c t i o n . However, f o r workers who n o r m a l l y wear g l o v e s or who r e g u l a r l y wash t h e i r hands, hand r i n s e s o r c o t t o n g l o v e m o n i t o r s may not a f f e c t the e x p e r i m e n t a l outcome. P r o p e r l y designed b i o l o g i c a l m o n i t o r i n g s t u d i e s are a f u t u r e p o s s i b i l i t y and p r o b a b i l i t y . As p r e v i o u s l y s t a t e d , the e x c r e t i o n k i n e t i c s o f p e s t i c i d e s and p e s t i c i d e m e t a b o l i t e s i n sweat and u r i n e and perhaps s a l i v a a f t e r a dermal exposure must be understood b e f o r e any b i o l o g i c a l f l u i d can be v a l i d a t e d as a dose e s t i m a t o r . When we understand human exposure r o u t e a b s o r p t i o n and e x c r e t i o n r o u t e k i n e t i c s and t h e i r i n t e r r e l a t i o n s h i p s , b i o l o g i c a l f l u i d m o n i t o r i n g w i l l a l l o w an a c c u r a t e c a l c u l a t i o n o f p e s t i c i d e exposure and i n t e r n a l dose. Research

Needs

There a r e s p e c i f i c q u e s t i o n s f u t u r e r e s e a r c h s h o u l d answer b e f o r e b i o l o g i c a l m o n i t o r i n g can be r e l i a b l y used to e s t i m a t e dose and risk. 1. 2. 3.

What are the r o u t e s and k i n e t i c s o f e x c r e t i o n i n humans f o l l o w i n g dermal exposure to a chemical? What i s the r e l a t i o n s h i p between i n t e r n a l dose and the r o u t e s and k i n e t i c s o f e x c r e t i o n i n humans? What p h y s i o l o g i c a l changes does a human undergo i n

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4.

5.

6. 7.

General

response t o a p e s t i c i d e and can these changes be r e l a t e d to dose? How do t h e i n t e r r e l a t i o n s h i p s between r o u t e s o f e x c r e t i o n change w i t h changes i n human p h y s i o l o g y , i . e . , heat s t r e s s , work l o a d , e t c . ? What a r e t h e ranges i n t o t a l d a i l y o u t p u t volumes o f t h e v a r i o u s human r o u t e s o f p e s t i c i d e e x c r e t i o n i n r e l a t i o n t o d i f f e r e n t working c o n d i t i o n s ? Can we use these ranges t o e s t i m a t e a dose from grab samples? Can we c o l l e c t i n t e r s t i t i a l f l u i d i n humans and measure p a r e n t and m e t a b o l i t e m o l e c u l e s t o e s t i m a t e dose? Do we see changes i n e x c r e t i o n p a t t e r n s and e x c r e t e d m e t a b o l i t e s i n human workers exposed t o a p e s t i c i d e day a f t e r day? Conclusions

Much o f o u r p r e s e n t d a t human u r i n e , b l o o d , s a l i v a , and o t h e r b i o l o g i c a l f l u i d s p r e c l u d e t h e i r use f o r d e t e r m i n i n g i n t e r n a l dose. A n o t a b l e e x c e p t i o n t o t h i s statement a r e t h e phenoxy h e r b i c i d e s . We s h o u l d remember, however, t h a t few b i o l o g i c a l m o n i t o r i n g experiments i n the l i t e r a t u r e were d e s i g n e d t o m o n i t o r dose. The u n c e r t a i n t y i n a dose e s t i m a t e based on b i o l o g i c a l m o n i t o r i n g g e n e r a l l y r e s u l t s from the l a c k o f b a s i c i n f o r m a t i o n on r e l e v a n t human b i o c h e m i s t r y and p h y s i o l o g y . We must proceed w i t h new b i o l o g i c a l m o n i t o r i n g experiments designed t o e s t i m a t e dose and we must be c o g n i z a n t o f the p o s s i b l e a p p l i c a t i o n o f d a t a never c o l l e c t e d . Biological m o n i t o r i n g f o r p e s t i c i d e dose e s t i m a t i o n i s a v i a b l e t e c h n o l o g i c a l f r o n t i e r , one which c o u l d s i m p l i f y r e g u l a t o r y problems. Science can and s h o u l d p r o v i d e t h i s t e c h n o l o g y . Acknowledgmen t s We a p p r e c i a t e the e x c e l l e n t r e v i e w s o f t h i s m a n u s c r i p t by Dr. M a r g u e r i t e Leng, Dow C h e m i c a l , Dr. C l a i r e F r a n k l i n , H e a l t h and W e l f a r e , Canada, and an anonymous r e v i e w e r . T h e i r s u g g e s t i o n s improved t h i s r e v i e w i n v e r y p o s i t i v e ways.

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Riley, R. T.; Kemppainen, B. W.; Norred, W. P. J. Toxicol. Environ. Hlth. 1985, 15, 769-77. Kolraodin-Hedman, B.; Hoglund, S.; Swenson, A.; Akerblom, M. Arch. Toxicol. 1983, 54, 267-73. Akerblom, M.; Kolmodin-Hedman, B.; Hoglund, S., in Hum. Welfare Environ., J. Miyamoto, Ed., Pergamon Press, New York, 1983, pp. 227-32. Nolan, R. J.; Freshour, N. L.; Kastl, P. E.; Saunders, J. H. Toxicol. Appl. Pharmacol. 1984,76,264-69. Nolan, R. J.; Rick, D. L.; Freshour, N. L.; Saunders, J. H. Toxicol. Appl. Pharmacol. 1984, 73, 8-15. Morgan, D. P.; Hetzler, H. L.; Slach, E. F.; Lin, L. I. Arch. Environ. Contam. Toxicol. 1977, 6, 159-73. Franklin, C. A.; Greenhalgh, R.; Maibach, H. I., in Hum. Welfare Environ., J. Miyamoto, Ed., Pergamon Press, New York, 1983, pp. 221-26. Wester, R.C.;Noonan 99-110. Dedek, W. In "Field Worker Exposure during Pesticide Application"; W. F. Tordoir, E. A. H. van Heenstra, Eds.; Elsevier, New York, 1980; pp. 47-50. Spittler, T. D.; Dewey, J. E.; Bourks, J. B. In "Determination and Assessment of Pesticide Exposure"; M. Siewierski, Ed.; Elsevier, New York, 1984; pp. 27-35. Jansen, H. T.; Dellinger, J. A. Neurotoxicol Teratol 1987, in press. Dellinger, J. A.; McKiernan, B.C.;Koritz, G. D.; Richardson, B. C. Neurotoxicol Teratol 1987,9,197-201. Dellinger, J. A.; Taylor, H. L.; Porges, S. W. Aviat Space Environ. Med. 1987, 58, 333-38. Birnbaum, S. G.; Richardson, B.C.;Dallinger, J. A. Pharmacol. Biochem. Behavior. 1988, 31, in press. Ferguson, D. B.; Botchway, C. A. Arch. Oral Biol. 1980, 25, 559-68. Stowe, C. M.; Plaa, G. L. Ann. Rev. Pharmacol. 1968, 8, 337-56. Ben Aryeh, H.; Gutman, D. Isr. J. Dent. Med. 1977, 26(2), 5-9. Ben Aryeh, H.; Gutman, D. Isr. J. Dent. Med. 1981, 29(1-2), 7-11. Chang, S. F.; Hanse, C. S.; Fox, J. M.; Ober, R. E. J. Chromatogr. 1981, 226(1), 79-89. Scott, N. R.; Chakraborty, J.; Marks, V. Ann. Clin. Biochem. 1984, 21(Pt2), 120-24. Gross, S. J.; Worthy, T. E.; Nerder, L.; Zimmermann, E. G.; Soares, J. R.; Lomax, P. J. Anal. Toxicol. 1985, 9(1), 1-5. McCurran, M. M.; Walberg, C. B.; Soares, J.; Gross, S. J.; Baselt, R. C. J. Anal. Toxicol. 1984, 8(5), 197-201. Ben Aryeh, H.; Laor, R.; Szargel, R.; Gutman, D.; Naon, H.; Pascal, M.; Hefetz, A. Isr. J. Med. Sci. 1984, 20(3), 197-201. Hambridge, E. M.; Krebs, N. F.; Jacobs, M. A.; Favier, A.; Guyette, L.; Ikie, D. N. Am. J. Clin. Nutr. 1983, 37, 429-42. Ben Aryeh, H.; Gutman, D. In Proceedings of the International

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

26

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Workshop at Luxembourg; Berlin, A., et al., Eds.; Martin Nijhoff Publishers, The Hague, 18-22 April 1977. 84. Ben Aryeh, H.; Bergman, S.; Gutman, D.; Barzilai, B.; Hammerman, H.; Szargel, R. Chest 1977, 72, 131-32. 85. Wotman, S.; Bigger, J. T., Jr.; Mandel, I. D.; Bartelston, H. J. N. E. J. Med. 1971, 285(16), 871. 86. Gilfrich, H. J. Klin. Wochenschr. 1981, 59(12), 617-21. 87. P'an A. Y. J. Toxicol. Environ. Health 1981, 7(2), 273-80. 88. Gervais, L.; Lacasse, Y.; Brodeur, J.; P'an A. Toxicol. Lett. 1981, B(l-2), 63-6. 89. Nilner, K.; Glantz, P. O. Swed. Dent. J. 1982, 6(2), 71-7. 90. Widerstom, L.; Bratthal, D., Scand. J. Dent. Res. 1984, 92, 33-7. 91. Jemmott, J. B.; Borysenko, M.; Chapman, R.; Borysenko, J. Z.; McClelland, D. C.; Meye, D. Lancet Apr. 1983, (8339), 1400-02. 92. Elkon, K. B.; Gharave Frankel, A. Clin 93. Turkes, A.; Pennington, G. W.; Griffiths, K. Eur. J. Cancer Oncol. 1984, 20(2), 291-92. 94. Read, G. F.; Wilson, D. W.; Campbell, F. C.; Holliday, H. W.; Blamey, R. W.; Griffiths, K. Eur. J. Cancer Clin. Oncol. 1983, 19(4), 477-83. 95. Read, G. F.; Bradley, J. A.; Wilson, D. W.; George, W. D.; Griffiths, K. Eur. J. Cancer Clin. Oncol. 1985, 21(1), 9-17. 96. Walker, R. F.; Wilson, D. W.; Read, G. F.; Riad-Fahmy, D. Int. J. Andol. 1980, 3(2), 105-20. 97. Walker, R. F.; Hughes, I. A.; Riad-Fahmy, D.; Read, G. F. Lancet Sept. 1978, 9, 585. 98. Walker, S.; Mustafa, A.; Walker, R. F.; Riad-Fahmy, D. Brit. J. Obstet. Gynecol. 1981, 88, 1009-15. 99. McGarricle, H. H.; Lachelin, G. C. L. B. J. Med. 1984, 289, 457 -59. 100. Robinson, J.; Walker, S.; Read, G. F.; Riad-Fahny, D. Lancet. May 16 1981, 1111-12. 101. Vining, R. F.; McGinley, R. A.; Symons, R. G., Clin. Chem. 1983, 29(10), 1752-56. 102. Vining, R. F.; McGinley, R. A.; Rice, B. V. J. Clin. Endocrinol. Metab. 1983a, 56, 454. 103. Zaki, K.; El Hak, R.; Amer, W.; Saleh, F.; El Faras, A.; Ragar, L.; Nour, H. Biomed. Biochim. Acta 1984, 43(6), 749-54. 104. Cusick, P. E.; Truran, P. L.; Read, G. F.; Alsotn, W. C. Ann. Clin. Biochem. 1984, 21, 22-5. 105. Lechner, W.; Marth, C.; Daxenbichler, G. Acta Endocrinol. 1985, 109, 266-68. 106. Skalsky, H. L.; Keeling, B. M.; Borzelleca, J. F. Toxicol. Appli. Pharmacol. 1979, 48(1 Part 2), A140. 107. Skalsky, H. L.; Lane, R. W.; Borzelleca, J. In Toxicol. Occupat. Med.; Deichmann, W. B., Ed.; Elsvier, New York, 1978, pp. 349-58. 108. Borzelleca, J.; Skalsky, H. L. J. Environ. Sci. Health [B] 1980, B15(6), 843-66. 109. Lindquist, L.; Augustinsson, K. B. Enzyme 1975, 20, 277. 110. Mahler, J. R.; Chauncey, H. H. J. Dent. Res. 1975, 36, 338.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1. NIGG AND STAMPER

Pesticide Dose Determination

27

111. Rosenberg, N. M.; Queen, R. M.; Stamper, J . H. Bull. Environ. Contam. Toxicol. 1985, 35, 68-72. 112. S e l l , C. R.; Maitlen, J. C.; Aller, W. A. Perspiration as an important physiological pathway for the elimination of 2, 4-diclorophenoxyacetic acid from the human body. Presented at Amer. Chem. Soc. Mtg, Las Vegas, NV, April 1, 1982. 113. Peck, C. C.; Conner, D. P.; Bolden, B. J.; Almirez, R. G.; Rowland, L. M.; Kwiatkowski, T. E.; McKelvin, B. A.; Bradley, C. R. Pharmacol. Skin 1987, 1, 201-8. 114. Peck, C.; Conner, D.; Bolden, B.; Kingsley, T . ; Mell, L.; Kwiatkowski, T.; Hill, V.; Ezra, D.; Dubois, A.; Rahim, M. A.; Murphy, G. Transdermal chemical collection: A novel research tool. Presented at Third Annual Symp. Skin Pharmacol. Soc., Karolinska Inst., Stockholm, Sweden, August 1-2, 1986. 115. Peck, C.; Conner, D.; Scheuplein, R.; Dedrick, R.; McDougal, J. Physiologically based pharmacokinetic modeling of chemical disposition in skin Pharmacol. Soc., Karolinsk 1-2, 1986. 116. Webster, H. L., in CRC Critical Reviews in Clinical Laboratory Sciences, CRC Press, Boca Raton, FL, 1983, 18, 113-338. 117. C o s t i l l , D. L . (Moderator) Ann. N.Y. Acad. Sci. 1977, 301, 183-88. 118. Armstrong, L. E.; Hubbard, R. W.; Szlyk, P. C.; Matthew, B. S.; Sils, I. V. Aviation, Space and Environ. Med. 1985, August, 765-70. 119. C o s t i l l , D. L.; Cote, R.; Fink, W. J.; Van Handel, P. Int. J. Sports Med. 1981, 2, 130-3 4. 120. Peck, C. C.; Lee, K.; Becker, C. E. J . Pharmacokinetics and Biopharmaceutics 1981, 9, 41-58. 121. Nigg, H. N.; Stamper, J . H. Arch. Environ. Contam. Toxicol. 1983, 12, 477-82. 122. Nigg, H. N.; Stamper, J . H . ; Queen, R. M. Arch. Environ. Contam. Toxicol. 1986, 15, 121-43. 123. Dowdy, S.; Weardon, S., in Stat, for Res., Wiley, New York, 1983, p. 196. RECEIVED July 5, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 2

Ways To Reduce Applicator Exposure to Pesticides J. E. Cowell, S. Dubelman, A. J. Klein, and K. Ohta Life Sciences Research Center, Environmental Science Department, Technology Division, Monsanto Agricultural Company, Chesterfield, MO 63198 Measurement of applicator exposure to pesticides has allowed scientist approaches to th sive dosimetry and b i o l o g i c a monitoring applicato exposure studies have been performed by our department on various pesticides. In these studies, four general areas have been identified which have an effect upon reduction of worker exposure. These four areas encompass: worker apparel, product packaging, application equipment and personal hygiene. Worker clothing and rubber gloves have been found effective i n reducing personal exposure. Small container size and closed transfer systems have been found to be important in mixer/loader exposure reduction, while tractors with closed cabs have led to reduction i n application exposure. F i n a l l y , important factors i n the reduction of applicator exposure have been found to be the workers' level of instruction for use of the pesticides and the workers' individual personal hygiene practices. Scientists have been interested i n the measurement of worker exposure to pesticides since the 1950s. In 1962, Durham and Wolfe reviewed, i n d e t a i l , the methods available for measurement of the exposure of workers to pesticides.(I) In 1985, a f a i r l y comprehensive review of the l i t e r a t u r e from 1951-1984 was conducted by Turnbull (2). Basically there are two approaches used to measure exposure, and these approaches have not changed radically throughout the years. The f i r s t approach, pioneered by Durham and Wolfe measures the external deposition or the amount of pesticide with which the worker's body comes into contact. This approach i s u t i l i z e d primarily when l i t t l e information on the pharmacokinetics (absorption, metabolism and excretion) of the chemical i s available. The second approach i s the monitoring of the worker's body fluids (usually urine or blood) for levels of the pesticide or metabolites or for changes i n enzyme a c t i v i t y . 0097-6156/89/0382-0028$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

2.

COWELL ET AL.

Ways To Reduce Applicator Exposure

T h i s has been c a l l e d b i o l o g i c a l m o n i t o r i n g . I f the pharmacokinet i c s o f a c h e m i c a l are known, t h e n as the World H e a l t h O r g a n i z a t i o n s t a t e s i n t h e i r 1982 p r o t o c o l , " B i o l o g i c a l m o n i t o r i n g p r o v i d e s a q u a n t i t a t i v e measure of the absorbed p e s t i c i d e r e s u l t i n g from exposure v i a a l l r o u t e s " ( 3 ) . Both approaches are v a l u a b l e i n t h a t each p r o v i d e s i n f o r m a t i o n t h a t the o t h e r does n o t . S i n c e most o f the exposure t o p e s t i c i d e i s from the dermal r o u t e , the Durham and Wolfe " p a t c h t e c h n i q u e " can e s t i m a t e the q u a n t i t y o f p e s t i c i d e t h a t comes i n t o c o n t a c t w i t h the s k i n . I t a l s o p r o v i d e s some i n f o r m a t i o n on the exposure a t s p e c i f i c s i t e s . T h i s has p r o v e n advantageous i n d e m o n s t r a t i n g avenues f o r exposure r e d u c t i o n ( 4 ) . The " b i o l o g i c a l m o n i t o r i n g t e c h n i q u e " a u t o m a t i c a l l y accounts f o r i n h a l a t i o n , o r a l exposure, c l o t h i n g p r o t e c t i o n and percutaneous a b s o r p t i o n w h i c h a l l have t o be assumed o r e s t i m a t e d v i a a d d i t i o n a l e x p e r i ments w i t h the p a t c h t e c h n i q u e ( 5 ) W i t h s u f f i c i e n t pharmacokin e t i c data, b i o l o g i c a l measure o f the t o t a l bod o f the a p p l i c a t o r ' s exposure. T h i s c a p a b i l i t y proves v e r y advantageous i n making r i s k assessments. A l t h o u g h some f e e l t h a t the two t e c h n i q u e s cannot be p e r formed c o n c u r r e n t l y ( 6 ) , o t h e r s i n c l u d i n g m y s e l f f e e l t h a t the t e c h n i q u e s are c o m p a t i b l e ( 7 - 9 ) . With p r o p e r p a t c h placement and c o o r d i n a t e d hand washes, the t e c h n i q u e s can be combined t o g i v e the g r e a t e s t amount o f i n f o r m a t i o n from a s i n g l e s t u d y . Consider the f o l l o w i n g p o i n t s : 1. The patches are a t t a c h e d t o the o u t e r c l o t h i n g . Thus any n o r m a l l y exposed s k i n such as f a c e , hands o r neck would not be o b s t r u c t e d from p o t e n t i a l dermal d e p o s i t i o n o r p e n e t r a t i o n . 2. I n many s t u d i e s (10-14), normal c l o t h i n g has approached 100% p r o t e c t i o n from dermal d e p o s i t i o n and t h e r e f o r e i n these c a s e s , patches would have no d e l e t e r i o u s impact upon b i o l o g i c a l monitoring. 3. About 10 t o 12 patches a t 100 square c e n t i m e t e r s p e r p a t c h are used on the a p p l i c a t o r . These patches amount t o o n l y 1000 t o 1200 square c e n t i m e t e r s o f body s u r f a c e area w h i c h i s o n l y a p p r o x i m a t e l y 6% of the average t o t a l body s u r f a c e a r e a . 4. Thus the w o r s t case would be t h a t the b i o l o g i c a l monit o r i n g i s 6% too low. However i n h a l a t i o n and o r a l exposure would be u n a f f e c t e d , thus a 1.06 c o r r e c t i o n f a c t o r may be i n a p p r o p r i a t e . 5. Hand washes w i t h a l c o h o l i c / a q u e o u s s o l u t i o n s remove the p e s t i c i d e d e p o s i t e d on the hands (I) (15-18). Soap and warm water r i n s e s have a l s o been used. I t has been s t a t e d t h a t hand washes may not be a c c u r a t e s i n c e they do not account f o r the c h e m i c a l a l r e a d y absorbed (19) . The t e c h n i q u e o f hand washing i s n o t i n c o m p a t i b l e w i t h the conduct of a b i o l o g i c a l m o n i t o r i n g s t u d y s i n c e these hand washes j u s t s i m u l a t e the normal p e r s o n a l hygiene p r a c t i c e of the a p p l i c a t o r washing h i s hands a f t e r he f i n i s h e s working. Measurement o f a p p l i c a t o r exposure t o p e s t i c i d e s by b o t h types o f s t u d i e s has been used t o e v a l u a t e approaches t o the r e d u c t i o n of exposure (20-22). Data developed by our department i n many d i f f e r e n t s t u d i e s employing the p a t c h o r b i o l o g i c a l m o n i t o r i n g t e c h n i q u e s o r a t times b o t h , w i l l be used t o d e s c r i b e avenues

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

29

30

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

f o r exposure r e d u c t i o n . These i n d i v i d u a l s t u d i e s w i l l n o t be d e s c r i b e d i n t h e i r e n t i r e t y as some s t u d i e s have a l r e a d y been p u b l i s h e d , o t h e r s i n c l u d i n g methodology a r e d e s c r i b e d i n a companion paper i n t h i s symposium ( 3 5 ) , and y e t o t h e r s a r e t o be published. Four g e n e r a l areas have been i d e n t i f i e d f o r r e d u c t i o n o f worker exposure: 1. Worker A p p a r e l 2. P r o d u c t P a c k a g i n g 3. A p p l i c a t i o n Equipment 4. P e r s o n a l Hygiene I n t h e worker a p p a r e l a r e a , c l o t h i n g i t s e l f reduces exposure (23). S e v e r a l s t u d i e s have been performed u t i l i z i n g p a t c h e s on the o u t s i d e and i n s i d e o f c l o t h i n g a p p r o p r i a t e l y o f f s e t so as t o not i n t e r f e r e w i t h p e n e t r a t i o n o f normal c l o t h i n g . C o r r e c t i o n f a c t o r s o b t a i n e d by d i v i d i n g t h e o u t s i d e p a t c h r e s i d u e by t h e i n s i d e p a t c h r e s i d u e , whe to 530 f o l d range (24-25) i n o u r s t u d i e s s i n c e 1983 and one p o s i t i v e e f f e c t o f t h i s v i d e o t a p i n g o f m i x e r / l o a d e r and a p p l i c a t i o n experiments has been t h e i d e n t i f i c a t i o n o f sources o f c o n t a m i n a t i o n . F o r i n s t a n c e , i n one study f o u r experiments t h a t c o n s i s t e d o f 2 i n d i v i d u a l s p e r f o r m i n g 2 r e p l i c a t e s o f m i x i n g / l o a d i n g and a p p l y i n g c h e m i c a l t o a 0.5 a c r e r i c e paddy each by a d i f f e r e n t mode, were viewed on tape and i t was observed t h a t some o f t h e i n s i d e p a t c h e s may have been contaminated upon removal from t h e a p p l i c a t o r ( 2 5 ) . D u r i n g r e p l i c a t e experiments t h e n e x t day w i t h t h e same i n d i v i d u a l s and c o n d i t i o n s , t h e i n s i d e p a t c h e s were c a r e f u l l y removed w i t h s o l v e n t r i n s e d f o r c e p s and t h e r e s u l t s o f these two s e t s o f e x p e r i m e n t a l a n a l y s e s can be seen i n T a b l e I :

Table I .

I n s i d e P a t c h A n a l y s e s (micrograms/square Backpack Spray - I n d i v i d u a l Number 1

centimeter)

DAY 2

DAY 1 Rep

1.

0.0134

Rep

1.

<0.00500

Rep

2.

0.00945

Rep

2.

<0.00500

Mechanical

Spreader - I n d i v i d u a l Number 2 DAY 2

DAY 1 Rep

1.

0.196

Rep

1,

<0.00500

Rep

2.

0.0434

Rep

2.

<0.00500

T h i s i s n o t meant t o i m p l y t h a t e v e r y i n s i d e gauze p a t c h that contains a residue i s a r e s u l t of contamination during removal, b u t o n l y t h a t c l o t h i n g o f f e r s e x c e l l e n t p r o t e c t i o n and

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

2.

COWELLETAL.

Ways To Reduce Applicator Exposure

c a r e s h o u l d be t a k e n t o a v o i d c o n t a m i n a t i o n i n t h e s e types o f experiments. Impermeable g l o v e s a r e another obvious p i e c e o f p r o t e c t i v e equipment which can be used t o reduce exposure. Exposure t o the forearms and hands has been found t o account f o r a p p r o x i m a t e l y 70-90% o r more o f the t o t a l dermal exposure. Some s t u d i e s have been performed w i t h c o t t o n g l o v e s worn on the o u t s i d e and i n s i d e of p r o t e c t i v e rubber g l o v e s (14) ( 2 5 ) . S o l v e n t e x t r a c t i o n and GC a n a l y s e s o f b o t h o u t s i d e and i n s i d e c o t t o n g l o v e s were performed. Comparison o f the a n a l y t i c a l r e s u l t s o f th e o u t s i d e v e r s u s the i n s i d e c o t t o n g l o v e s have y i e l d e d g l o v e p r o t e c t i o n f a c t o r s i n our s t u d i e s o f 130 t o 233 f o l d w h i c h compares f a v o r a b l y w i t h a n o t h e r l i t e r a t u r e r e p o r t e d v a l u e o f 220 f o l d ( 2 1 ) . P e n e t r a t i o n s t u d i e s w i t h v a r i o u s g l o v e m a t e r i a l s have p r o v i d e d d a t a as t o which m a t e r i a l s are the most i m p e r v i o u s t o c h e m i c a l p e n e t r a t i o n . However use o f i m p e n e t r a b l e g l o v e materi a l s i n exposure s t u d i e exposure. Cotton glove s t i l l c o n t a i n e d d e t e c t a b l e r e s i d u e s (26-27). The r e s u l t s o f s o l v e n t e x t r a c t i o n and GC a n a l y s i s o f i n d i v i d u a l gauze m a t e r i a l worn underneath each rubber g l o v e r e v e a l e d one hand had s i g n i f i c a n t l y h i g h e r d e p o s i t i o n t h a n the o t h e r ( 2 4 ) , as shown i n T a b l e II:

T a b l e I I . Hand D e p o s i t i o n (micrograms/square R i g h t Hand

centimeters)

L e f t Hand

S u b j e c t 1.

1.63

0.724

(2.3X)

S u b j e c t 2.

11.6

389

(34X)

S u b j e c t 3.

18.6

4.14

(4.5X)

S u b j e c t 4.

0.976

20.7

(21X)

A l t h o u g h too few r e p l i c a t e s were performed to a l l o w s t a t i s t i c a l i n t e r p r e t a t i o n , w i t h h e l p from the v i d e o t a p e s and v i s u a l o b s e r v a t i o n i t was surmised t h a t the d e p o s i t i o n was a r e s u l t o f the g l o v e removal p r o c e s s as one r u b b e r - g l o v e d hand removed one rubber g l o v e and the c o t t o n - g l o v e d hand removed t h e o t h e r rubber g l o v e . I n e f f e c t , most rubber g l o v e m a t e r i a l s a r e adequate f o r the d u r a t i o n o f worker exposures t o most p e s t i c i d e s . Because o f t h i s p o t e n t i a l r o u t e o f exposure, EPA i s now a d v i s i n g t h a t t h e o u t s i d e o f rubber g l o v e s be washed b e f o r e removal. In the p r o d u c t p a c k a g i n g a r e a , dermal d e p o s i t i o n data have been developed which seem t o i n d i c a t e p o u r i n g o f l a r g e r and more d i f f i c u l t t o handle c o n t a i n e r s i s a cause o f g r e a t e r c h e m i c a l exposure. From f i v e s t u d i e s , g e n e r i c p a t c h d a t a were c o m p i l e d from o n l y the m i x i n g / l o a d i n g o f 10-20 g a l l o n s o f h e r b i c i d e s from two d i f f e r e n t s i z e c o n t a i n e r s (12) (14) (26-28). C o n s i d e r the

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

31

32

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

two s e t s o f p a t c h data i n T a b l e I I I w h i c h have been n o r m a l i z e d f o r an average body weight and pounds o f c h e m i c a l l o a d e d :

T a b l e I I I . C o n t a i n e r S i z e Comparison (Dermal D e p o s i t i o n i n j j g / k g / l b a p p l i e d )

Container Size

Replicates

5.0 g a l l o n

8

2.5 g a l l o n

15

Mean Deposition

Range

S.D.

0.118

0.150

0.0107-0.279

0.00830

0.0169

0.000104-0.0544

Use o f t h e 5 g a l l o h i g h e r dermal d e p o s i t i o containers. A l l t h e examples used t o t h i s p o i n t have been from p a t c h d a t a . The remainder o f t h e examples c i t e d w i l l be b i o l o g i c a l m o n i t o r i n g o f worker's u r i n e . P r e r e q u i s i t e p h a r m a c o k i n e t i c s t u d i e s have determined t h e d u r a t i o n o f e x c r e t i o n o f the p e s t i c i d e and/or m e t a b o l i t e s and t h e percentage o f t h e a d m i n i s t e r e d dose t h a t i s p r e s e n t i n t h e u r i n e . Thus a l l u r i n e specimens were collected f o r the period of excretion plus a baseline i n t e r v a l . The worker's u r i n a r y m e t a b o l i t e l e v e l s were n o r m a l i z e d f o r body w e i g h t , amount o f c h e m i c a l handled and c o r r e c t e d f o r t h e amount e x c r e t e d i n t h e u r i n e t o e s t i m a t e t h e t o t a l body dose (29) (35). I n t h e n e x t example, Table IV, b i o l o g i c a l m o n i t o r i n g o f worker's u r i n e was used t o measure t h e d i f f e r e n c e s i n t o t a l body doses o f workers u s i n g a c o m p l e t e l y c l o s e d t r a n s f e r m i n i - b u l k package system f o l l o w e d by a p p l i c a t i o n t o 20 a c r e s o f c r o p l a n d and an open pour m i x e r - l o a d i n g f o l l o w e d by a p p l i c a t i o n t o t h e same amount o f crop acreage (29). C l o s e d t r a n s f e r systems have been found t o reduce m i x e r / l o a d e r exposure i n many o f o u r s t u d i e s (26-28) and t h e Shuttle™ system used i n t h i s example seems t o be among the most e f f i c i e n t (10).

Table IV.

Package

Replicates

Mini-bulk (closed system) 2.5 g a l containers (open pour)

Product Packaging Mean T o t a l Body Dose (mg/kg/lb Applied)

Comparison

Range (mg/kg/lb Applied)

S.,D. 7

16

1.8 X 10"

7

4.7 X 10~

8

7.8 X 10"

6

9.1 X i o "

6

0.0-1.8 X 10"

6

0.0-2.3 X 10"

5

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

2.

33

Ways To Reduce Applicator Exposure

COWELLETAL.

As can be seen from t h e d a t a i n T a b l e IV and as o t h e r s have observed (21) ( 3 0 ) , the c l o s e d t r a n s f e r does reduce the body dose o f the m i x e r / l o a d e r p e r s o n n e l . I n the a r e a o f a p p l i c a t i o n equipment, t h e r e a r e many t y p e s of s e l e c t i v e equipment ( r e c i r c u l a t i n g s p r a y e r s , s h i e l d e d s p r a y e r s , ropewick a p p l i c a t o r s , b a n d i n g a p p l i c a t o r s , e t c . ) which d e l i v e r the p e s t i c i d e s s p e c i f i c a l l y t o t h e p e s t s w i t h the s i d e b e n e f i t o f p o s s i b l y l o w e r i n g a p p l i c a t o r exposure. Data are b e i n g developed w i t h s h i e l d e d s p r a y e r s , w h i c h show a s i g n i f i c a n t exposure r e d u c t i o n ( 3 1 ) . An example o f t h i s i s a backpack s p r a y e r wand w i t h a s m a l l can s u r r o u n d i n g t h e n o z z l e t h a t ensures t h a t the s p r a y c o n t a c t s the i n t e n d e d f o l i a g e , not a d j a c e n t p l a n t s o r the a p p l i c a t o r . Data a r e c u r r e n t l y b e i n g completed f o r t h i s example ( 3 2 ) . Another f a c t o r t h a t r e s u l t s i n r e d u c t i o n o f exposure i s the use o f the c l o s e d cab t r a c t o r . A comparison o f the absorbed dose i n workers u s i n g a c l o s e d cab t r a c t o r w i t h t h a t o f workers not u s i n g a cab i s p r e s e n t e pour t a n k - f i l l p r o c e d u r A g a i n t h e r e a r e too few experiments f o r much s t a t i s t i c a l i n t e r p r e t a t i o n , b u t as can be seen from the ranges t h a t t h e r e i s n o t v e r y much o v e r l a p . The d a t a are s u g g e s t i v e t h a t c l o s e d cab t r a c t o r s o f f e r more p r o t e c t i o n from exposure d u r i n g a p p l i c a t i o n t h a n open s e a t t r a c t o r s . The l a s t a r e a o f r e d u c t i o n o f worker exposure i s based on the worker h i m s e l f and h i s p e r s o n a l h a b i t s and h y g i e n e . A b i o l o g i c a l m o n i t o r i n g s t u d y was performed w i t h an open pour m i x e r - l o a d i n g and a p p l i c a t i o n ( 1 2 ) . The major d i f f e r e n c e from a p r e v i o u s s t u d y was t h a t the workers were g i v e n s a f e t y i n s t r u c t i o n s i n the form o f a Farmer E d u c a t i o n Program ( 3 4 ) . As can be seen i n the T a b l e V I , t h i s makes a one o r d e r o f magnitude d i f f e r ence i n the means.

T a b l e V.

Tractor

Subjects

A p p l i c a t i o n Equipment

Mean T o t a l Body Dose (mg/kg/lb Applied)

Comparison

Range (mg/kg/lb Applied)

S.D.

No cab (Cockshutt)

4

5.0 X 1 0 "

5

4.2 X 1 0 "

C l o s e d cab ( J . Deere 8450)

8

7.8 X 1 0 "

6

9.1 X

5

10"

5

2.0 X 10" -1.1 X 10

6

0.0-2.3 X 10

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

34

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Table VI.

Exposure Study No s p e c i a l directions Farmer education program

Worker I n s t r u c t i o n L e v e l Comparison

Subjects

Mean T o t a l Body Dose (mg/kg/lb Applied)

8

7.8 X 1 0 "

6

9.1

X 10~

6

0.0-2.3 X

10'

20

.1 X 1 0 "

7

8.6

X 10"

7

0.0-2.7 X

10"

Range (mg/kg/lb Applied)

S.D.

5

C u l t u r a l hygiene/wor i n exposure r e d u c t i o n s t i o n of a r i c e h e r b i c i d e t o a paddy. I n one c a s e , a m e c h a n i c a l s p r e a d e r was used w i t h the o p e r a t o r equipped w i t h rubber g l o v e s and rubber paddy shoes. I n the o t h e r case, the worker a p p l i e d the g r a n u l e s w i t h h i s bare hands and waded through the paddies i n h i s bare f e e t . The obvious s p e c u l a t i o n i s t h a t the bare-handed, b a r e - f o o t e d worker would have a v e r y l a r g e exposure when compared t o the more p r o t e c t e d worker. From b i o l o g i c a l m o n i t o r i n g exposure s t u d i e s of these two types o f a p p l i c a t i o n s ( 1 3 ) , the f o l l o w i n g data l i s t e d i n T a b l e V I I were developed:

Table V I I .

Case

Subjects

Rotary Spreader Hand Broadcast

3

Worker Hygiene Comparison

Mean T o t a l Body Dose (mg/kg/lb Applied)

Range (mg/kg/lb Applied)

9.80

X 10~

4

7.3 X 10" -1.45 X

4

10"

3

1.82

X 10"

3

8.9 X 10" -2.70 X

4

10"

3

Why were the exposures i n these two cases not v a s t l y d i f f e r e n t ? W e l l , f o r two reasons: 1) the a c t i v e i n g r e d i e n t i n the g r a n u l e d i d not r e a d i l y p a r t i t i o n t o any g r e a t e x t e n t from the g r a n u l e m a t e r i a l t o the s k i n ( i . e . , dermal p e n e t r a t i o n o f the a c t i v e i s low) ( 3 6 ) , and 2) the c u l t u r a l p r a c t i c e o f the worker was t h a t upon c o m p l e t i o n o f the a p p l i c a t i o n , the worker washed h i s hands and f e e t i n the u n t r e a t e d i r r i g a t i o n d i t c h a l o n g s i d e the paddy. T h i s p r a c t i c e reduced the worker's c h e m i c a l c o n t a c t

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

COWELL ET AL.

Ways To Reduce Applicator Exposure

time (exposure) to a minimum and therefore not too different from a well protected worker. In conclusion, we have seen in our studies utilizing both techniques for measuring worker exposure, that the following are effective means for exposure reduction: 1. Normal long-sleeved cotton or cotton/polyester blend shirt, long-trouser - typical work clothing and rubber gloves provide good protection. 2. Closed transfer systems which convey chemical via hose or pipe from container to spray tank are the best. When open pouring containers are used, small, easier to handle containers are preferred. 3. Improvement of application equipment such as target delivery applicators and closed cab tractors appear to reduce exposure. 4. The instruction and personal hygiene/work practice of the worker can be very LITERATURE CITED 1. 2. 3. 4.

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6. 7. 8. 9. 10. 11. 12.

Durham, W. F. and Wolfe, H. R. Bull. Wld. Hlth. Org. 1962, 26, 75-91. Turnbull, G. J. Occupational Hazards of Pesticide Use. Taylor and Francis: London, 1985. Field Surveys of Exposure to Pesticide. World Health Organization. 1982. Standard Protocol: Technical Mono­ graph N 7. Geneva, Switzerland. Reinert, J. C.; Nielsen, A. P.; Lunchick, C.; Hernandez, O. and Mazzetta, D. M. The United States Environmental Protec­ tion Agency's Guidelines For Applicator Exposure Monitoring. Toxicology Letters, 1986, 33, 183-191. Cowell, J. E. Applicator Exposure To Pesticides. Proceed­ ings of National Academy of Science-Czechoslovakian Academy of Science Workshop on Agricultural Development and Environ­ mental Research, Ceske Budejovice, Czechoslovakia. 1987, (in press). Ackerman, J. W. August 5, 1986. U.S. Environmental Pro­ tection Agency Correspondence. Franklin, C. A. Can. J. Physiol. Pharmacol. 1984, 62, 1037-39. Wojeck, G. A.; Price, J. F.; Nigg, H. N. and Stamper, J. H. Arch. Environ. Contamin. Toxicol. 1983, 12, 65-70. Lavy, T. L.; Shepard, J. S. and Mattice, J. D. J. Agr. Food Chem. 1980, 28, 626-30. Cowell, J. E.; Danhaus, R. D.; Kunstman, J. L.; Hackett, A. G.; Oppenhuizen, M. E. and Steinmetz, J. R. Arch. Envi­ ron. Contam. Toxicol. 1987, 16, 327-332. Leavitt, J. R. C.; Gold, R. E.; Holcslaw, T. and Tupy, D. Arch. Environm. Contam. Toxicol. 1982, 11, 57-62. Danhaus, R. G.; Kunstman, J. L.; Oppenhuizen, M. E. and Steinmetz, J. R. Operator Exposure from Open-Pour Transfer and Application of Lasso® EC, Lasso® MT™ and Lasso® WDG Herbicides. Monsanto Report Number MSL-5398, 1986. Submit-

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

13. 14.

15. 16. 17. 18. 19.

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2. COWELLETAL. 29. 30. 31.

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Cowell, J. E. and Dubelman, S. In Pesticide Science and Technology; Greenhalgh, R. and Roberts, T. R., Eds; Blackwell Scientific Publications: Oxford, 1987, 573-578. Dubelman, S., Lauer, R., Arras, D. D., and Adams, S. A. J. Agric. Food Chem. 1982, 30, 528-532. Kramer, R. M. Herbicide Applicator Exposure to Glyphosate During Application of Roundup® Herbicide and Field Reentry. Monsanto Report Number MSL-0288, 1978. Submitted to U.S. Environmental Protection Agency, EPA Accession No. 233914. Lavy, T. L.; Mattice, J. D.; Steinmetz, J. R. and Cowell, J. E. 1988-9. (Study in progress). Klein, A. J.; Bade, T. R.; Smith, R. G. and Rupel, F. L. Urinary Excretion of Alachlor Residue Following Normal Application of Lasso® of Lasso® Micro-Tech™ Herbicide Under Commericial Conditions in Indiana. Monsanto Report Number MSL-4207, 1984. Submitted to U.S Environmental Protection Agency, EPA Accessio Monsanto Agricultura to the Correct Handling of Lasso® Herbicide. 1985. Dubelman, S. and Cowell, J. E. Biological Monitoring Technology for Measurement of Applicator Exposure. ACS Sym­ posium Series (in press). 1987. Adams, S. A. Butachlor Dermal Penetration Study. Monsanto Report Number MSL-3102, 1983.

RECEIVED March 14, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

37

Chapter 3

Estimating Exposure to Pesticides in Epidemiological Studies of Cancer Aaron Blair, Shelia Hoar Zahm, Kenneth P. Cantor, and Patricia A. Stewart Environmental Epidemiology Branch, National Cancer Institute, Landow Building, Bethesda, MD 20892

Epidemiologic studies of cancer and pesticides have proven d i f f i c u l t becaus f limitation i assessment procedures multiple exposure lengt posure and initial symptoms of cancer. Biochemical monitoring coupled with traditional methods of exposure assessment based on job histories and duties offers the opportunity to evaluate the r e l i a b i l i t y of, and to improve, exposure assessment procedures. Pesticides are designed to be toxic to organisms which are considered undesirable. Because of this ability to i n f l i c t damage on living organisms, there has been concern regarding their effect on humans. The International Agency for Research on Cancer, an agency which reviews and evaluates the carcinogenicity of chemicals, has concluded that there i s sufficient evidence for the carcinogenicity of chlordane, chlordecone, heptachlor, hexachlorobenzene, mirex and toxaphene in laboratory animals (1) and that limited evidence exists for tetrachlorinphos and diallate (2). Epidemiologic studies of cancer and pesticides, however, have proven d i f f i c u l t (3). This paper briefly reviews procedures used to evaluate pesticide exposures in epidemiologic studies of cancer and suggests ways of improving our effort in this area. Some of the problems are typical of most epidemiologic studies of cancer, i.e. the long period between f i r s t exposure and i n i t i a l evidence of disease and the need for large numbers of study subjects. A particular vexing problem has been the difficulty in documenting exposures. Information on the type of pesticides handled and levels of exposure is crucial for evaluation of exposure-response patterns which i s a c r i t i c a l criterion for identifying etiologic factors. Accurate assessment of exposure is important because exposure misclassification reduces estimates of relative risks and dampens dose-response gradients. In addition, misclassification increases the chances of attributing elevated cancer risk among persons exposed to pesticides to the wrong agent. This chapter not subject to U.S. copyright Published 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

3.

BLAIR ETAL.

Epidemiological Studies of Cancer

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I t i s r e l a t i v e l y easy t o i d e n t i f y persons i n o c c u p a t i o n s where exposure t o p e s t i c i d e s i s common and/or h e a v i e r than the g e n e r a l p o p u l a t i o n , but documentation or measurements of exposure t o s p e c i f i c p e s t i c i d e s are seldom a v a i l a b l e . Furthermore, even i f some m o n i t o r i n g data or o t h e r documentation of exposure are a v a i l a b l e , i t i s d i f f i c u l t to evaluate r i s k s associated with s p e c i f i c p e s t i c i d e s because most a p p l i c a t o r s have c o n t a c t w i t h a v a r i e t y of p e s t i c i d e s and because v a r i a t i o n i n work p r a c t i c e s can g r e a t l y a f f e c t d e l i v e r e d dose. Although the l a t e n t p e r i o d ( i . e . the time from f i r s t exposure t o d i a g n o s i s of d i s e a s e ) a s s o c i a t e d w i t h p e s t i c i d e s i s not g e n e r a l l y known, i t i s l e n g t h y f o r most c h e m i c a l c a r c i n o g e n s which f u r t h e r c o m p l i c a t e s exposure e v a l u a t i o n . P r e c i s e d e t e r m i n a t i o n of r e c e n t exposures i s not n e c e s s a r i l y r e l e v a n t t o the i n i t i a t i o n of cancer, s i n c e the most important exposures may be those t h a t o c c u r r e d 20 or y e a r s b e f o r e the development of d i s e a s e . D e s p i t e these d i f f i c u l t i e s t u d i e s of persons h a v i n m a n u f a c t u r e r s , a p p l i c a t o r s , and farmers suggest t h a t exposure t o p e s t i c i d e s may i n c r e a s e t h e i r r i s k of c a n c e r . S t u d i e s of persons exposed t o p e s t i c i d e s i n manufacturing o p e r a t i o n s and as a p p l i c a t o r s have shown r e l a t i v e r i s k s of l u n g t a n c e r r a n g i n g from 1.3 to 1.9 (49 ) . In some s t u d i e s , r i s k s i n c r e a s e w i t h d u r a t i o n of exposure to n e a r l y 3 - f o l d among persons exposed f o r 20 or more y e a r s ( 4 , 8 ) . Numerous s t u d i e s from around the w o r l d have a l s o noted excesses f o r some cancers among farmers^, . p a r t i c u l a r l y tumors of the l y m p h a t i c and h e m a t o p o i e t i c system (3, 10-15). D e s p i t e a c o n s i d e r a b l e number of studies suggesting that occupation* having contact with p e s t i c i d e s may e x p e r i e n c e e l e v a t e d r i s k s f o r c e r t a i n c a n c e r s , the t a s k of i d e n t i f y i n g the s p e c i f i c p e s t i c i d e s i n v o l v e d i n these a s s o c i a t i o n s has proven d i f f i c u l t . Recent s t u d i e s , however, have suggested t h a t p h e n o x y a c e t i c a c i d h e r b i c i d e s may be i n v o l v e d (10-16). To improve our c a p a b i l i t i e s of i d e n t i f y i n g cancer r i s k s a s s o c i a t e d w i t h s p e c i f i c p e s t i c i d e s , we need t o improve methods f o r e v a l u a t i n g r e l e v a n t h i s t o r i c a l exposures and to assess t h e i r r e l i a b i l i t y and validity. Methods C u r r e n t l y Used t o E v a l u a t e Exposures C a s e - c o n t r o l and cohort methods have both been used i n e p i d e m i o l o g i c s t u d i e s of cancer and p e s t i c i d e s . Although the procedures f o r e v a l u a t i n g exposures need not d i f f e r by study d e s i g n , employment r e c o r d s have g e n e r a l l y been the major source of exposure i n f o r mation i n cohort s t u d i e s , whereas c a s e - c o n t r o l s t u d i e s have tended to r e l y on i n t e r v i e w s of s u b j e c t s to o b t a i n i n f o r m a t i o n on p e s t i c i d e exposure. Members of cohort s t u d i e s , however, c o u l d be i n t e r v i e w e d and employment or o t h e r exposure r e c o r d s c o u l d be o b t a i n e d f o r s u b j e c t s i n c a s e - c o n t r o l s t u d i e s . I n f o r m a t i o n from more than one source has been sought i n some s t u d i e s , i . e . , exposure r e c o r d s f o r some s u b j e c t s were o b t a i n e d i n c a s e - c o n t r o l s t u d i e s i n Sweden (12) and Kansas (11) and i n t e r v i e w s have r e c e n t l y been completed w i t h s e l e c t e d s u b j e c t s i n a c o h o r t study of F l o r i d a a p p l i c a t o r s ( 4 ) .

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Cohort S t u d i e s . Employment r e c o r d s used t o i d e n t i f y persons exposed to p e s t i c i d e s f o r h i s t o r i c a l cohort s t u d i e s (4-9, 16-19) t y p i c a l l y c o n t a i n p e r s o n a l i d e n t i f i e r s (name, s o c i a l s e c u r i t y number, and date of b i r t h ) and employment i n f o r m a t i o n , such as dates of employment and j o b t i t l e s . These r e c o r d s seldom, however, c o n t a i n s u f f i c i e n t i n f o r m a t i o n t o r e c o n s t r u c t a d e t a i l e d h i s t o r y of p e s t i c i d e exposure a s s o c i a t e d w i t h t h e j o b . F o r example, t h e cohort of l i c e n s e d p e s t i c i d e a p p l i c a t o r s from F l o r i d a (4) was e s t a b l i s h e d from l i c e n s i n g r e c o r d s maintained by t h e s t a t e . The r e c o r d s c o n t a i n e d , f o r each i n d i v i d u a l , i n f o r m a t i o n on s p e c i f i c y e a r s l i c e n s e d , company where employed, and j o b t i t l e . However, they l a c k e d i n f o r m a t i o n on each p e s t i c i d e a p p l i c a t i o n , which would be needed t o r e c o n s t r u c t a d e t a i l e d exposure h i s t o r y . We have r e c e n t l y completed i n t e r v i e w s of n e x t - o f - k i n of deceased cancer cases and a group of c o n t r o l s from t h i s cohort. I n these i n t e r v i e w s we o b t a i n e d i n f o r m a t i o n on j o b c h a r a c t e r i s t i c s t h a t s h o u l d h e l p i n e v a l u a t i n g p e s t i c i d e exposure, such as g e n e r a l use of a p p l i e d , frequency of s k i commonly a s s o c i a t e d w i t h p e s t i c i d e p o i s o n i n g . We d i d not ask f o r i n f o r m a t i o n c o n c e r n i n g i n d i v i d u a l a p p l i c a t i o n s because a c c u r a t e r e c a l l of such d e t a i l by the n e x t - p f - k i n seemed u n l i k e l y . Although i n f o r m a t i o n o b t a i n e d from these i n t e r v i e w s may improve our unders t a n d i n g of a s u b j e c t ' s g e n e r a l exposure, t h e l a c k of d e t a i l on d i l u t i o n r a t e s , volume of m i x t u r e s used, frequency of a p p l i c a t i o n , and d i r e c t measures of exposure p r e v e n t s t h e development of quant i t a t i v e e s t i m a t e s of exposure t o s p e c i f i c p e s t i c i d e s , or t o pesticides i n general. The N a t i o n a l Cancer I n s t i t u t e r e c e b t l y i n i t i a t e d a cohort m o r t a l i t y study of employees of ChemLawn, a major lawn care company. Again, employment r e c o r d s c o n t a i n i n f o r m a t i o n on j o b t i t l e and years employed. Because ChemLawn has s t a n d a r d i z e d p e s t i c i d e f o r m u l a t i o n s and a p p l i c a t i o n procedures f o r v a r i o u s g e o g r a p h i c r e g i o n s of t h e c o u n t r y , i t may be p o s s i b l e t o develop a s e m i - q u a n t i t a t i v e e s t i m a t e of exposure t o p e s t i c i d e s f o r employees w i t h i n these r e g i o n s u s i n g ambient a i r and b i o c h e m i c a l m o n i t o r i n g data a v a i l a b l e from t h e company. The e x i s t e n c e of separate lawn care and t r e e / s h r u b d i v i s i o n s p r o v i d e s another source of exposure i n f o r m a t i o n . There has been l i t t l e c r o s s o v e r between d i v i s i o n s i n r e c e n t y e a r s , thus the s e p a r a t i o n p r o v i d e s t h e o p p o r t u n i t y t o i d e n t i f y a group of employees exposed t o i n s e c t i c i d e s , but not h e r b i c i d e s , s i n c e t r e e and shrub d i v i s i o n employees do not a p p l y h e r b i c i d e s . D e s p i t e t h i s r e l a t i v e l y r i c h source of exposure i n f o r m a t i o n , i t w i l l not be p o s s i b l e t o r e c o n s t r u c t i n d i v i d u a l h i s t o r i e s of a p p l i c a t i o n s . I n s t e a d we must r e l y upon a g e n e r a l r e c o n s t r u c t i o n of exposure based on t h e u s u a l work p r a c t i c e s recommended by the company. N e i t h e r the study of F l o r i d a a p p l i c a t o r s , nor of ChemLawn employees, o f f e r s t h e o p p o r t u n i t y t o e s t i m a t e s p e c i f i c d a i l y exposures e x p e r i e n c e d by i n d i v i d u a l cohort members. A r e c o r d - k e e p i n g system t h a t may p r o v i d e such d e t a i l has r e c e n t l y been brought t o our a t t e n t i o n . County Noxious Weed Departments i n Kansas a r e charged w i t h c o n t r o l l i n g weeds on p r i v a t e and p u b l i c l a n d s . These departments appear t o r e t a i n a d e t a i l e d r e c o r d of each p e s t i c i d e a p p l i c a t i o n . Recorded i n the a p p l i c a t i o n r e c o r d s i s t h e nai&e of t h e a p p l i c a t o r , h e r b i c i d e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

3.

BLAIR ET AL.

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used, d i l u t i o n , g a l l o n s a p p l i e d , acres t r e a t e d , time and date of t r e a t m e n t , c l i m a t i c c o n d i t i o n s , and type of spray equipment. Although these r e c o r d s r e p r e s e n t a c o n s i d e r a b l e improvement over those a v a i l a b l e i n the s t u d i e s p r e v i o u s l y d e s c r i b e d , they are not supported by h i s t o r i c a l m o n i t o r i n g d a t a . Measurement of l e v e l s a s s o c i a t e d w i t h c u r r e n t o p e r a t i o n s may p r o v i d e important q u a n t i t a t i v e d a t a , i f we can e s t a b l i s h t h a t c u r r e n t o p e r a t i o n s are s i m i l a r t o those of the p a s t . C a s e - c o n t r o l S t u d i e s . I n f o r m a t i o n on p e s t i c i d e exposures i n casec o n t r o l s t u d i e s i s t y p i c a l l y o b t a i n e d by i n t e r v i e w of s u b j e c t s , or t h e i r n e x t - o f - k i n (10-15, 20, 21). We have conducted s t u d i e s . o f cancer i n r u r a l areas where the q u e s t i o n n a i r e s have focused on s p e c i f i c p e s t i c i d e s used by farmers (11,13). In these s t u d i e s we sought l i f e t i m e h i s t o r i e s of s p e c i f i c p e s t i c i d e s used, e a r l i e s t and l a s t year of use, number of days per year t y p i c a l l y used type of a p p l i c a t i o n equipment, us ciated with p e s t i c i d e poisoning c l o t h i n g i s changed. Although these i n t e r v i e w s o b t a i n i n f o r m a t i o n on s p e c i f i c p e s t i c i d e s used, they do not p r o v i d e i n f o r m a t i o n on each application. In a d d i t i o n , no m o n i t o r i n g of exposures has been conducted i n c o n j u n c t i o n w i t h the i n t e r v i e w s . Thus, i n f o r m a t i o n from q u e s t i o n n a i r e s i n our c a s e - c o n t r o l s t u d i e s resembles t h a t assembled i n most cohort s t u d i e s i n t h a t d e t a i l e d a p p l i c a t i o n h i s t o r i e s cannot be r e c o n s t r u c t e d . Although i t i s p o s s i b l e t o perform some exposure-response e v a l u a t i o n s u s i n g i n f o r m a t i o n o b t a i n e d from these i n t e r v i e w s , i t i s c o m p l i c a t e d by the i n c r e a s i n g i n a b i l i t y of respondents t o r e c a l l d e t a i l s of exposure w i t h the passage of time, and by the p o t e n t i a l f o r d i f f e r e n c e s i n r e c a l l between cases and c o n t r o l s . D i f f e r e n t i a l r e c a l l i s the more s e r i o u s problem and may occur because cancer cases and t h e i r n e x t - o f - k i n may have spent more time t r y i n g to r e c a l l past exposure t o p e s t i c i d e s than person w i t h o u t cancer. T h i s type of r e c a l l b i a s tends t o c r e a t e a s s o c i a t i o n s where none e x i s t . The a b i l i t y t o r e c a l l the d e t a i l s r e g a r d i n g p e s t i c i d e use may a l s o fade w i t h time. Unfortun a t e l y because of t h i s problem, the a c c u r a c y of the i n f o r m a t i o n r e p o r t e d i s weakest f o r e a r l i e r uses of p e s t i c i d e s , the v e r y time p e r i o d t h a t i s c o n s i d e r e d most important f o r d i s e a s e s w i t h l o n g l a t e n c i e s such as c a n c e r . On the o t h e r hand, a g e n e r a l f a i l u r e to r e c a l l use of p e s t i c i d e s by a l l s u b j e c t s (both cases and c o n t r o l s ) would tend t o have an e f f e c t o p p o s i t e of t h a t of d i f f e r e n t i a l r e c a l l b i a s and would g e n e r a l l y reduce e s t i m a t e s of r i s k a s s o c i a t e d w i t h p e s t i c i d e exposure. T h i s type of r e c a l l problem would not be an e x p l a n a t i o n f o r any excesses observed. Immediate Needs To Improve Exposure E v a l u a t i o n s The g o a l of exposure e v a l u a t i o n i s t o d e v e l o p d e t a i l e d measures of d e l i v e r e d dose over the e n t i r e p e r i o d of exposure. D e l i v e r e d dose, however, i s dependent upon p h a r m a c o k i n e t i c s and r e q u i r e s more b i o l o g i c i n f o r m a t i o n than i s g e n e r a l l y a v a i l a b l e . A i r measurements a r e sometimes a v a i l a b l e . A i r m o n i t o r i n g , however,, p r i m a r i l y

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p r o v i d e s an i n d i c a t i o n of dose r e c e i v e d by i n h a l a t i o n , which f o r p e s t i c i d e s may be i n s i g n i f i c a n t compared t o t h a t d e l i v e r e d d e r m a l l y . A l t h o u g h the a v a i l a b i l i t y of a i r m o n i t o r i n g and b i o c h e m i c a l m o n i t o r i n g data i s growing, these d a t a are u s u a l l y l i m i t e d o r n o n e x i s t e n t f o r u n r e g u l a t e d c h e m i c a l s not c l e a r l y shown t o be c a r c i n o g e n s , or t o p r e s e n t o t h e r h e a l t h problems. I n g e n e r a l , however, u n l e s s we are c o n t e n t t o w a i t f o r years u n t i l such m o n i t o r i n g data are assembled, immediate e f f o r t s a r e needed t o improve the q u a l i t y and r e l i a b i l i t y of the e v a l u a t i o n of p e s t i c i d e exposure i n e p i d e m i o l o g i c s t u d i e s where m o n i t o r i n g d a t a are l i m i t e d . Assuming t h a t we must c o n t i n u e to use, at l e a s t f o r the immediate f u t u r e , i n f o r m a t i o n o b t a i n e d from i n t e r v i e w or p e r s o n n e l r e c o r d s t o e s t i m a t e exposures, we s h o u l d make every attempt t o e v a l u a t e the r e l i a b i l i t y of these procedures and t o c o r r e l a t e them w i t h measured l e v e l s of exposure. Several p o s s i b i l i t i e s t o a c c o m p l i s h t h i s come t o mind. Simple R e t e s t R e l i a b i l i t retest r e l i a b i l i t y i s a we are unaware of such s t u d i e s r e g a r d i n g i n t e r v i e w s on p e s t i c i d e use. Most i n t e r v i e w s t u d i e s r o u t i n e l y i n c l u d e a s m a l l number of r e i n t e r v i e w s , p r i m a r i l y as a check on the i n t e r v i e w e r s . These c o u l d e a s i l y be expanded t o i n c l u d e c r u c i a l q u e s t i o n s r e g a r d i n g p e s t i c i d e use t o develop i n f o r m a t i o n on the r e l i a b i l i t y of s u b j e c t r e c a l l . E p i d e m i o l o g i s t s have conducted many such r e l i a b i l i t y s t u d i e s f o r o t h e r i n t e r v i e w items such as tobacco use and d i e t , but we have not a d e q u a t e l y c o n s i d e r e d r e p o r t e d us© of p e s t i c i d e s . Comparison of I n f o r m a t i o n from D i f f e r e n t Sources. Confidence i n exposure e s t i m a t e s i s improved when t h e r e i s agreement between e v a l u a t i o n s from d i f f e r e n t e x p e r t s or between e v a l u a t i o n s based on d i f f e r e n t i n f o r m a t i o n s o u r c e s . S e v e r a l p o s s i b i l i t i e s f o r such comparisons e x i s t . 1.

I n a c a s e - c o n t r o l study of lymphoma and s o f t - t i s s u e sarcoma i n Kansas (11), we attempted t o c o r r o b o r a t e farmers' r e p o r t e d p e s t i c i d e use by c o n t a c t i n g t h e i r p e s t i c i d e s u p p l i e r s . When a v a i l a b l e , s u p p l i e r ' s r e c o r d s were reviewed t o document p e s t i c i d e purchases, o t h e r w i s e we r e l i e d upon the memory of the s u p p l i e r f o r c o r r o b o r a t i o n . O v e r a l l , agreement between farmer and s u p p l i e r r e g a r d i n g p e s t i c i d e purchases was not h i g h , i . e . about 50%. T h i s i s not s u r p r i s i n g g i v e n the number of p e s t i c i d e s purchased, the l o n g p e r i o d of time over which farmers had made such purchases, the l a c k of h i s t o r i c a l purchase r e c o r d s r e t a i n e d by many s u p p l i e r s , the l a r g e number of s u p p l i e r s g e n e r a l l y a v a i l a b l e t o farmers, and the c l o s i n g of e s t a b l i s h ments t h a t had p r e v i o u s l y s u p p l i e d farmers w i t h p e s t i c i d e s . There was l i t t l e d i f f e r e n c e i n agreement r a t e s , however, between cases and c o n t r o l s . For 2,4-D, agreement was 83% among cases of non-Hodgkin's lymphoma and 74% among c o n t r o l s . T h i s i s c o m f o r t i n g i n t h a t at l e a s t r i s k s a s s o c i a t e d w i t h h e r b i c i d e use seen i n t h i s study c o u l d not be a t t r i b u t e d t o r e c a l l b i a s . As expected, agreement was b e t t e r f o r more r e c e n t , r a t h e r than f o r e a r l i e r y e a r s . L e v e l of agreement a l s o v a r i e d by type of p e s t i c i d e and was b e t t e r f o r h e r b i c i d e s tha» f o r i n s e c t i c i d e s .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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In these comparisons i t i s not always c l e a r whether t h e r e p o r t by t h e s u b j e c t o r s u p p l i e r i s c o r r e c t , s i n c e e i t h e r t h e memory or t h e r e c o r d s of the s u p p l i e r c o u l d a l s o be i n c o m p l e t e . Such d u p l i c a t e assessments of exposure p r o v i d e t h e o p p o r t u n i t y t o e v a l u a t e the i n f l u e n c e of d i f f e r e n t r e p o r t s of exposure on the e s t i m a t e of r e l a t i v e r i s k . I n t h i s study a d j u s t i n g the d a t a f o r h e r b i c i d e use as r e p o r t e d by s u p p l i e r s had l i t t l e e f f e c t on e s t i m a t e s of r e l a t i v e r i s k f o r non-Hodgkin s lymphoma. F u r t h e r e v a l u a t i o n of these d a t a and a d d i t i o n a l comparisons a r e needed t o assess t h e magnitude and type of r e p o r t i n g e r r o r s from s u b j e c t s and t h e i n f l u e n c e of these e r r o r s on r i s k e s t i m a t e s . 1

2.

The a v a i l a b i l i t y of d e t a i l e d r e c o r d s on i n d i v i d u a l h e r b i c i d e a p p l i c a t i o n s from County Noxious Weed Departments i n Kansas o f f e r s the o p p o r t u n i t y t o compare i n f o r m a t i o n on h e r b i c i d e use o b t a i n e d from c u r r e n from r e c o r d s prepare would be e s p e c i a l l y u s e f u l i n e v a l u a t i n g memory l o s s over time and the s p e c i f i c a s p e c t s of h e r b i c i d e a p p l i c a t i o n t h a t a r e most s u s c e p t i b l e t o i n a c c u r a t e r e c a l l , i n c l u d i n g names of p e s t i c i d e s , a p p l i c a t i o n r a t e s , f o r m u l a t i o n s , f r e q u e n c i e s of a p p l i c a t i o n , and use of p r o t e c t i v e equipment. Other o p p o r t u n i t i e s of t h i s type may e x i s t elsewhere.

3.

Comparison d a t a from a p p l i c a t i o n r e c o r d s or i n t e r v i e w i n g w i t h b i o c h e m i c a l m o n i t o r i n g r e s u l t s o f f e r s the g r e a t e s t p o t e n t i a l t o e v a l u a t e t h e r e l i a b i l i t y o f , dnd t o improve, our exposure assessment t e c h n i q u e s . Biochemical monitoring for p e s t i c i d e exposure has been conducted on s m a l l p o p u l a t i o n s f o r a number of years and many new methods f o r b i o c h e m i c a l m o n i t o r i n g a r e appearing. M o n i t o r i n g studies, g e n e r a l l y i n d i c a t e t h a t t h e type of a p p l i c a t i o n equipment used and procedures f o l l o w e d at t h e time of a p p l i c a t i o n c o r r e l a t e w i t h t h e exposure l e v e l measured. For e p i d e m i o l o g i c s t u d i e s of cancer i t would be h e l p f u l t o know how w e l l i n d i v i d u a l s can r e c a l l p e r t i n e n t d e t a i l s r e g a r d i n g a p p l i c a t i o n episodes t h a t took p l a c e s e v e r a l years i n t h e p a s t . R e - i n t e r v i e w of p a r t i c i p a n t s i n e a r l y b i o c h e m i c a l m o n i t o r i n g s t u d i e s c o u l d assess how w e l l i n d i v i d u a l s can r e c a l l important events s u r r o u n d i n g these a p p l i c a t i o n s e p i s o d e s . I f accurate, d a t a from r e c e n t i n t e r v i e w s c o u l d be used t o develop models t o p r e d i c t exposure l e v e l s measured a t t h e time of t h e a p p l i c a t i o n . We a r e i n t e r e s t e d i n e x p l o r i n g o p p o r t u n i t i e s f o r d e v e l o p i n g such c o l l a b o r a t i v e p r o j e c t s w i t h r e s e a r c h e r s who may have b i o c h e m i c a l m o n i t o r i n g d a t a o b t a i n e d s e v e r a l y e a r s ago.

4.

Another approach t o e v a l u a t e i n d i v i d u a l r e p o r t s of p e s t i c i d e use o b t a i n e d by q u e s t i o n n a i r e s would be t o assemble a p a n e l of e x p e r t s t o a s s e s s t h e p l a u s i b i l i t y of t h e types of p e s t i c i d e s used and t h e methods of a p p l i c a t i o n r e p o r t e d by s t u d y s u b j e c t s . T h i s would be a p o p u l a t i o n , r a t h e r than an i n d i v i d u a l , comparison. O b v i o u s l y , i t would g e n e r a l l y not be p o s s i b l e t o determine whether o r not a p a r t i c u l a r i n d i v i d u a l used a p a r t i c u l a r p e s t i c i d e i n a p a r - t i e u l a r y e a r . I t a i g h t be p o s s i b l e , however t o r

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

e v a l u a t e whether o r not i t was l i k e l y t h a t a c e r t a i n percentage of s u b j e c t s would have used a p a r t i c u l a r p e s t i c i d e i n a s p e c i f i c year f o r a p a r t i c u l a r purpose. T h i s i s p r o b a b l y t h e weakest approach f o r c o r r o b o r a t i n g i n d i v i d u a l r e p o r t s , because as i m p l a u s i b l e as a r e p o r t e d p r a c t i c e may seem, i t may i n f a c t be e x a c t l y what happened. T h i s e v a l u a t i o n of the p l a u s i b i l i t y of r e p o r t s i s o f t e n performed by t h e study of i n v e s t i g a t o r s , but i t would work b e t t e r i f e n t o m o l o g i s t s , a g r i c u l t u r a l s c i e n t i s t s , t o x i c o l o g i s t s , and i n d u s t r i a l h y g i e n i s t s knowledgeable i n use of p e s t i c i d e s perform the a c t i v i t y . U l t i m a t e Needs f o r E v a l u a t i o n Of P e s t i c i d e Exposures The u l t i m a t e g o a l of exposure e v a l u a t i o n i n e p i d e m i o l o g i c s t u d i e s of cancer i s t o i d e n t i f y t h e d e l i v e r e d dose of the a c t i v e c h e m i c a l t o the t a r g e t t i s s u e a t the time r e l e v a n t f o r tumor i n i t i a t i o n o r p r o m o t i o n . The d i f f i c u l t i e l o g i c s t u d i e s of cancer C o u p l i n g b i o c h e m i c a l measures w i t h t r a d i t i o n a l exposure e v a l u a t i o n procedures used i n e p i d e m i o l o g i c s t u d i e s of cancer o f f e r s the best o p p o r t u n i t y f o r i m p r o v i n g our assessment of h i s t o r i c p e s t i c i d e e x p o s u r e s . Although the u t i l i t y of b i o c h e m i c a l measures has been c l e a r l y demonstrated f o r a s s o c i a t i n g acute symptoms w i t h p e s t i c i d e exposure, s t u d i e s of cancer impose a d d i t i o n a l r e q u i r e m e n t s . As d i s c u s s e d above, the maior l i m i t a t i o n , of b i o c h e m i c a l measures i s t h a t they a r e seldom a v a i l a b l e f o r t h e time p e r i o d of g r e a t e s t i n t e r e s t . For e a r l y stage c a r c i n o g e n s , t h e time between f i r s t exposure and d i a g n o s i s of d i s e a s e i s t y p i c a l l y 20 o r more y e a r s . U n f o r t u n a t e l y , many measures of p e s t i c i d e s o r t h e i r m e t a b o l i t e s i n body f l u i d s or t i s s u e s r e f l e c t r e l a t i v e l y r e c e n t exposures and must be e v a l u a t e d c a r e f u l l y t o assess whether o r not they a r e s i m i l a r t o , or r e f l e c t , those from e a r l i e r t i m e s . Changes i n the p r e f e r r e d p e s t i c i d e f o r a p a r t i c u l a r p e s t , i n c o n c e n t r a t i o n and a p p l i c a t i o n procedures, and i n work p r a c t i c e s make e v a l u a t i o n of exposure t o specific pesticides d i f f i c u l t . B i o c h e m i c a l measurements c a n , however, h e l p i n r e l a t i n g i n t e r m i t t e n t exposures e x p e r i e n c e by workers t o exposures i n b i o a s s a y s . B i o c h e m i c a l measures c o u l d , of c o u r s e , be u t i l i z e d i n p r o s p e c t i v e s t u d i e s . P r o s p e c t i v e cancer s t u d i e s , however, r e q u i r e v e r y l a r g e p o p u l a t i o n s which would need t o be f o l l o w e d many y e a r s . Single b i o c h e m i c a l measures i n p r o s p e c t i v e s t u d i e s , a l t h o u g h u s e f u l , would not by themselves p r o v i d e s u f f i c i e n t i n f o r m a t i o n t o e v a l u a t e c u m u l a t i v e dose, because they would p r o v i d e an e s t i m a t e of exposure f o r a s h o r t p e r i o d of time. Given t h e c o s t and c o m p l e x i t y of sample c o l l e c t i o n and a n a l y s i s , repeat measurements on l a r g e numbers of study s u b j e c t s may not be p r a c t i c a l . I n c l u s i o n of b i o c h e m i c a l m o n i t o r i n g i n c a s e - c o n t r o l s t u d i e s o f f e r s the advantage of much s m a l l e r numbers of s u b j e c t s than a r e necessary i n p r o s p e c t i v e s t u d i e s . A major drawback t o c a s e - c o n t r o l s t u d i e s i s t h a t t h e d i s e a s e p r o c e s s i t s e l f , o r i t s t r e a t m e n t , may a f f e c t t h e c o n c e n t r a t i o n of t h e c h e m i c a l b e i n g measured.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

3. BLAIR ET AL.

Epidemiological Studies of Cancer

45

Another l i m i t a t i o n i n most e p i d e m i o l o g i c s t u d i e s of p e s t i c i d e s and cancer i s t h e d i f f i c u l t y i n a s s e s s i n g t h e independent e f f e c t s of s p e c i f i c p e s t i c i d e s . B i o c h e m i c a l m o n i t o r i n g i s no more u s e f u l i n t a c k l i n g t h i s problem than t h e more t r a d i t i o n a l exposure e v a l u a t i o n methods, s i n c e t h e fundamental problem i s m u l t i p l e exposures and i d e n t i f i c a t i o n of which agent i s r e s p o n s i b l e f o r t h e d i s e a s e , not the p r e c i s i o n w i t h which measurements a r e made. We have not addressed many p e r t i n e n t t e c h n i c a l i s s u e s i n c l u d i n g r e l i a b i l i t y of t h e l a b o r a t o r y a n a l y s e s , d i f f i c u l t y i n p r e s e r v i n g and t r a n s p o r t i n g t h e samples, e f f e c t s of i n t e r f e r e n c e s from o t h e r e n v i r o n m e n t a l exposures, and h a l f - l i f e and metabolism of t h e substances. O b v i o u s l y some p e s t i c i d e s a r e r a p i d l y e l i m i n a t e d from the body, w h i l e o t h e r s may be measured y e a r s a f t e r exposure. These are v e r y important i s s u e of p e s t i c i d e s w i t h l o n i n d i c a t i o n of c u m u l a t i v e exposure. Conclusions B i o c h e m i c a l measures can supplement t r a d i t i o n a l methods of exposure e v a l u a t i o n used i n e p i d e m i o l o g i c s t u d i e s o f cancer and exposure t o p e s t i c i d e s . Such measures can p r o v i d e more s o l i d e s t i m a t e s o f d e l i v e r e d dose than e s t i m a t e s based on p a t c h e s , on ambient a i r measures, on those s t r i c t l y from i g b d e s c r i p t i o n s , o r on s u b j e c t r e p o r t s of p e s t i c i d e use. The U n i t e d number of b i o c h e m i c a l measures l i k e l y t o be a v a i l a b l e (fcn o n l y a sample o f t h e study p o p u l a t i o n , f o r o n l y a few p e s t i c i d e s , f o r o n l y a few p o i n t s i n time) i m p l i e s t h a t o t h e r procedures f o r e v a l u a t i n g p e s t i c i d e exposure w i l l s t i l l be r e q u i r e d . B i o c h e m i c a l measures can be v e r y u s e f u l i n d e v e l o p i n g more r e l i a b l e procedures t o e s t i m a t e exposures based on work p r a c t i c e s by r e l a t i n g d i f f e r e n c e s i n exposure a s s o c i a t e d w i t h tank s i z e , type of spray system, use o f p r o t e c t i v e equipment and o t h e r i n f o r m a t i o n t h a t may be o b t a i n e d from work h i s t o r y records or i n t e r v i e w . E f f o r t s t o evaluate the r e l i a b i l i t y and v a l i d i t y of procedures combining d a t a from v a r i o u s sources a r e needed.

Literature Cited 1. International Agency for Research on Cancer. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 20. Lyon, France. 1979. 2. International Agency for Research on Cancer. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 30. Lyon, France. 1983. 3. Blair, A.; Malker, H.; Cantor, K.P.; Burmeister, L.; Wiklund, K. Scand. J. Work Environ. Health. 1985, 11, 397. 4. Blair, A.; Grauman, D.J.; Lubin, J.H.; Fraumeni, J.F., Jr. J. Nat. Cancer Inst. 1983, 71, 31. 5. Kauppinen, T.P.; Partenen, T.J.; Nurminen, N.M.; Nickels, J.I.; Hernberg, S.G.; Hakulinen, J.R.; Pukkala, E.I.; Savonen, E.T. _Br. J. Industr. Med. 1986, 43, 84.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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6. Wang, H.H.,; MacMahon, B. J. Occupat. Med. 1979, 21, 741. 7. Ditraglia, D.; Brown, D.P.; Namekata, T.; Iverson, N. Scand. J. Work Environ. Health 1981, 7 (Suppl. 4), 140. 8. Barthel, E. J. Toxic. Environ. Health. 1981, 8, 1027. 9. Wang, H.H.; MacMahon, B. J. Occupat. Med. 1979, 21, 745. 10. Hardell, L.; Eriksson, M.; Lenner, P.; Lundgren, E. Br. J. Cancer. 1981, 43, 169. 11. Hoar, S.K.; Blair, A.; Holmes, F.; Boysen, C.; Robel, R.J.; Hoover, R.; Fraumeni, J.F., Jr. J. Amer. Med. Assoc. 1986, 256, 1141. 12. Hardell, L.; Sandstrom, A. Br. J. Cancer. 1979, 39, 711. 13. Blair, A.; Cantor, K.P.; Zahm, S.H.; Burmeister, L.; Van Lier, S.; Gibson, R.; Schuman, L. Proceedings of Pesticides and Groundwater. St. Paul, MN. October, 1986. 14. Woods, J.S.; Polissar, L.; Severson, R.K.; Heauser, L.S.; Kulander, B.G. J. Nat. Cancer Inst. 1987, 78, 899. 15. Vineis, P.; Terracini E.; Donna, A.; Maffi Comba, P. Scand. J. Work Environ. Health. 1987, 13, 9. 16. Lynge, E. Br. J. Cancer. 1985, 52, 259. 17. Riihimaki, V.; Asp, S.; Hernberg, S. Scand. J. Work Environ. Health. 1982, 8, 37. 18. Ott, M.G.; Holder, B.B.; Olson, R.D. J. Occupat. Med. 1980, 22, 47. 19. Axelson, O.; Sundell, L.; Andersson, K.; Edling, C.; Hogstedt, C.; Kling, H. Scand. J. Work Environ. Health. 1980, 7, 73. 20. Smith, A.H.; Pearce, N.E.; Fisher, D.O.; Giles, H.J.; Teague, C.A.; Howard, J.K. J. Nat. Cancer Inst. 1984, 73, 1111. 21. Pearce, N.E.; Smith, A.J.; Howard, J.K.; Sheppard, R.A.; Giles, H.J.; Teague, C.A. Br. J. Industr. Med. 1986, 43, 75. RECEIVED July 5, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter

4

Evaluation of Field Worker Exposure to Chlordimeform by Using Urine Monitoring K. Balu Agricultural Division, Ciba-Geigy Corporation, Greensboro, NC 27419 Chlordimeform, the active ingredient of Galecron 4E, i s an effective insecticide-ovicide used i n cotton. Chlordimeform i s readil in urine followin CIBA-GEIGY Corporation has conducted extensive urine monitoring of f i e l d workers for several years under f i e l d use conditions which employ aerial applications on cotton. Over 38,000 urine samples have been analyzed for chlordimeform and metabolites during 1978-1984 for approximately 4,600 f i e l d workers. These data provide a significant s t a t i s t i c a l v a l i d i t y for assessing the f i e l d worker exposure during actual use.

Chlordimeform, N(4-chloro-o-tolyl)N,N-dimethy1 forraamidine, the active ingredient of Galecron 4E, i s an effective insecticideovicide used in cotton. CIBA-GEIGY Corporation conducted extensive urine monitoring of f i e l d workers during the years 1978-1984 growing seasons under f i e l d use conditions using aerial applications of chlordimeform on cotton. Over 38,600 urine samples were analyzed during this period for approximately 4,600 f i e l d workers. The purpose of the urine monitoring of f i e l d workers was to provide a measure of the daily worker exposure and adherence to safe work practices. This report summarizes the results of these studies along with additional supportive data on the urine monitoring of chlordimeform. STUDY DESIGN AND LIMITATIONS Because the worker exposure of chlordimeform was conducted under actual f i e l d use conditions for a large number of f i e l d workers, the urine monitoring program had a few necessary limitations which were a) urine residues were determined on a grab sample rather than on a 0097-6156/89/0382-0047$06.00/0 © 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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24-hour t o t a l c o l l e c t i o n f o r the f i e l d worker, b) t o t a l d a i l y u r i n e output of i n d i v i d u a l workers was not measured, and c) exposure of f i e l d workers d u r i n g the work p e r i o d was v a r i a b l e . T h i s i s s i g n i f i c a n t l y d i f f e r e n t from a c o n t r o l l e d f i e l d experiment where i n d i v i d u a l work a c t i v i t y would be e x t e n s i v e l y m o n i t o r e d f o r an exposure of short d u r a t i o n . The exposure e s t i m a t e o b t a i n e d from a s i n g l e grab sample i s only approximate f o r a f i e l d worker. However, when a l a r g e number of samples are a n a l y z e d i n the m o n i t o r i n g program ( e . g . , 13,779 u r i n e samples a n a l y z e d from 866 f i e l d workers i n 1978 a l o n e ) , the exposure e s t i m a t e s become m e a n i n g f u l . However, i t i s r e c o g n i z e d t h a t c o l l e c t i o n of u r i n e samples were not randomized i n a t r u e s t a t i s t i c a l sense and hence an unknown b i a s may e x i s t i n the s t a t i s t i c a l i n t e r p r e t a t i o n of the d a t a . D a i l y exposure v a l u e s f o r f i e l d workers are expected to show a range of v a l u e s d i s t r i b u t e d around mean and median v a l u e s . Some of the h i g h e r exposure v a l u e s may be caused by u n c o n t r o l l e d v a r i a b l e s suc t i o n s , exposure t o contaminate v a l u e f o r exposure i s more r e l e v a n t f o r r i s k e s t i m a t i o n of c h l o r dimeform than i n d i v i d u a l e x c u r s i o n s from the mean. The c o n c e n t r a t i o n of c h l o r d i m e f o r m and i t s m e t a b o l i t e s i n a u r i n e specimen w i l l f l u c t u a t e a c c o r d i n g to normal p h y s i o l o g i c a l v a r i a b l e s . U r i n e volume i s i n f l u e n c e d by f l u i d i n t a k e and nonu r i n a r y f l u i d l o s s e s which are i n f l u e n c e d by c l i m a t i c c o n d i t i o n s , i n d i v i d u a l h e a l t h s t a t u s , p h y s i c a l a c t i v i t y , e t c . Hence, the conc e n t r a t i o n of c h l o r d i m e f o r m and i t s m e t a b o l i t e s i n u r i n e c o l l e c t e d a r b i t r a r i l y d u r i n g the day p r o v i d e o n l y a q u a l i t a t i v e i n d i c a t i o n of the exposure on a p a r t i c u l a r day. Attempts were made i n i t i a l l y to o b t a i n the f i r s t u r i n e v o i d of the morning; however, because of the l a r g e number of people i n v o l v e d i n the m o n i t o r i n g program, the time of s a m p l i n g c o u l d not be s t r i c t l y c o n t r o l l e d . The u r i n a r y e l i m i n a t i o n of d e r m a l l y absorbed c h l o r d i m e f o r m as a s i n g l e dose i s expected to f o l l o w the plasma l e v e l and approximate a log normal d i s t r i b u t i o n as a f u n c t i o n of time as shown by the e x c r e t i o n curve i n F i g u r e 1 ( 1 ) . I n c o n t r a s t , the u r i n a r y e x c r e t i o n curve f o r a c o n t i n u o u s exposure i s expected to show a l a r g e p l a t e a u . The exposure t o f i e l d workers h a n d l i n g c h l o r d i m e f o r m under f i e l d c o n d i t i o n s i s expected to be i n t e r m i t t e n t w i t h v a r y i n g amounts of dose depending on the work a c t i v i t y . The e x c r e t i o n curve under t h i s c i r c u m s t a n c e w i l l r e s u l t i n broader peak but a l s o t r o u g h s , i . e . , something i n between the sharp peak and the p l a t e a u . Hence, u r i n e sample taken at any time d u r i n g the day i s expected to be u n p r e d i c t a b l e as to whether i t r e p r e s e n t s a peak or a t r o u g h i n the e x c r e t i o n pattern. Dermal a b s o r p t i o n s t u d i e s on human v o l u n t e e r s i n which c h l o r dimeform was a p p l i e d up to n i n e hours d e r m a l l y , showed t h a t the m a j o r i t y of the absorbed dose i s e x c r e t e d i n the u r i n e r a p i d l y w i t h i n 24 hours w i t h a peak maximum of 8-12 h o u r s . D e t a i l s of these studies are presented i n unpublished reports (1,2). A l s o , u r i n e m o n i t o r i n g s t u d i e s conducted by Maddy e t . a l . (3) on C a l i f o r n i a farm workers i n 1982 have shown c l o s e agreement between c h l o r d i m e f o r m c o n c e n t r a t i o n s measured i n u r i n e specimens c o l l e c t e d the f o l l o w i n g morning. Under c e r t a i n c i r c u m s t a n c e s when s i g n i f i c a n t exposures are encountered through a c c i d e n t a l s p i l l s , t h e r e c o u l d be d i f f e r e n c e s i n

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BALU

Worker Exposure to Chlordimeform

Hours

A f t e r

Treat-merit

Figure 1. Excretion curve for chlordimeform. Single-dose dermal application to human.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

49

50

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

the u r i n a r y r e s i d u e s i n the evening of the day of exposure and the next m o r n i n g . However, i t s h o u l d be r e c o g n i z e d t h a t c o l l e c t i o n of u r i n e samples at a s p e c i f i e d i n t e r v a l i s i m p r a c t i c a l when d e a l i n g w i t h a l a r g e number of f i e l d workers f o r a l o n g p e r i o d . METHODS F i e l d Monitoring The l a b e l d i r e c t i o n s f o r G a l e c r o n 4E s p e c i f i e d f o r m i x i n g and l o a d i n g , u s i n g c l o s e d system equpment, p e r s o n a l p r o t e c t i v e c l o t h i n g requirements and p r e c a u t i o n s were f o l l o w e d d u r i n g the s t u d y . Bulk h a n d l i n g systems were employed f o r c l o s e d - s y s t e m s t o r a g e and t r a n s f e r of G a l e c r o n 4E through 1981. Bulk system was l a r g e l y r e p l a c e d w i t h an automated C a p t a i n Crunch® d e v i c e u s i n g 5 - g a l l o n c o n t a i n e r s at l a t e r p e r i o d s to meet the c l o s e d system r e q u i r e m e n t s Protective clothin 4E l a b e l to reduce worke a e r i a l a p p l i c a t i o n s . These p r o t e c t i v e c l o t h i n g requirements i n c l u d e d heavy-duty, l o n g - s l e e v e , one-piece work s u i t ; c l o t h caps; rubber or neoprene-type g l o v e s ; and heavy-duty work b o o t s . S p e c i a l p r o t e c t i v e c l o t h i n g and equipment s p e c i f i e d f o r c l e a n i n g or s p i l l s i t u a t i o n s i n c l u d e d complete w a t e r p r o o f r u b b e r i z e d or p l a s t i c r a i n s u i t s w i t h head c o v e r i n g s and approved p e s t i c i d e r e s p i r a t o r s . I t should be noted t h a t these p r o t e c t i v e c l o t h i n g and equipment requirements may not have been s t r i c t l y f o l l o w e d under a c t u a l use c o n d i t i o n s . However, because of c l o s e d - s y s t e m r e q u i r e m e n t s , the m i x i n g and l o a d i n g o p e r a t i o n p r o v i d e s v e r y l i t t l e o p p o r t u n i t y f o r exposure. Exposures to m i x e r / l o a d e r s and a p p l i c a t o r s at the s i t e where thought to be p r i m a r i l y caused by the cleanup o p e r a t i o n of the a i r c r a f t , inadequate use of g l o v e s d u r i n g adjustments of the spray n o z z l e and o t h e r i n d i r e c t causes. CIBA-GEIGY C o r p o r a t i o n a l s o conducted e x t e n s i v e worker exposure s t u d i e s i n 1982 under an E x p e r i m e n t a l Use Permit f o r a e r i a l a p p l i c a t i o n s of c h l o r d i m e f o r m i n o i l u s i n g u l t r a - l o w - v o l u m e (ULV) s p r a y s . Because the study was conducted under a c t u a l use c o n d i t i o n s , use of o i l as the o n l y c a r r i e r d u r i n g the growing season was not p r a c t i c a l and most p a r t i c i p a t i n g a e r i a l a p p l i c a t i o n s i t e s a p p l i e d G a l e c r o n 4E i n both o i l and w a t e r . F i e l d worker exposure d u r i n g t h i s p e r i o d i s thought to have o c c u r r e d d u r i n g clean-up of a i r c r a f t and equipment d u r i n g s w i t c h i n g of the c a r r i e r s . Laboratory

Studies

L a b o r a t o r y s t u d i e s have shown t h a t c h l o r d i m e f o r m i s r e a d i l y absorbed and e x c r e t e d i n u r i n e f o l l o w i n g dermal, o r a l and i n h a l a t i o n exposure. O r a l d o s i n g of r i n g ^ C - l a b e l l e d c h l o r d i m e f o r m to r a t s or mice showed t h a t a p p r o x i m a t e l y 85% and 74%, r e s p e c t i v e l y , of the a p p l i e d r a d i o a c t i v i t y was e x c r e t e d i n the u r i n e d u r i n g the i n i t i a l 24 hours ( 4 ) . These r e s u l t s are i n good agreement w i t h the d a t a o b t a i n e d by Knowles and Sengupta (5) i n which 88% of the a p p l i e d dose i n the r a t was r e c o v e r e d i n u r i n e .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

4.

BALU

Worker Exposure to Chlordimeform

51

An a b s o r p t i o n / e x c r e t i o n study was conducted i n 1978 i n which a human v o l u n t e e r r e c e i v e d a s i n g l e dermal dose o f G a l e c r o n 4E i n water f o r a d u r a t i o n o f n i n e hours ( 1 ) . T h i s study showed t h a t c h l o r d i m e f o r m and i t s m e t a b o l i t e s a r e r a p i d l y e x c r e t e d i n the u r i n e . M a j o r i t y of the m e t a b o l i t e s o f c h l o r d i m e f o r m appeared as c o n j u g a t e s which were r e l e a s e d as 4 - c h l o r o - o - t o l u i d i n e by base h y d r o l y s i s . A n a l y s e s of 48-hour u r i n e sample by the 4 - c h l o r o - o _ - t o l u i d i n e common m o i e t y method accounted f o r a p p r o x i m a t e l y 40% of the d e r m a l l y absorbed dose. T h i s formed the b a s i s f o r the method t o determine the exposure o f c h l o r d i m e f o r m t o f i e l d w o r k e r s . CIBA-GEIGY C o r p o r a t i o n conducted an a d d i t i o n a l dermal study i n 1983 u s i n g e i g h t human v o l u n t e e r s who r e c e i v e d a s i n g l e dose o f G a l e c r o n 4E i n water f o r a p e r i o d o f f o u r hours ( 2 ) . T h i s study a l s o confirmed that an average o f 38.3% o f the absorbed dose was accounted f o r by u r i n e a n a l y s e s f o r c h l o r d i m e f o r m and i t s metabol i t e s c o n t a i n i n g the 4 - c h l o r o - o - t o l u i d i n e common m o i e t y The h a l f l i f e for excretion varie hours w i t h an average o The e x c r e t i o n p a t t e r n found i n the human study i s c o n s i s t e n t w i t h t h a t found i n the r a t and the mouse study u s i n g o r a l d o s i n g (4). Based on the r e s u l t s o f ^ C - m e t a b o l i s m s t u d i e s on a n i m a l s , the m a j o r i t y of the u n d e t e c t e d p o r t i o n r e p r e s e n t s m e t a b o l i t e s i n the u r i n e which the a n a l y t i c a l method does not r e c o v e r . A b u i l d u p o f c h l o r d i m e f o r m r e s i d u e s i n any t i s s u e i s u n l i k e l y , s i n c e animal s t u d i e s have shown e x c l u s i v e l y the e n t i r e r a d i o a c t i v e dose i n the excreta. A n a l y t i c a l Procedure The u r i n e samples c o l l e c t e d i n the f i e l d worker exposure s t u d i e s were a n a l y z e d f o r t o t a l c h l o r d i m e f o r m r e s i d u e s c o n t a i n i n g the 4 - c h l o r o - o - t o l u i d i n e common moiety ( 6 ) . I n t h i s method, the u r i n e sample was h y d r o l y z e d t o 4 - c h l o r o - o - t o l u i d i n e by h e a t i n g w i t h sodium hydroxide. The h y d r o l y z e d u r i n e was c o o l e d and p a r t i t i o n e d w i t h hexane and an a l i q u o t o f the o r g a n i c phase was analyzed by gas chromatography or h i g h p r e s s u r e l i q u i d chromatography (HPLC). The l i m i t o f d e t e c t i o n f o r the method was 0.05 ppm, expressed i n c h l o r d i m e f o r m e q u i v a l e n t s . The method f o r the a n a l y s e s of c h l o r d i m e f o r m and m e t a b o l i t e s i n u r i n e and o t h e r u n p u b l i s h e d r e p o r t s c i t e d i n the r e f e r e n c e s w i l l be a v a i l a b l e on r e q u e s t . E s t i m a t i o n o f Worker Exposure Worker exposure v a l u e s were c a l c u l a t e d from the u r i n e r e s i d u e s u s i n g some reasonable a s s u m p t i o n s . Assuming an average e x c r e t i o n volume of 1,500 ml/day, 90% e l i m i n a t i o n i n u r i n e (10% e l i m i n a t e d o t h e r than u r i n e , p r i m a r i l y feces based on animal data) and 40% d e t e c t i o n o f c h l o r d i m e f o r m r e s i d u e s i n u r i n e by the t o t a l method (based on the a c c o u n t a b i l i t y i n the human v o l u n t e e r s t u d i e s ) , the u r i n a r y r e s i d u e s , i n ppm, was c o r r e l a t e d w i t h the dermal dose u s i n g the following relationship:

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

52

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

100 Body Burden = ( U r i n e R e s i d u e , ppm) X 1,500 ml X

100 X

90

40

= ( U r i n e R e s i d u e , ppm) X 4.16 mg/day Where the body burden i s the amount of the dermal dose absorbed through the s k i n . Assuming an average weight of 70 kg f o r a f i e l d w o r k e r , the average exposure e s t i m a t e f o r the f i e l d worker u s i n g c h l o r d i m e f o r m i s c a l c u l a t e d from the u r i n e m o n i t o r i n g d a t a as Body Burden = ( U r i n e R e s i d u e , ppm) X .0595 mg/kg/day RESULTS AND

DISCUSSION

The number of samples, worker p r o f i l e s for mixer/loader d u r i n g the y e a r s 1978-1984 are summarized i n T a b l e s I and I I . These r e s u l t s show a remarkable s i m i l a r i t y i n the d i s t r i b u t i o n p r o f i l e s during d i f f e r e n t years. TABLE I :

SUMMARY OF PERCENT DAILY VALUE DISTRIBUTION FOR MIXER/ LOADERS EXPOSED TO CHLORDIMEFORM DURING 1978-1984

Year

1978

No. of Samples No. of Workers

8,223 352

1

1979

1

4,408 597

1980

2,886 650

PPM Range <0.05-0.10 0.1 -0.3 0.3 -0.5 0.5 -1.0 1.0 -5.0 >5.0

1981

1

1

1982

2

1984

2

1,986 381

505 34

96 7

55 18 6.8 7.7 10 1.6

52 10 7.4 12 18 1.4

53 28 6.3 10 2.1 0

Total 18,104 2,021

% 783 163 5.6 0.5

46 23 10 11 9.2 1.9

51 22 8.1 8.5 9.1 1.0

1-Used water as a c a r r i e r U s e d both water and o i l c a r r i e r i n ULV ( w i t h 1984 p r e d o m i n a t e l y using o i l - U L V ) . ^Combined v a l u e s f o r <0. 3 ppm ,and 0.3--1.0 ppm., r e s p e c t i v e l y 2

The r e s u l t s of the 1982 worker exposure study showed a t w o - f o l d i n c r e a s e i n exposure v a l u e s exceeding 0.5 ppm to m i x e r / l o a d e r s (Table I ) compared to the p r e v i o u s y e a r s , when w a t e r only was used as a c a r r i e r . However, d u r i n g the 1982 s t u d y p e r i o d , most of the a p p l i c a t i o n s i t e s s w i t c h e d between water and o i l c a r r i e r s and e x c u r s i o n s i n exposure might have been caused by the a d d i t i o n a l equipment h a n d l i n g when s w i t c h i n g between water and o i l as a c a r r i e r . I n exposure s t u d i e s conducted i n 1984 (where p r e d o m i n a n t l y o i l - U L V was u s e d ) , the d i s t r i b u t i o n p r o f i l e was q u i t e s i m i l a r t o the y e a r s i n which only water was used as a c a r r i e r . A l s o , the 1984

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

53

Worker Exposure to Chlordimeform

4. BALU

v a l u e s showed d a i l y u r i n e r e s i d u e s g e n e r a l l y c l o s e to the d e t e c t i o n l i m i t and v e r y few exceeding 1 ppm. Hence, i t appears from the 1984 study and e a r l i e r d a t a t h a t c h l o r d i m e f o r m exposure to f i e l d workers u s i n g c l o s e d systems and p r o t e c t i v e c l o t h i n g was p r o b a b l y caused by u n c o n t r o l l a b l e f a c t o r s , such as s i t e c o n t a m i n a t i o n s , work h a b i t s , or cleanup o p e r a t i o n s . The median f o r the d a i l y v a l u e d i s t r i b u t i o n f o r f i e l d workers i s l e s s than 0.1 ppm; 52-70% o f the d a i l y u r i n e v a l u e s were below 0.1 ppm ( T a b l e I I ) . TABLE I I :

SUMMARY OF PERCENT DAILY VALUE DISTRIBUTION (PPM) FOR PILOTS AND OTHER WORKERS EXPOSED TO CHLORDIMEFORM DURING 1978- •1984

Year

1978

No. of Samples No. of Workers

5,556 514

PPM

1

1979

1

1980

1

Range

<0.05-0.10 0.1 -0.3 0.3 -0.5 0.5 -1.0 1.0 -5.0 >5.0

1981

1

1982

2

1984

2

Total

% 90

4

6.9 3.0 0.3

4

66 19 5.3 4.8 4.1 0.4

63 21 5.4 5.7 4.5 0.7

68 17 4.7 5.8 4.4 0.4

74 11 7.7 2.9 4.2 0.3

74 20 3.7 1.2 1.2 0

1-Used water as a c a r r i e r . U s e d both water and o i l c a r r i e r i n ULV ( w i t h 1984 p r e d o m i n a t e l y using o i l - U L V ) . ^Combined v a l u e s f o r <0.3 ppm and 0.3-1.0 ppm, r e s p e c t i v e l y . 2

These r e s u l t s a l s o show t h a t a p p r o x i m a t e l y 75% of the d a i l y u r i n e v a l u e s f o r a l l f i e l d workers were below 0.3 ppm. Only 6-7% of a l l the samples f o r the f i e l d workers (10.1% or 3.8% of the m i x e r / l o a d e r s or p i l o t s and o t h e r w o r k e r s , r e s p e c t i v e l y ) showed d a i l y u r i n e v a l u e s exceeding 1 ppm. I t i s i m p o r t a n t to note t h a t a l t h o u g h u r i n e v a l u e s exceeded 1 ppm f o r some f i e l d workers on some days, these v a l u e s f o r the i n d i v i d u a l workers decreased on subsequent days. Hence, c o n s i d e r i n g o n l y the maximum d a i l y exposure f o r a f i e l d worker w i l l o v e r - e s t i m a t e exposure on a s e a s o n a l b a s i s . The median v a l u e f o r the d a i l y v a l u e d i s t r i b u t i o n found i n the CIBA-GEIGY u r i n e m o n i t o r i n g s t u d i e s of G a l e c r o n i s i n c l o s e agreement w i t h the d a i l y average v a l u e of 0.13 ppm o b t a i n e d by Maddy et a l . (3) f o r worker exposure to c h l o r d i m e f o r m i n the I m p e r i a l V a l l e y i n C a l i f o r n i a d u r i n g the 1982 c o t t o n growing season. A s e a s o n a l c u m u l a t i v e v a l u e d i s t r i b u t i o n f o r a l l the f i e l d workers d u r i n g the y e a r s 1978 and 1979 i s summarized i n Table I I I . The c u m u l a t i v e s e a s o n a l exposure v a l u e f o r a f i e l d worker i s the p r o j e c t e d sum of a l l the average d a i l y u r i n e v a l u e s d u r i n g the e n t i r e growing season.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

54

TABLE I I I : CUMULATIVE VALUE DISTRIBUTION (PPM) FOR ALL FIELD WORKERS AS MEASURED FROM URINE SAMPLES Cumulative Range (ppm)

No. of Workers

1978 % Distribution

No. of Workers

1979 % Distribution

<5 5-10 10-20 20-30 30-60 >60

530 175 100 34 21 6

61.2 20.2 11.5 3.9 2.4 0.7

1,015 259 185 78 48 38

73.3 15.6 11.2 4.7 2.9 2.3

866

100.0

1,623

100.0

T o t a l No. of Personnel

These d a t a show t h a t 61.2-73.3 d u r i n g these y e a r s showed a t o t a l c u m u l a t i v e s e a s o n a l v a l u e of <5 ppm. For the purpose of c a l c u l a t i o n of the c u m u l a t i v e v a l u e s , a l l the u r i n e sarnies c o n t a i n i n g r e s i d u e s below the 0.05 ppm s c r e e n i n g l e v e l were a s s i g n e d a v a l u e of 0.05 ppm and hence the t o t a l cumulat i v e v a l u e i s expected to be c o n s e r v a t i v e l y h i g h e r than they would be u s i n g the a c t u a l v a l u e s f o r samples <0.05 ppm. A s m a l l percent (3.1-5.2%) of the f i e l d workers showed s e a s o n a l c u m u l a t i v e v a l u e s e x c e e d i n g 30 ppm. C a l i f o r n i a Department of Food and A g r i c u l t u r e (CDFA) conducted e x t e n s i v e u r i n e m o n i t o r i n g of c h l o r d i m e f o r m i n C a l i f o r n i a d u r i n g the 1982-1985 growing seasons. These d a t a were p r e s e n t e d by Maddy et a l . (7) at the 6th I n t e r n a t i o n a l Conference on P e s t i c i d e Chemistry (IUPAC) a t Ottawa, Canada. I n the f o u r y e a r s of m o n i t o r i n g , a p p r o x i m a t e l y 8,800 f i e l d a p p l i c a t i o n s of c h l o r d i m e f o r m were made at 0.25 l b s . a i / a c r e on an average s i z e of 80 a c r e s , and a p p r o x i m a t e l y 200 f i e l d workers were m o n i t o r e d . These s t u d i e s showed t h a t the c h l o r d i m e f o r m u r i n e r e s i d u e s f o r the f i e l d workers averaged 0.09 ppm, and d i d not i n c r e a s e d u r i n g the work season. The l e v e l of c h l o r d i m e f o r m r e s i d u e s i n u r i n e a t the end of the work s h i f t was s i m i l a r to t h a t found 8 to 10 hours l a t e r . The u r i n e m o n i t o r i n g d a t a i n the CDFA s t u d i e s are i n good agreement w i t h the e x t e n s i v e d a t a o b t a i n e d by CIBA-GEIGY on u r i n e m o n i t o r i n g of the f i e l d workers exposed to c h l o r d i m e f o r m . CONCLUSIONS Based on l a b o r a t o r y animal metabolism and s t u d i e s w i t h human v o l u n t e e r s , the amount of t o t a l c h l o r d i m e f o r m r e s i d u e s c o n t a i n i n g the 4 - c h l o r o - o _ - t o l u i d i n e common moiety r e c o v e r e d i n the u r i n e f o l l o w i n g dermal a p p l i c a t i o n of c h l o r d i m e f o r m i s a p p r o x i m a t e l y 40% of the d e r m a l l y absorbed dose. These s t u d i e s have a l s o shown t h a t c h l o r d i m e f o r m and i t s m e t a b o l i t e s are r a p i d l y e x c r e t e d i n the u r i n e . T h i s approach has been used by CIBA-GEIGY C o r p o r a t i o n to m o n i t o r exposure of a l a r g e number of f i e l d workers and a p p l i c a t o r s .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

4. BALU

Worker Exposure to Chlordimeform

55

The e x t e n s i v e u r i n e m o n i t o r i n g conducted by CIBA-GEIGY p r o v i d e s a l a r g e d a t a base f o r e v a l u a t i n g worker exposure to c h l o r d i m e f o r m under a c t u a l use c o n d i t i o n s . The r e p r o d u c i b i l i t y o f the u r i n a r y d i s t r i b u t i o n p r o f i l e s of c h l o r d i m e f o r m i n these d a t a a l l o w f o r e v a l u a t i o n of the f i e l d worker exposure to c h l o r d i m e f o r m under a c t u a l use c o n d i t i o n s .

REFERENCES 1. 2. 3.

4.

Camp, H. B., ABR-78005, "Chlordimeform Residues in Human Urine Following Dermal Applications," CIBA-GEIGY Corporation, Greensboro, NC, January 1978. Nixon, W. B., B. E. Neal, "Chlordimeform Residues in Human Urine Following Dermal Applications," July 11, 1983. Maddy, K. T., P. H. Kurtz, Dennis Gibbons, Linda O'Connell, "Preliminary Report on Health Monitoring of Pest Control Workers Exposed t 1982 Cotton Growin Pest Management, California Department of Food and Agriculture, Sacramento, CA, April 1, 1983. Ifflaander, U., Project Report 39/77, "Comparison of the Urinary Metabolite Pattern of Mice and Rats After Oral Applica­ tion of C-Chlordimeform," CIBA-GEIGY Limited, Basle, Switzerland, August 18, 1987. Knowles, C. O., Sen Gupta, A. K., J. Econ. Entomol., 1970, 63, 856-9. Cheung, M., "Chlordimeform Methods for Biological Monitoring," CIBA-GEIGY Corporation, Greensboro, NC. Presented at ACS Symposium on Biological Monitoring, September 1987, New Orleans (Not Published), available on request. Maddy, K. L., J. B. Knaak, D. B. Gibbons, "Monitoring of the Urine of Pesticide Applicators in California for Residues of Chlordimeform and Its Metabolites," 1982-1985, Sixth Interna­ tional Conference of Pesticide Chemistry (IUPAC), Ottawa, Canada, 1986. 14

5. 6.

7.

RECEIVED June 24, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 5

Convenient Field Sampling Method for Monitoring Volatile Compounds in Exhaled Breath Michael S. Morgan, Gary S. Phillips, and Eileen M . Kirkpatrick Department of Environmental Health, University of Washington, Seattle, WA 98195 A modified half-face air purifying respirator was developed as a technique for large-scale monitoring that will be acceptabl and relatively sensitive through the respirato por throug two-part cartridge. The f i r s t part contained four layers of activated charcoal cloth which adsorbed organic solvent vapors; the cloth was subsequently desorbed for gas chromatographic analysis. The second part contained 70 g of 8-12 mesh molecular sieve whose weight gain (water) was proportional to the volume of air exhaled. The proportionality was determined by independent measurement of volume in subjects while at rest and while exercising. Controlled atmospheres containing toluene at relative humidities near saturation were passed through the cartridge at steady flows equal to resting human ventilatory rates. Toluene recovery was 70% for simulated breath concentrations of 0.75 mg/m to 60 mg/m (0.20 ppm to 16 ppm). Normal adults volunteered to breathe toluene in air at 150 mg/m (40 ppm) or 340 mg/m (90 ppm) for four hours (TLV = 375 mg/m or 100 ppm, 8 hr TWA). About eighteen hours later their breath was sampled, to simulate a worksite measurement at the start of the next shift. Samples of at least 140 liters were taken from each subject, and the mean exhaled concentrations were 344 μg/m and 600 μg/m (0.23% and 0.18% of exposure TWA), respectively, in good agreement with literature reports using more complicated methods not appropriate for field application. The device can be used to collect samples of up to 300 liters in 30 minutes, and i t s sensitivity and selectivity are then limited only by the desorption and analysis of the charcoal adsorbent. The lower limit of quantitation for toluene, with conventional GC analysis, was about 65 μg/m (18 ppb). The technique can be applied to most solvents capable of adsorption on charcoal, and w i l l permit sampling large numbers of workers with minimal job disruption. In a limited f i e l d application, automobile body painters showed levels of toluene in their exhaled breath that could not be 3

3

3

3

3

3

3

3

0097-6156/89/0382-0056$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5.

MORGAN E T A L .

Volatile Compounds in Exhaled Breath

57

explained by inhalation exposure alone; observations of frequent skin contact with the solvent suggested that important skin absorption also occurred, and the breath levels reflected the workers' total solvent exposure. Among methods available for biological monitoring, exhaled breath analysis offers potential advantages as i t i s less intrusive than drawing blood and often gives a better reflection of blood composition than does urinalysis. The appearance of volatile compounds in exhaled breath after industrial exposure has been demonstrated in several instances (1-7). Table I shows some examples, together with solubility data which suggest means of identifying other compounds as candidates for this form of biological monitoring. However, breath analysis has not become widespread in the workplace because of practical problems in sampling large numbers o Because concentrations o the air is moist, previous researc has relied eithe o elaborate and expensive equipment or on sampling methods subject to moisture-related loss of contaminant. Further, evaluating the results of breath sampling requires consideration of the complex excretion kinetics of most volatile compounds and of the effects of respiratory dead space on the composition of exhaled breath. The last point has led to efforts to sample alveolar air by procedures requiring much cooperation from the worker. This work addresses some of these problems. A portable, simplified breath sampling system has been developed which is suitable for mass screening, is capable of collecting a relatively large sample, and gives good performance in sampling moist air. Table I. Materials Shown to be Present in Exhaled Breath after Industrial Exposure

Compounds

—Partition Coefficients— Found in Blood/Air Fat/Air QU/Ajr Breath 3 t

Benzene Toluene Xylene Methyl Ethyl Ketone Ethylene Oxide Trichloroethylene Chloroform Methylchloroform Carbon Tetrachloride

6-8 10-16 26-38 200 90 8-10 8-10 3 2-6

425 962 140 560-660 280 250 360

492 1470 3700 260 700-940 400-560

A

20 hours 20 hours 20 hours 2 hours 0 hours 20 hours 0 hours 20 hours 6 hours

Ref. 15,24 12 25 26 27 5 28 29 30

& Time measured from the end of the exposure period The primary value in breath sampling lies in the fact that alveolar gas is very nearly in equilibrium with arterial blood (Figure 1). Mixed expired air, however, is not equal in composition to alveolar air, because of the addition of air from the respiratory dead space. Nevertheless, for samples taken over periods of ten minutes or more, such that a l l the expired air i s

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

c o l l e c t e d and i t s volume measured, t h e r e i s u s u a l l y a p r e d i c t a b l e r e l a t i o n s h i p between a l v e o l a r and mixed e x p i r e d a i r , so t h a t t h e l a t t e r can s t i l l be used t o make e s t i m a t e s of t h e b l o o d l e v e l s of t r a c e compounds.(1-3) A more s e r i o u s p r o b l e m i n s a m p l i n g e i t h e r a l v e o l a r o r mixed e x p i r e d a i r i s t h e e f f e c t of t h e passage o f t i m e a f t e r exposure has ended. Models and e x p e r i m e n t a l measurements of e x c r e t i o n k i n e t i c s of v o l a t i l e compounds have shown t h a t w i t h i n two t o f o u r hours a f t e r t h e end of exposure, t h e b r e a t h l e v e l s d e c r e a s e r a p i d l y , and are s t r o n g l y c o r r e l a t e d w i t h t h e c o n c e n t r a t i o n i n t h e i n h a l e d a i r a t t h e end of exposure.(4-6) At longer times post-exposure, the breath l e v e l s are l e s s time dependent, and a r e more c l o s e l y p r o p o r t i o n a l t o t h e time w e i g h t e d average exposure, o r body burden, of t h e compound.(H) The use of b r e a t h a n a l y s i s i n m o n i t o r i n g p e s t i c i d e exposure has not y e t been r e p o r t e d , but t h i s g e n e r a l approach has p o t e n t i a l v a l u e f o r c e r t a i n k i n d s of p e s t i c i d e f o r m u l a t i o n s . Table I I p r e s e n t s some examples of p e s t i c i d e components which have physicochemical propertie i n part, i n breath. Suitabl have s o l u b i l i t y p r o p e r t i e s i n b l o o d and body f a t t h a t a r e c l o s e t o t h e ranges g i v e n i n Table I . Many of t h e fumigants used i n a g r i c u l t u r e meet t h e s e r e q u i r e m e n t s , and a few have a c t u a l l y been found i n t h e b r e a t h of workers exposed i n e x p e r i m e n t a l o r industrial settings. In a d d i t i o n , many of t h e v e h i c l e s used i n p e s t i c i d e f o r m u l a t i o n s a r e good c a n d i d a t e s f o r b r e a t h m o n i t o r i n g , and examples a r e i n c l u d e d i n Table I I . T a b l e I I . P e s t i c i d e Components w i t h P h y s i c o c h e m i c a l P r o p e r t i e s F a v o r i n g t h e Use of E x h a l e d B r e a t h f o r B i o l o g i c a l M o n i t o r i n g Compound

Volatile

Chloropicrin Methylisocyanate Ethylene D i c h l o r i d e HCN Sulfuryl Fluoride Vapam Dichloropropylene Dichloropropane Methyl Chloroform E t h y l e n e Oxide Naphthalene p-Dichlorobenzene M e t h y l Bromide Methyl Isothiocyanate Diesel fuel Kerosene Methanol Petroleum d i s t i l l a t e s Xylene Toluene

X X X X X X X X X X X X X X X X X X X X

L i p i d Soluble

X

X X X X X X X X X X X X X

B r e a t h Data Available

E^fs.

X

28

X X

29 27

X

31

X X

25 12

The g o a l of t h e work d e s c r i b e d here was t o d e s i g n and demonstrate a f i e l d - p o r t a b l e t e c h n i q u e f o r s a m p l i n g mixed e x p i r e d

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5.

MORGAN ETAL.

Volatile Compounds in Exhaled Breath

a i r f o r v o l a t i l e compounds, a t t i m e s l o n g a f t e r exposure when t h e b r e a t h l e v e l i s i n d i c a t i v e of body burden. The use of mixed e x p i r e d a i r r e p r e s e n t s a c o n c e s s i o n t o t h e need f o r s i m p l i c i t y i n s a m p l i n g , but t h e method s h o u l d s t i l l g i v e a r e s u l t u s e f u l i n b i o l o g i c a l monitoring. The s a m p l i n g method was d e m o n s t r a t e d i n human v o l u n t e e r s l o n g a f t e r a r e l a t i v e l y low i n h a l a t i o n exposure (as low as 20% of t h e o c c u p a t i o n a l T h r e s h o l d L i m i t V a l u e , TLV), as a s e v e r e t e s t of performance. R e s u l t s w i l l a l s o be p r e s e n t e d f o r f i e l d s t u d i e s w i t h workers exposed t o s o l v e n t s i n t h e a u t o m o b i l e repair industry. M a t e r i a l s and Methods Sampler D e s i g n and

Performance

The sampler i s based on a h a l f - f a c e , d u a l c a r t r i d g e r e s p i r a t o r whose i n h a l a t i o n p o r t s a r e f i t t e d w i t h s t a n d a r d a i r p u r i f y i n g e l e m e n t s . ( 1 ) On t h e e x h a l a t i o for sampling exhaled a i r c a r t r i d g e i s based on: a.) q u a n t i t a t i v e a d s o r p t i o n of o r g a n i c v a p o r s and subsequent a n a l y s i s by s t a n d a r d methods, and b.) d e t e r m i n a t i o n of t h e a i r volume sampled by t a k i n g advantage of t h e n e a r l y i n v a r i a n t c o n c e n t r a t i o n of water vapor i n e x p i r e d a i r among i n d i v i d u a l s o v e r a wide range of a c t i v i t y l e v e l s . (8 9) The s a m p l i n g c a r t r i d g e i s shown i n cutaway v i e w i n F i g u r e 2. The upper s e c t i o n c o n t a i n s f o u r l a y e r s of a c t i v a t e d c h a r c o a l c l o t h mounted i n p a i r s s e p a r a t e d by t e f l o n g a s k e t s . This adsorbent i s a woven m a t e r i a l ( C h a r c o a l C l o t h L i m i t e d , Maidenhead, U n i t e d Kingdom) h a v i n g p r o p e r t i e s s i m i l a r t o g r a n u l a r c h a r c o a l , e x c e p t t h a t i t s c o l l e c t i o n performance, based on s t u d i e s w i t h v o l a t i l e a n e s t h e t i c s , i s r e p o r t e d t o be much b e t t e r at h i g h w a t e r vapor c o n t e n t . (1Q.) The lower s e c t i o n c o n t a i n s 70 g of 8 - 12 mesh m o l e c u l a r s i e v e of 3 A p o r e s i z e which c o l l e c t s t h e w a t e r vapor (maximum c a p a c i t y 14 g water vapor p e r 100 g a d s o r b e n t ) . The o r g a n i c v a p o r s c o l l e c t e d on t h e c h a r c o a l c l o t h were r e c o v e r e d by d e s o r p t i o n of p a i r e d l a y e r s i n 10 ml carbon d i s u l f i d e f o r one hour. The e l u e n t was a n a l y z e d by gas chromatograph u s i n g a flame i o n i z a t i o n d e t e c t o r f o l l o w i n g recommended p r o c e d u r e s . (JL1) G e n e r a l l y , t h e c l o t h l a y e r p a i r s were a n a l y z e d s e p a r a t e l y t o p e r m i t d e t e c t i o n of b r e a k t h r o u g h p a s t t h e f i r s t p a i r of l a y e r s . The c o l l e c t e d water vapor was d e t e r m i n e d g r a v i m e t r i c a l l y . The c a r t r i d g e was f a b r i c a t e d from p o l y v i n y l c h l o r i d e p i p e w i t h s t a i n l e s s s t e e l screens t o r e t a i n the adsorbents. The l o a d e d c a r t r i d g e weighed about 180 ± 10 g, and t h u s added about 60% t o t h e weight of t h e r e s p i r a t o r . Resistance t o a i r f l o w through the c a r t r i d g e was l e s s t h a n 2 cm H 2 O / L / S a t f l o w r a t e s up t o 1 L/s. The s a m p l i n g c a r t r i d g e was t e s t e d f o r r e c o v e r y e f f i c i e n c y w i t h c o n t r o l l e d atmospheres d e s i g n e d t o s i m u l a t e human b r e a t h cont a i n i n g low l e v e l s of o r g a n i c v a p o r . Toluene was s e l e c t e d f o r t h i s s t u d y as i t i s used i n l a r g e q u a n t i t i e s i n i n d u s t r y and s t u d i e s of i t s t o x i c o k i n e t i c s have been r e p o r t e d by s e v e r a l a u t h o r s . (2 12-15) The atmospheres were g e n e r a t e d i n a 90 l i t e r g l a s s m i x i n g chamber e q u i p p e d w i t h a c o o l e d - m i r r o r dew p o i n t hygrometer and a p h o t o i o n i z a t i o n d e t e c t o r . The d e t e c t o r was c a l i b r a t e d b e f o r e and a f t e r t h e s t u d y u s i n g a c a p i l l a r y d i f f u s i o n tube (1£). Compressed b r e a t h i n g q u a l i t y a i r was p a s s e d t h r o u g h a h i g h e f f i c i e n c y f i l t e r and a bed of g r a n u l a r c h a r c o a l b e f o r e e n t e r i n g t h e chamber a t 40 L/min. Water was a t o m i z e d i n t o the chamber, and l i q u i d t o l u e n e was metered v i a m o t o r - d r i v e n s y r i n g e t h r o u g h a h e a t e d m e t a l t u b e . r

r

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

mixed expired air

t

dead s p a c e

xalveolar air:* venous blood

arterial blood

arterial conten

F i g u r e 1 — Schematic diagram o f t h e r e l a t i o n s h i p among venous b l o o d , a r t e r i a l b l o o d , a l v e o l a r a i r and mixed e x p i r e d a i r , w i t h r e s p e c t t o c o n c e n t r a t i o n o f a v o l a t i l e m a t e r i a l . "p" r e f e r s t o t h e p a r t i a l p r e s s u r e o f t h e v o l a t i l e component o f i n t e r e s t .

F i g u r e 2 — Cutaway view o f t h e b r e a t h s a m p l i n g c a r t r i d g e showing t h e s e c t i o n f o r o r g a n i c s o l v e n t vapor c o l l e c t i o n and t h e s e c t i o n f o r m o i s t u r e c o l l e c t i o n ; t h e two s e c t i o n s nest t i g h t l y t o g e t h e r when i n use. The s o l v e n t s e c t i o n i s t h r e a d e d t o f i t a s t a n d a r d r e s p i r a t o r c a r t r i d g e h o l d e r which i s mounted i n t h e exhaust p o r t o f t h e mask.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5. MORGAN ETAL.

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Volatile Compounds in Exhaled Breath

Both r e l a t i v e h u m i d i t y (RH) and s o l v e n t c o n c e n t r a t i o n were m o n i t o r e d c o n t i n u o u s l y . The g e n e r a t o r was c a p a b l e o f p r o d u c i n g s t a b l e atmospheres a t between 5% and 95% RH and c o n t a i n i n g between 0.38 mg/m (0.1 ppm) and 1500 mg/m (400 ppm) o f t o l u e n e . Recovery d a t a were o b t a i n e d o v e r a range o f RH and a i r t e m p e r a t u r e s , whose s e l e c t i o n was based on p r e l i m i n a r y measurements o f the a i r s t r e a m l e a v i n g r e s p i r a t o r s w h i l e b e i n g worn. C a r t r i d g e s were c h a l l e n g e d w i t h 10 L/min a i r c o n t a i n i n g t o l u e n e a t l e v e l s between 0.75 mg/m (0.20 ppm) and 150 mg/m (40 ppm) f o r 30 minutes based on the r e s u l t s o f t o x i c o k i n e t i c s t u d i e s . To d e t e r m i n e t h e r e l a t i o n s h i p between water vapor c o l l e c t e d and the volume o f e x p i r e d a i r sampled, a group o f s i x v o l u n t e e r s wore t h e r e s p i r a t o r w h i l e a t r e s t o r w h i l e w a l k i n g on a t r e a d m i l l a t v a r i o u s work l e v e l s . The r e s u l t i n g pulmonary v e n t i l a t i o n r a t e s ranged from 4 L/min t o 28 L/min. E x p i r e d a i r was p a s s e d t h r o u g h the sampling c a r t r i d g e i n s e r i e s w i t h a c a l i b r a t e d pneumotachometer. The f l o w s i g n a l from t h e pneumotachometer was integrated electronicall volume. The weight g a i was t h e n compared t o the independent measurement o f volume. The s a m p l i n g p e r i o d s were between f o u r t e e n and twenty m i n u t e s . 3

3

3

3

Performance i n C o n t r o l l e d Human Exposures As a d e m o n s t r a t i o n o f the d e v i c e under r e a l i s t i c c o n d i t i o n s o f use, h e a l t h y a d u l t male v o l u n t e e r s were r e c r u i t e d f o r a l a b o r a t o r y s t u d y . A f t e r g i v i n g i n f o r m e d c o n s e n t , each s u b j e c t was sampled f o r t h e b a s e l i n e l e v e l o f t o l u e n e i n e x p i r e d b r e a t h . Then a i r a t 50% RH c o n t a i n i n g t o l u e n e a t 150 mg/m (40 ppm) o r 340 mg/m (90 ppm) was p a s s e d t h r o u g h a mask c o v e r i n g t h e s u b j e c t ' s nose and mouth a t 35 L/min, f o r f o u r h o u r s . Between 17 and 20 hours l a t e r , c o r r e s p o n d i n g t o the s t a r t o f t h e next work s h i f t , a b r e a t h sample was t a k e n and a n a l y z e d . The sampling p r o c e d u r e c o n s i s t e d of f i r s t w e a r i n g t h e r e s p i r a t o r w i t h o u t the s p e c i a l s a m p l i n g c a r t r i d g e f o r seven t o t e n minutes t o a l l o w a i r t e m p e r a t u r e and RH i n s i d e t h e mask t o s t a b i l i z e . Then the s a m p l i n g c a r t r i d g e was a t t a c h e d and t h e s u b j e c t c o n t i n u e d t o b r e a t h e a t r e s t f o r 15 t o 20 min. The c a r t r i d g e was t h e n d i s a s s e m b l e d and t h e a d s o r b e n t s were a n a l y z e d w i t h i n one hour. R o u t i n e sampling o f l a b o r a t o r y ambient a i r i n d i c a t e d t h a t t o l u e n e l e v e l s were always l e s s t h a n 10 |ig/m . The o b s e r v e d b r e a t h c o n c e n t r a t i o n s were c o r r e c t e d f o r t h e d i l u t i o n e f f e c t o f t h e r e s p i r a t o r c a v i t y dead space. I n a s e p a r a t e e x p e r i m e n t , the c o r r e c t i o n f a c t o r f o r each s u b j e c t was e s t i m a t e d by measuring the c o n c e n t r a t i o n o f carbon d i o x i d e i n mixed e x p i r e d a i r c o l l e c t e d : a.) a t t h e r e s p i r a t o r o u t l e t p o r t and b.) a t the mouth. I t was assumed t h a t the d i l u t i o n o f e x p i r e d c a r b o n d i o x i d e by the r e s p i r a t o r dead space a p p r o x i m a t e s c l o s e l y the d i l u t i o n o f e x p i r e d t r a c e organics. 3

3

3

Performance i n I n d u s t r i a l S e t t i n g s E l e v e n p a i n t e r s employed i n f i v e a u t o m o b i l e body r e p a i r and r e p a i n t i n g shops were t h e n s t u d i e d , w i t h t h e i n t e n t o f d e t e r m i n i n g whether o r not b r e a t h c o n c e n t r a t i o n s o f t o l u e n e used as a s o l v e n t compared t o t r a d i t i o n a l measurements o f t h e s o l v e n t i n b r e a t h i n g zone ambient a i r . B r e a t h samples were c o l l e c t e d f o r f i f t e e n m i n u t e s b e f o r e the b e g i n n i n g o f each s h i f t f o r f i v e c o n s e c u t i v e days i n each p a i n t e r . D u r i n g t h e s a m p l i n g p e r i o d no p a i n t i n g was done, and s t a n d a r d o r g a n i c vapor c a r t r i d g e s were a t t a c h e d t o t h e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

i n l e t p o r t s o f t h e s a m p l i n g r e s p i r a t o r , t o ensure t h a t no exposure occurred. D u r i n g each s h i f t t o l u e n e c o n c e n t r a t i o n s i n each worker's b r e a t h i n g zone were measured, as t h e 8 hour t i m e - w e i g h t e d average, u s i n g a p a s s i v e d o s i m e t e r i n c o r p o r a t i n g a c t i v a t e d charcoal.(12) Samples were c o l l e c t e d u s i n g t h e Model 530-11 d o s i m e t e r o f SKC I n c . , F u l l e r t o n , CA. The c h a r c o a l elements were d e s o r b e d i n 2.0 m l carbon d i s u l f i d e and t h e e l u e n t a n a l y z e d by t h e procedure c i t e d e a r l i e r . Recovery e f f i c i e n c y f o r d o s i m e t e r s was t a k e n from m a n u f a c t u r e r ' s l i t e r a t u r e . A l l p a i n t e r s were o b s e r v e d d u r i n g a t l e a s t p a r t o f each s h i f t , t o determine t h e use o f g l o v e s and r e s p i r a t o r y p r o t e c t i o n , and t o e s t i m a t e t h e e x t e n t o f s k i n contact with l i q u i d paint solvent. Results O v e r a l l r e c o v e r y o f t o l u e n e from t h e c h a r c o a l c l o t h was c a l c u l a t e d as t h e mass d e t e r m i n e d a f t e r c o l l e c t i o n and d e s o r p t i o n , d i v i d e d by t h e mass p r e s e n t e d t o t h toluene concentration. Result III. The r e c o v e r y dropped a t c o n c e n t r a t i o n s above 60 mg/m (16 ppm), presumably due i n p a r t t o b r e a k t h r o u g h . A t 60 mg/m o r l e s s , t h e r e c o v e r y c o e f f i c i e n t of v a r i a t i o n was l e s s t h a n 15%. 3

Table I I I O v e r a l l Recovery o f Toluene from Toluene C o n c e n t r a t i o n (mg/m ) 3

Temp <°C)

EE (%)

Charcoal Cloth No. o f Layers

%Recovered

69. 1 ± 6.2 (n = 7) 63. 6 ± 7.5 (n = 6) 70. 5 ± 7.5 (n = 6) 68. 8 ± 5.2 (n = 5) 67. 0,70.0 (n = 2) 38. 0,25.0 (n = 2)

0.75

22

80

4

1.30

30

90

4

2.00

30

90

4

7.4

22

80

4

60.0

22

80

6

150.

22

80

6

A

B

A

Mean + s t a n d a r d d e v i a t i o n (no. o f r e p l i c a t e runs) B r e a k t h r o u g h d e t e c t e d on second p a i r o f l a y e r s i n each r e p l i c a t e run

B

The r e s u l t s f o r t h e comparison o f e x h a l e d a i r volume and water vapor c o l l e c t e d a r e g i v e n i n F i g u r e 3. There was a good c o r r e l a t i o n between t h e two, d e s p i t e t h e f a c t t h a t among t h e s u b j e c t s t h e r e was c o n s i d e r a b l e v a r i a t i o n i n t i d a l volume, b r e a t h i n g p a t t e r n and r a t e o f v e n t i l a t i o n . The d a t a suggest t h a t t h e r e i s a s i m p l e , r e l i a b l e r e l a t i o n s h i p between weight g a i n o f t h e d e s i c c a n t and e x h a l e d volume which can be a p p l i e d as t h e c a l i b r a t i o n f o r t h e sampler when used under t h e c o n d i t i o n s d e s c r i b e d . From l i n e a r r e g r e s s i o n , t h i s c a l i b r a t i o n i s : M o i s t u r e g a i n (g) = 0.0268 x E x p i r e d volume (L) + 0.73 g. The c o r r e l a t i o n c o e f f i c i e n t (r) was 0.9789.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5.

MORGAN ETAL.

63

Volatile Compounds in Exhaled Breath 1

When t h e s u b j e c t s b r e a t h was sampled 17 t o 20 hours a f t e r c o n t r o l l e d exposure, t o l u e n e was r e a d i l y q u a n t i t a t e d i n each p e r s o n , w h i l e no t o l u e n e was d e t e c t e d i n background b r e a t h samples and b l a n k s . I n t h e p o s t - e x p o s u r e samples, t h e amount of t o l u e n e found on t h e second p a i r of c h a r c o a l c l o t h l a y e r s was l e s s t h a n 5% of t h e t o t a l r e c o v e r e d . Table IV summarizes t h e r e s u l t s f o r a l l s u b j e c t s g i v e n c o n t r o l l e d exposure, and i n d i c a t e s t h e range of sample volumes and r e s u l t i n g t o l u e n e c o n c e n t r a t i o n s , c o r r e c t e d f o r r e c o v e r y e f f i c i e n c y and f o r r e s p i r a t o r dead space. The t o l u e n e l e v e l s a f t e r exposure t o 150 mg/m , showed an o v e r a l l mean e x h a l e d c o n c e n t r a t i o n t h a t was 0.23% of t h e exposure c o n c e n t r a t i o n i n h a l e d t h e p r e v i o u s day. The r e s u l t s f o r t h e exposures t o 335 mg/m gave a mean e x h a l e d c o n c e n t r a t i o n of 0.18% of t h a t b r e a t h e d on t h e p r e v i o u s day. 3

3

Table IV. R e s u l t s f o r 4 Hour C o n t r o l l e d Exposure of Huma

Subject

Exposure C o n c e n t r a t i o n s (mg/m3) Hours A f t e r

Sample Volume (L)

Breath Toluene (mg/m )

1 2 3 4 5 6 7 8 9

145 152 160 148 147 155 140 147 156

18 20 18 17 18 19 19 19 18

174 311 231 184 214 200 237 137 200 mean std.dev.

0.246 0.356 0.389 0.309 0.453 0.246 0.211 0.606 0.282 0.344 0.125

10 11 12 13 14 15 16 17

338 331 338 342 335 338 338 327

19 20 20 19 19 21 19 20

168 125 118 164 83 155 149 207 mean s t d dev.

0.465 0.438 0.436 0.638 0.434 1.14 0.664 0.600 0.602 0.238

A

A

3

C o r r e c t e d f o r r e c o v e r y e f f i c i e n c y and f o r mask dead

space.

The r e s u l t s of the f i e l d s t u d y i n a u t o m o b i l e p a i n t e r s a r e shown i n f i g u r e 4, where t h e c o n c e n t r a t i o n of t o l u e n e found i n b r e a t h i s p l o t t e d a g a i n s t t h e ambient a i r c o n c e n t r a t i o n measured on t h a t worker's l a p e l d u r i n g t h e p r e v i o u s day's s h i f t . Also shown a r e t h e e s t i m a t e d l e v e l s of t o l u e n e i n b r e a t h t h a t would be o b s e r v e d i f t h e p r e v i o u s s h i f t ambient a i r c o n c e n t r a t i o n s had been e q u a l t o the TLV, and t o o n e - h a l f t h i s l e v e l . These b r e a t h l e v e l s were e s t i m a t e d from t h e r e s u l t s of t h e c o n t r o l l e d l a b o r a t o r y exposures.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

64

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

12 T

10 SUBJ A 8 ••

o

O WEIGHT GAIN, g

6 ••

A

•

• •

*A«

A

SUBJB SUBJC SUBJD SUBJE

A SUBJF

VOLUME, liters

F i g u r e 3 — R e l a t i o n s h i p between m o l e c u l a r s i e v e weight g a i n and e x h a l e d volume measured by an independent method (see t e x t ) i n v o l u n t e e r s u b j e c t s , measured a t 20C and 40% RH ambient conditions.

5 -r

•

A1

•

A2

A

B1

A

C1

X

C2

X

D1

-

D2

4 -•

3 Breath, mg/m3 2 +

•

E1

o

E2

•

E3

•

E4

• 1 x-

0 " A W X * 0

h

_A

20

§_+ 40

—I 60

80

100

120

Previous Day Ambient Air, mg/m3

— TLV —- 0.5 TLV

F i g u r e 4 — B r e a t h c o n c e n t r a t i o n s o f t o l u e n e i n a u t o m o b i l e body p a i n t e r s a t t h e s t a r t o f each s h i f t , p l o t t e d a g a i n s t t h e t i m e w e i g h t e d average a i r c o n c e n t r a t i o n d u r i n g t h e p r e v i o u s s h i f t . Legend i d e n t i f i e s i n d i v i d u a l p a i n t e r s . The h o r i z o n t a l l i n e s i n d i c a t e e s t i m a t e d b r e a t h l e v e l s c o r r e s p o n d i n g t o exposure a t t h e TLV and o n e - h a l f t h e TLV f o r t o l u e n e i n a i r .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5.

MORGAN ETAL.

65

Volatile Compounds in Exhaled Breath

Discussion In d e s i g n i n g t h e b r e a t h s a m p l i n g c a r t r i d g e t h e p r i m a r y g o a l was t o measure t o l u e n e i n b r e a t h a t t h e s t a r t o f t h e next s h i f t . This was b a s e d on t h e p r e d i c t i o n s from t o x i c o k i n e t i c models, and l i m i t e d e x p e r i m e n t a l d a t a , showing t h a t one must w a i t a t l e a s t two t o f o u r hours a f t e r t h e end o f exposure b e f o r e t h e b r e a t h l e v e l o f s o l v e n t r e f l e c t s t h e i n t e g r a t e d dose over t h e p r e v i o u s s h i f t ( o r s h i f t s ) , r a t h e r t h a n t h e most r e c e n t c o n c e n t r a t i o n i n h a l e d . ( 4 - 6 ) S i n c e i t appears i m p r a c t i c a l t o ask workers t o remain a t work u n t i l two t o f o u r hours a f t e r t h e s h i f t ends, t h e f i r s t chance t o o b t a i n t h e d e s i r e d sample i s 16 t o 20 hours a f t e r t h e s h i f t ends. Then t h e b r e a t h l e v e l s o f s o l v e n t a r e e x p e c t e d t o be l e s s t h a n 1% of t h e p r e v i o u s time-weighted-average exposure c o n c e n t r a t i o n . ( A ) The s a m p l i n g and a n a l y t i c a l procedure must t h e r e f o r e have a q u a n t i t a t i o n range t h a t extends much lower t h a n t h a t r e q u i r e d o f methods f o r s a m p l i n g workroom a i r f o r contaminants a t c o n c e n t r a t i o n s near t h Threshold L i m i t Values. The performance o f t h e s a m p l i n g c a r t r i d g e was s a t i s f a c t o r y , c o n s i d e r i n g t h e c o n d i t i o n s employed: v e r y m o i s t e x p i r e d a i r c o n t a i n i n g t o l u e n e a t c o n c e n t r a t i o n s between 0.75 mg/m and 60 mg/m , as compared t o t h e PEL o f 750 mg/m and t h e TLV o f 375 mg/m which a r e u s u a l l y measured a t RH l e v e l s l e s s t h a n 75%. G r a n u l a r forms o f a c t i v a t e d c h a r c o a l have been r e p o r t e d t o show s e r i o u s d e g r a d a t i o n i n c a p a c i t y and b r e a k t h r o u g h t i m e a t RH l e v e l s above 80%. (Ul) I n a d d i t i o n , c o l l e c t i o n e f f i c i e n c y o f s o l v e n t s on g r a n u l a r c h a r c o a l i s known t o be a d v e r s e l y a f f e c t e d by low s a m p l i n g f l o w r a t e s . (X2.) Recovery i s a l s o lower f o r vapor l o a d i n g as compared t o l i q u i d l o a d i n g . (20 21) I t s h o u l d be n o t e d t h a t r e c o v e r y e f f i c i e n c y was determined f o r a range o f a i r c o n c e n t r a t i o n s which was above t h e l e v e l s found i n b r e a t h . The a c c u r a c y o f b r e a t h l e v e l s r e p o r t e d i s thus u n c e r t a i n t o t h e degree that the recovery e f f i c i e n c y i s uncertain. 3

3

3

3

r

The r e l a t i o n s h i p between m o i s t u r e c o l l e c t e d and t h e volume of b r e a t h e x h a l e d was a p p r o x i m a t e l y 27 mg/L. T h i s i s i n good agreement w i t h t h e work o f o t h e r s who have r e p o r t e d v a l u e s between 25 and 31 mg/L u s i n g more s o p h i s t i c a t e d methods.{5U2Z) Those authors a l s o reported that the moisture content of exhaled breath i s m o d e r a t e l y dependent on ambient temperature and RH. V a r i a t i o n i n b r e a t h m o i s t u r e i s v e r y s m a l l f o r temperature o r RH above room c o n d i t i o n s , b u t i t becomes i m p o r t a n t below 10C o r below 20% RH. Under such c o n d i t i o n s t h e volume measuring p o r t i o n o f t h e b r e a t h sampler d e s c r i b e d here would have t o be r e c a l i b r a t e d . When t h e sampler i s used as r e p o r t e d i n t h i s work, t h e lower l i m i t o f q u a n t i t a t i o n f o r t o l u e n e i n b r e a t h i s 65 |lg/m (0.018 ppm). T h i s i s based on a sample volume o f 240 l i t e r s , c o r r e c t i o n f o r dead space, d e s o r p t i o n o f t h e c l o t h i n 10 ml o f carbon d i s u l f i d e w i t h a r e c o v e r y e f f i c i e n c y o f 70%, i n j e c t i o n o f 1.0 JIl e l u a t e and a r a t h e r modest gas chromatograph q u a n t i t a t i o n l i m i t o f 0.87 ng. With t h e e x c e p t i o n o f dead space, each o f t h e s e f a c t o r s i s s u b j e c t t o improvement so t h a t a much b e t t e r d e t e c t i o n l i m i t c o u l d be a c h i e v e d r e a d i l y . However, a b r e a t h l e v e l o f 65 |lg/m would c o r r e s p o n d t o a p r e v i o u s day's exposure t o t o l u e n e a t an e i g h t hour t i m e - w e i g h t e d average o f about 15 mg/m , o r 4% o f t h e p r e s e n t TLV. Thus even w i t h moderate a n a l y t i c a l s e n s i t i v i t y , r e l a t i v e l y low exposures may be m o n i t o r e d w i t h t h i s t e c h n i q u e . 3

3

3

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

The f i n d i n g s from t h e c o n t r o l l e d human exposure may be compared t o o t h e r p u b l i s h e d r e p o r t s o f b r e a t h s a m p l i n g f o r t o l u e n e i n which p l a s t i c bags, g l a s s t u b e s , o r more e l a b o r a t e methods (not s u i t a b l e f o r w o r k p l a c e s c r e e n i n g ) were used. F i g u r e 5 shows t h e d a t a from t h i s s t u d y , c o r r e c t e d f o r d i l u t i o n by t h e r e s p i r a t o r y dead space (assuming a p h y s i o l o g i c a l dead space o f 30% o f t i d a l volume) and mask dead space. The f i g u r e a l s o shows r e s u l t s from o t h e r s t u d i e s . ( 2 , 1 2 - 1 5 ) A l l c o n c e n t r a t i o n s have been n o r m a l i z e d t o t h e i n h a l e d dose c a l c u l a t e d as t h e p r o d u c t o f exposure concent r a t i o n and d u r a t i o n . Note t h a t o n l y one p r e v i o u s s t u d y i n c l u d e d d a t a a t 18 hours p o s t - e x p o s u r e , and t h a t v a l u e i s c l o s e t o t h e r e s u l t from t h i s work. A t e a r l i e r s a m p l i n g t i m e s , t h e d a t a from o t h e r s t u d i e s show c o n s i d e r a b l e v a r i a t i o n . That v a r i a t i o n might be a t t r i b u t e d t o d i f f e r e n c e s i n sampling technique o r t o v a r i a b l e e x c r e t i o n k i n e t i c s at short post-exposure times. F i e l d measurements o f t o l u e n e i n t h e b r e a t h o f p a i n t e r s indicated that the breath l e v e l correlated with inhalation exposure o n l y when t h e mg/m , and t h e n t h e c o r r e l a t i o c o n c e n t r a t i o n s were d e r i v e d from p a s s i v e d o s i m e t e r s , whose performance under f i e l d c o n d i t i o n s has n o t been f u l l y v a l i d a t e d . (12.) The e x t e n t t o which u n c e r t a i n t y i n ambient c o n c e n t r a t i o n c o n t r i b u t e d t o t h e lack of c o r r e l a t i o n w i t h b r e a t h data i s not known. O b s e r v a t i o n o f t h e work p r a c t i c e s employed i n t h e w o r k p l a c e s r e v e a l e d t h a t t h e r e was s i g n i f i c a n t s k i n exposure i n many employees, and t h a t r e s p i r a t o r usage was h i g h l y v a r i a b l e among them. These f a c t o r s would confound any r e l a t i o n s h i p e x p e c t e d between i n h a l a t i o n exposure,as e s t i m a t e d by a i r c o n c e n t r a t i o n , and b r e a t h l e v e l s . On t h e o t h e r hand, t h e l e v e l s of t o l u e n e f o u n d i n t h e b r e a t h o f a l l workers c o r r e s p o n d e d t o e q u i v a l e n t a i r b o r n e exposures a t a s i g n i f i c a n t p r o p o r t i o n o f t h e h e a l t h g u i d e l i n e , and suggest t h a t t h e workers were r e c e i v i n g i m p o r t a n t exposure by a r o u t e o t h e r t h a n i n h a l a t i o n . The most l i k e l y e x p l a n a t i o n appears t o be s k i n c o n t a c t , which was f r e q u e n t l y observed. I f t h i s f i n d i n g i s confirmed i n wider s t u d i e s o f workers who a r e exposed by b o t h r o u t e s , t h e v a l u e o f b i o l o g i c a l m o n i t o r i n g w i l l be r e i n f o r c e d . The s a m p l i n g method used i n t h i s work o f f e r s s e v e r a l advantages o v e r o t h e r t e c h n i q u e s . The s a m p l i n g r e s p i r a t o r would not be i n t i m i d a t i n g t o t h e m a j o r i t y o f workers exposed t o s o l v e n t s , as many a r e u n d o u b t e d l y f a m i l i a r w i t h a i r p u r i f y i n g r e s p i r a t o r s a l r e a d y . The s a m p l i n g and a n a l y t i c a l t e c h n i q u e s a r e r e l a t i v e l y s i m p l e and i n e x p e n s i v e , and t h u s many workers can be m o n i t o r e d s i m u l t a n e o u s l y . The a b i l i t y t o sample a l a r g e volume o f e x p i r e d a i r g i v e s t h e method v e r y good s e n s i t i v i t y . Finally, l e a k a g e o f e x p i r e d a i r around t h e f a c e s e a l produces no e r r o r as t h i s l o s t a i r does n o t r e a c h t h e a d s o r b e n t o r d e s i c c a n t i n t h e c a r t r i d g e . T h e r e f o r e t h e d e v i c e measures o n l y t h e volume o f a i r from which o r g a n i c vapor i s a l s o c o l l e c t e d . There a r e some f e a t u r e s which c o u l d l i m i t t h e v a l u e o f t h i s approach under c e r t a i n c i r c u m s t a n c e s . The sampler as d e s c r i b e d i s r a t h e r heavy, and s a m p l i n g f o r l o n g e r t h a n f i f t e e n t o twenty minutes may prove u n c o m f o r t a b l e . Use o f l i g h t e r weight m a t e r i a l s i n c o n s t r u c t i n g t h e c a r t r i d g e s h o u l d reduce t h i s problem. The need t o c o r r e c t f o r t h e e f f e c t o f mask dead space i s a d i s a d v a n t a g e r e l a t i v e t o use o f bags o r o t h e r methods p e r m i t t i n g s a m p l i n g d i r e c t l y a t t h e mouth. The range o f c o r r e c t i o n f a c t o r s i n a d u l t males a t r e s t was r e l a t i v e l y narrow, however, so t h a t u s i n g a group mean c o r r e c t i o n f o r dead space would have produced a 3

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Volatile Compounds in Exhaled Breath

MORGAN ET AL.

•

0.1000

67

Carlsso

T

•

• Nomiyama 0.0100 Cb/C

A • > A A

• Brugnone A

Sato

• This Study

• t 0.0010

0.0001 -I 0

1 2

1 4

1 6

1 8

1 10

1 12

1 14

1 16

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HOURS AFTER EXPOSURE

F i g u r e 5 — B r e a t h t o l u e n e a f t e r c o n t r o l l e d l a b o r a t o r y exposure (Cb), n o r m a l i z e d t o dose e x p r e s s e d as exposure c o n c e n t r a t i o n (C) m u l t i p l i e d by exposure d u r a t i o n ( t ) i n h o u r s . Note t h a t t h e o r d i n a t e i s a l o g a r i t h m i c s c a l e . R e s u l t s a r e shown f o r t h i s s t u d y and f o r f i v e o t h e r s , i d e n t i f i e d by f i r s t a u t h o r o f report.

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deadspace error of less than 15% for any individual in our group of subjects. The technique also does not permit the estimation of true alveolar concentration, but there i s some doubt that any of the published methods could accomplish this in the f i e l d . When the features of this device are weighed, i t may be an attractive means of monitoring the body burdens of v o l a t i l e organic compounds in a wide variety of industries and processes. This preliminary study has shown that the respirator-based technique for sampling mixed expired breath appears to be a potentially useful method for pre-shift monitoring. Its use in practice w i l l require measuring the efficiency of recovery of compounds of interest from the sampling cartridge. Some data are available now for toluene and additional data are being developed for the xylene isomers, methyl ethyl ketone and methyl isobutyl ketone. Other sampling conditions, such as temperature, relative humidity and presence of possible interfering compounds, may affect sampler performance in applications such as pesticide biomonitoring. More extensiv which present biologica for parent compounds or , compare analysis in the same exposed workers. Acknowledgment s Supported by Grant No. 5 R01 OH01490-02 from the National Institute for Occupational Safety and Health Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

12. 13.

Wilson. H.K. Scand. J. Work Environ. Health. 1986, 12:174192. Veulemans, H.; Masschelein, R. Int. Arch. Occup. Environ. Health1978, 42:91-103. Berlin, M.; Gage, J.C.; Gullberg, B.; Holm, S.; Knutsson, p.; Tunek, A. Scand. J. Work Environ. Health. 1980, 6:104-111. Fiserova-Bergerova, V.; Vlach, J.; Singhal, K. Br. J. Ind. Med. 1974, 31:45-52. Fernandez, J.G.; Droz, P.O.; Humbert, B.E.; Caperos, J.R.; Br. J. Ind. Med. 1977, 34:43-55. Stewart, R.D. In Essays in Toxicology. W.L. Hayes, Ed. Academic Press, New York 1974, pp 121-148. Sherwood, R.J.; Carter, F.W.G. Ann. Occup. Hyg. 1970, 13:125-146. Mitchell, J.W.; Nadel E.R.; Stolwijk J.A.J. J. Appl. Physiol. 1972, 32:474-476. Caldwell, P.B.; Gomez, D.M.; F r i t t s , H.W. J. Appl. Physiol. 1969, 26:82-88. Maggs, F.A.P.; Smith, M.E. Anaesthesia. 1976, 31: 30-40. E l l e r , P.M. Ed. NIOSH Manual of Analytical Methods; 3rd Ed. U.S. Dept. of Health and Human Services Publication 1984, No. 84-100. Cincinnati, pp 1500.1-1500.4. Carlsson, A. Scand. J. Work Environ. Health. 1982, 8:4359. Brugnone, F.; DeRosa, E.; Perbellini, L.; Bartolucci, G.B.; Pasini, F.; Faccini, G. Appl. Ind. Hyg. 1986, 1:21-24.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5. MORGAN ETAL. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Volatile Compounds in Exhaled Breath

Sato. A.; Nakajima, T.; Fujiwara, Y.; Hirosawa, K. Int. Arch. Arbeitsmed. 1974,33:169-182. Nomiyama, K.; Nomiyama, H. Int. Arch. Arbeitsmed. 1974, 32: 85-91. Altshuller, A.P; Cohen, I.R. Anal. Chem. 1960, 32, 802-810. Rose, V.E.; Perkins, J.L. Am. Ind. Hyg. Assoc. J. 1982, 43, 605-621. Nelson, G.O.; Correia A.N.; Harder, C.A. Am. Ind. Hyg. Assoc. J. 1976, 37:280. Levine, M.S.; Schneider, M. Am. Ind. Hyg. Assoc. J. 43:423. Krajewski, J.; Gromiec, J.; Dobecki, M. Am. Ind. Hyg. Assoc. J. 1980, 41:531. Dommer, R.A.; Melcher, R.G. Am. Ind. Hyg. Assoc. J. 1978, 39:240. Burch, G.E. Arch Int Med 1945 76:315 Sato, A.; Nakajima 1987, 13, 81-93. Sato, A.; Nakajima, T. Br. J. Ind. Med. 1979,36,231-234. Riihimaki, V . Scand. J. Work Environ. Health. 1979, 5, 143-150. Perbellini, L.; Brugnone, F.; Mozzo, P.; Cocheo, V.; Caretta, D. Int. Arch. Occup. Environ. Health. 1984, 54, 73-81. Brugnone, F.; Perbellini, L,; Faccini, G.; Pasini, F. Am J. Ind. Med. 1985, 8, 67-72. Morgan, A.; Black, A.; Belcher, D.R. Ann. Occup. Hyg. 1970, 13. 219-233. Monster, A.C. Int. Arch. Occup. Environ. Health. 1979, 42, 311-317. Stewart, R.D.; Gay, H.H.; Erley, D.S.; Hake, C.L.; Peterson, J.E. J. Occup. Med. 1961, 43, 586-590. Dutkewicz, B. In Industrial and Environmental Xenobiotics; Fouts, J.R.; Gut, J.; Eds.; Excerpta Medica: Amsterdam, 1978, pp 106-109.

RECEIVED May 4, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 6

Validation of Environmental Monitoring by Biological Monitoring Fluorescent Tracer Technique and Patch Technique Richard A. Fenske New Jersey Agricultural Experiment Station, Department of Environmental Science, Rutgers University, New Brunswick, NJ 08903

Environmenta sure allows pathways and appropriate exposure reduction strategies. Biological monitoring studies which c o l l e c t t o t a l urinary metabolites from a s i n g l e exposure episode provide a r e l a t i v e index of exposure for evaluating the v a l i d i t y of environmental monitoring methods. Essential validation study design factors include selection of an appropriate p e s t i c i d e for b i o l o g i c a l monitoring, and elimination of extraneous and i n t e r f e r i n g exposures. Most studies which have r e ­ ported poor c o r r e l a t i o n s between patch estimates of dermal exposure and metabolite excretion e x h i b i t d e f i c i e n c i e s in one or more of these design factors. A recent study of malathion exposure during a i r b l a s t applications demonstrated a high correla­ tion between fluorescent tracer measure­ ments of dermal deposition and malathion metabolite excretion (r=.84). Patch depo­ s i t i o n values were a l s o c o r r e l a t e d with excretion (r=.71). Environmental and biological monitoring are complementary approaches to characterizing human exposure to chemicals in occupational and general populations. The combination of these two techniques provides a comprehensive evalua­ tion of exposure magnitude and the pathways by which exposure occurs. Biological monitoring has been an im­ portant part of pesticide exposure assessment for nearly forty years due to the importance of the dermal route of exposure (JL). Cholinesterase and urinary metabolite monitoring have often been conducted in conjunction with estimates of dermal deposition to determine the r e l a t i v e 0097-6156/89/0382-0070$06.00/0 ° 1989 American Chemical Society

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c o n t r i b u t i o n of dermal exposure to t o t a l exposure, and t o determine the accuracy o f dermal exposure measurements. This paper examines the g e n e r a l p r i n c i p l e s i n v o l v e d i n f i e l d v a l i d a t i o n o f e n v i r o n m e n t a l m o n i t o r i n g methods/ and r e v i e w s s t u d i e s w h i c h have e m p l o y e d b i o l o g i c a l monitoring as a r e l a t i v e index of p e s t i c i d e exposure to evaluate dermal exposure estimates. Studies employing the p a t c h t e c h n i q u e a r e compared t o a r e c e n t study w h i c h m e a s u r e d p e s t i c i d e d e p o s i t i o n on t h e s k i n w i t h fluorescent t r a c e r s and video imaging. The s t r e n g t h s a n d l i m i t a t i o n s of these techniques a r e d i s c u s s e d together w i t h suggestions regarding future areas f o r research i n p e s t i cide exposure assessment. Human E x p o s u r e A s s e s s m e n t

Methods

Environmental monitorin i.e./ amount o f m a t e r i a l r e a c h i n g a body b a r r i e r and a v a i l a b l e f o rabsorption. A i rsampling/ patch monitoring and handwashing a r e e x a m p l e s o f e n v i r o n m e n t a l monitoring methods employed r o u t i n e l y i n p e s t i c i d e exposure a s s e s s ment. Such t e c h n i q u e s a l l o w study o f the r e l a t i v e importance of routes of exposure and p r o v i d e the f o u n d a t i o n for the development of appropriate c o n t r o l strategies. Since environmental m o n i t o r i n g t e c h n i q u e s n e e d n o t be compound-specific/ t h e y h a v e o f t e n been e m p l o y e d t o meas u r e e n t i r e c l a s s e s o f c o m p o u n d s (e.g./ a i r s a m p l i n g o f v o l a t i l e organics)/ or to provide a generic examination of p r o d u c t i o n processes (e.g./ a u t o b o d y p a i n t i n g o r pesticide applications). F i n a l l y / environmental monitoring i s t h e o n l y m e a n s o f a s s e s s i n g e x p o s u r e f o r t h e many c h e m i c a l s w h i c h do n o t h a v e a r e l i a b l e b i o l o g i c a l e n d p o i n t t o measure. B i o l o g i c a l m o n i t o r i n g p r o v i d e s a measure o f a b s o r p t i o n i n body t i s s u e o r f l u i d s / or i n v o l v e s measuring change i n a p h y s i o l o g i c a l parameter. Such m o n i t o r i n g i n t e g r a t e s exposure from a l l r o u t e s and a l l o w s e s t i m a t i o n of an absorbed dose. S i n c e a dose e s t i m a t e i s the u l t i mate g o a l o f any e x p o s u r e a s s e s s m e n t / a w e l l - c o n d u c t e d b i o l o g i c a l monitoring procedure can provide important information unattainable with environmental monitoring. C l e a r l y both of these approaches a r e e s s e n t i a l t o reducing exposures and s e t t i n g a p p r o p r i a t e o c c u p a t i o n a l and environmental h e a l t h standards. U n d e r many c i r c u m s t a n c e s t h e y c a n be c o n d u c t e d s i m u l t a n e o u s l y t o p r o v i d e a c o m p r e hensive exposure assessment. The A m e r i c a n C o n f e r e n c e o f Governmental I n d u s t r i a l Hygienists considers biological m o n i t o r i n g t o be a n i m p o r t a n t s u p p l e m e n t t o e n v i r o n m e n t a l m o n i t o r i n g / and has r e c e n t l y e s t a b l i s h e d B i o l o g i c a l Expos u r e I n d i c e s t o p r o v i d e g u i d a n c e i n t h i s a r e a (2^). Simil a r b i o l o g i c a l e x p o s u r e g u i d e l i n e s have been d e v e l o p e d i n Europe/ where b i o l o g i c a l m o n i t o r i n g i s an e s t a b l i s h e d e x p o s u r e a s s e s s m e n t p r o c e d u r e (3^).

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Exposure

Assessment

Method

Validation

The r e l a t i v e a c c u r a c y o f a n e x p o s u r e a s s e s s m e n t t e c h n i q u e c a n be e s t a b l i s h e d b y d e m o n s t r a t i n g i t s a b i l i t y t o m o n i t o r changes i n exposure c o n s i s t e n t l y . Measurements p r o d u c e d by t h e t e c h n i q u e a r e c o m p a r e d t o a r e l i a b l e m e a s u r e of exposure or exposure p o t e n t i a l . The a b s o l u t e a c c u r a c y o f a m e t h o d i s n o r m a l l y d e m o n s t r a t e d by c a l i b r a t i n g t h e measurement s y s t e m a g a i n s t a known s t a n d a r d . I f s u c h a standard i s not a v a i l a b l e / a c c u r a c y i s d e t e r m i n e d by c a l c u l a t i o n of e r r o r or uncertainty. V a l i d a t i o n of dermal e x p o s u r e a s s e s s m e n t methods has p r o v e n p r o b l e m a t i c . Laboratory f a c i l i t i e s which create w e l l - d e f i n e d exposures a r e commonly employed t o e v a l u a t e r e s p i r a t o r y measurement t e c h n i q u e s (e.g./ c o n t r o l l e d t e s t a t m o s p h e r e s a n d inhalat i o n chambers). No s u c h p r o c e d u r e s a r e a v a i l a b l e f o r t h e study of dermal exposure be c o n d u c t e d i n t h e be c o n t r o l l e d . As a r e s u l t / t h e d e r m a l e x p o s u r e a s s e s s ment methods i n c u r r e n t use h a v e n e v e r been v a l i d a t e d i n terms of a b s o l u t e a c c u r a c y . Dermal exposure validation s t u d i e s h a v e i n s t e a d f o c u s e d on r e l a t i v e a c c u r a c y by comparing environmental monitoring data to a r e l a t i v e index of exposure. Two f i e l d study parameters are often employed as r e l a t i v e i n d i c a t o r s of e x p o s u r e : work r a t e and active ingredient applied. I m p l i c i t i n the use of such v a r i a b l e s i s an a s s u m p t i o n t h a t a s t r o n g a s s o c i a t i o n e x i s t s between the v a r i a b l e and e x p o s u r e ; i . e . / e x p o s u r e i s proportional to time worked or amount of material handled. Work r a t e i s commonly e m p l o y e d i n i n d u s t r i a l s e t t i n g s w h e r e e x p o s u r e p o t e n t i a l i s p r e d i c t a b l e (e.g./ a m b i e n t a i r l e v e l s i n a f a c t o r y ) / and where work a c t i v i t y is routine. In a recent study of timber m i l l workers exposed to t e t r a c h l o r o p h e n o l / f o r example/ i t was d e m o n s t r a t e d t h a t s k i n d e p o s i t i o n o f t e t r a c h 1 o r o p h e n o l was d i r e c t l y r e l a t e d t o the amount o f time worked (4). This a s s o c i a t i o n was e x p e c t e d s i n c e w o r k e r s p u l l e d a n d sorted t i m b e r a t a r a t e c o n t r o l l e d by t h e s p e e d o f a c o n v e y o r belt. Such work c o n d i t i o n s c o n t r a s t s h a r p l y w i t h p e s t i c i d e m i x i n g and a p p l i c a t i o n i n a g r i c u l t u r e / where b o t h e x p o s u r e p o t e n t i a l and work r a t e a r e h i g h l y v a r i a b l e . A 1979 s t u d y o f s i x t e e n m i x e r / a p p l i c a t o r s demons t r a t e d t h a t w h e n w o r k e r s e m p l o y t h e i r own e q u i p m e n t and w o r k a t t h e i r own s p e e d / w o r k r a t e i s h i g h l y variable (J5). R e g r e s s i o n a n a l y s i s r e s u l t e d i n no significant c o r r e l a t i o n b e t w e e n t i m e w o r k e d a n d 48 h r e x c r e t i o n o f a z i n p h o s m e t h y l m e t a b o l i t e s (r=.43). However/ a s t r o n g c o r r e l a t i o n (r=.77) was f o u n d when t h e a m o u n t o f a c t i v e i n g r e d i e n t h a n d l e d by e a c h w o r k e r a n d 48 h r e x c r e t i o n were compared. S e v e r a l other i n v e s t i g a t o r s have conf i r m e d t h e s e r e s u l t s (6). Most r e c e n t l y a study of 2/4-D a p p l i c a t o r s i n Canada found s t r o n g c o r r e l a t i o n s between

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the u r i n a r y e x c r e t i o n o f e i g h t w o r k e r s a n d amount s p r a y e d ( r = . 9 0 ) / a s w e l l a s w i t h s p r a y i n g t i m e ( r = . 9 3 ) (8). T h e a s s o c i a t i o n o f amount o f a c t i v e i n g r e d i e n t a p p l i e d a n d exposure suggests that this parameter i suseful a s a r e l a t i v e i n d i c a t o r o f exposure p o t e n t i a l . W o r k r a t e may be a n a p p r o p r i a t e i n d i c a t o r i n some c a s e s / b u t c a n b e highly variable for pesticide applications. Study Design f o r B i o l o g i c a l

Monitoring

Validation

Most p e s t i c i d e s t u d i e s aimed a t v a l i d a t i n g dermal exposure measurements have employed b i o l o g i c a l m o n i t o r i n g a s a r e l a t i v e index o f exposure. An i d e a l v a l i d a t i o n s t u d y design would attempt t o demonstrate a strong correspondence between dermal exposure a n d i n t e r n a l dose. In lieu o f a m e a s u r e o f i n t e r n a l dose f o r humans/ t h e u s e o f urinary metabolite excretio dose h a s g a i n e d w i d e s p r e a a p p l i e d t o x i c o l o g y (£). T h i s approach assumes a l i n e a r r e l a t i o n s h i p b e t w e e n e x p o s u r e a n d a b s o r b e d d o s e / a n d may n o t be a p p r o p r i a t e f o r a l l compounds. Most s u c c e s s f u l v a l i d a t i o n s t u d i e s a r e based on a "single exposure" design. S u b j e c t s work f o r a d e f i n e d exposure peciod/ and t o t a l urine samples are then c o l lected until metabolite excretion i s negligible. The duration of urine c o l l e c t i o n i s a function of the k i n e t i c s o f the a b s o r p t i o n ande x c r e t i o n o f the p e s t i c i d e applied. I fv i r t u a l l y a l l m e t a b o l i t e s are c o l l e c t e d / t h e amount o f p e s t i c i d e m e t a b o l i t e s e x c r e t e d i sa d i r e c t r e f l e c t i o n o f thes i n g l e period exposure/ though n o t n e c e s s a r i l y a measure o f a b s o r b e d dose. Three c r i t e r i a can be e m p l o y e d t o e v a l u a t e the soundness o f a v a l i d a t i o n s t u d y d e s i g n : (1) c o n t r o l o f e x t r a n e o u s e x p o s u r e b e f o r e a n d a f t e r t h e s t u d y p e r i o d / (2) c o n t r o l o f i n t e r f e r i n g e x p o s u r e d u r i n g t h e s t u d y p e r i o d / a n d (3) s e l e c t i o n o f a n appropriate pesticide for biological monitoring. Extraneous Exposure. S u b j e c t s a r e asked t oa v o i d expos u r e p r i o r t o t h es t u d y f o r a time s u f f i c i e n t t o a l l o w u r i n a r y m e t a b o l i t e l e v e l s o f the s t u d y compound o r i n t e r f e r i n g compounds t o r e a c h b a c k g r o u n d l e v e l s . Following the a p p l i c a t i o n p e r i o d no a d d i t i o n a l e x p o s u r e i s a l l o w e d u n t i l urine c o l l e c t i o n i s completed. This protocol i s d i f f i c u l t t o implement i f w o r k e r s must m a i n t a i n a r e g u l a r work s c h e d u l e . F u r t h e r m o r e / e v e n i f w o r k e r s do n o t m i x o r a p p l y p e s t i c i d e s b e f o r e a n d a f t e r t h e s t u d y / t h e y may come i n t o c o n t a c t w i t h c o n t a m i n a t e d e q u i p m e n t o r r e u s e exposed clothing. I n t e r f e r i n g Exposure. Biological monitoring integrates exposure from a l l sources. I f the e n v i r o n m e n t a l m o n i t o r ing technique under study measures o n l y a p o r t i o n o f t h e p o t e n t i a l e x p o s u r e / t h e n o t h e r e x p o s u r e s s h o u l d be m i n i mized. When d e r m a l e x p o s u r e i s o f i n t e r e s t / f o r e x a m p l e /

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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o r a l a n d r e s p i r a t o r y e x p o s u r e s h o u l d be c o n t r o l l e d f o r the study p e r i o d . S i m i l a r l y / a s t u d y of hand e x p o s u r e would r e q u i r e p r o t e c t i o n of a l l other dermal s u r f a c e s . Pesticide Selection. The c e n t r a l d e c i s i o n i n d e s i g n i n g a v a l i d a t i o n study i s the c h o i c e of the p e s t i c i d e to monitor. Compound s e l e c t i o n r e q u i r e s k n o w l e d g e o f the e x c r e t i o n k i n e t i c s f o l l o w i n g d o s i n g / i d e a l l y by t h e d e r m a l r o u t e i n humans. The c o m p o u n d s h o u l d be r a p i d l y e x c r e t e d i n the u r i n e e i t h e r unchanged or as a major m e t a b o l i t e . T a b l e I p r e s e n t s a b s o r p t i o n and e x c r e t i o n d a t a i n humans r e p o r t e d by F e l d m a n n a n d M a i b a c h f o r f o u r o f t h e compounds which have been employed i n v a l i d a t i o n s t u d i e s to d a t e (1_0). T h e s t u d y i n v o l v e d a n i n t r a v e n o u s d o s e f o l l o w e d by a s i n g l e t o p i c a l a p p l i c a t i o n l e f t on t h e s k i n f o r 24 h o u r s . T o t a l u r i n e was c o l l e c t e d f o r 5 d a y s i n each case. The intravenou pounds e x h i b i t e d n e a r l f o r m a l a t h i o n t o 14 h o u r s f o r e t h i o n . The p e r c e n t a g e of t h e d e r m a l d o s e e x c r e t e d r a n g e d f r o m 3.3% f o r e t h i o n t o 9.7% f o r parathion/ or n e a r l y t h r e e - f o l d . Column 3 of Table I was c a l c u l a t e d f r o m the 5 day e x c r e t i o n d a t a p r e s e n t e d i n the study. The v a l u e s a r e t h e percentages o f t h e t o t a l m e t a b o l i t e s w h i c h w e r e e x c r e t e d on t h e f i n a l day o f c o l l e c t i o n . L e s s t h a n 2% o f t h e m a l a t h i o n was e x c r e t e d o n d a y 5, w h e r e a s m o r e t h a n 1 1 % o f 2/4-D was e x c r e t e d on t h e f i n a l d a y o f s a m p l i n g . Data of t h i s k i n d a l l o w b a s i c c o m p a r i s o n s among p e s t i c i d e s i n t h e s e l e c t i o n of s u i t a b l e c a n d i d a t e s f o r b i o l o g i c a l m o n i t o r i n g . Table

I.

Compound

Dermal of

A b s o r p t i o n and E x c r e t i o n C h a r a c t e r i s t i c s F o u r P e s t i c i d e s i n Humans*

Half-life** (hours)

Ethion 2/4-D Para t h i o n Malathion

14 13 8 3

Data adapted from Half-life following

Patch Technique

Percent Dermal Dose Excreted 3.3 5.8 9.7 8.2

Day as

5 Excretion Percent of Total 8.0 11.1 7.2 1.5

; mean o f 6 s u b j e c t s intravenous administration

Va1idation

The p a t c h t e c h n i q u e has b e e n t h e m o s t w i d e l y e m p l o y e d method f o r e s t i m a t i n g dermal exposure s i n c e i t s f o r m a l p u b l i c a t i o n by i n v e s t i g a t o r s f r o m t h e U.S. P u b l i c H e a l t h S e r v i c e (1^1). I t i s now a n a p p r o v e d p r o t o c o l f o r b o t h t h e W o r l d H e a l t h O r g a n i z a t i o n a n d t h e U.S. Environmental P r o t e c t i o n Agency (12-13). However/ the method has n e v e r been v a l i d a t e d / a n d t h e r e a r e many i n v e s t i g a t o r s who

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

FENSKE

Validation of Environmental Monitoring

75

c o n s i d e r p a t c h t e c h n i q u e data i n a d e q u a t e (14-16). This j u d g m e n t i s b a s e d on t h e q u e s t i o n a b l e a c c u r a c y o f t h e method/ b u t a l s o stems from the r e p e a t e d o b s e r v a t i o n t h a t p a t c h d a t a do n o t c o r r e l a t e w i t h u r i n a r y m e t a b o l i t e e x ­ c r e t i o n data. A r e v i e w of p u b l i s h e d s t u d i e s which have attempted t o d e m o n s t r a t e a c o r r e l a t i o n between d e r m a l e x p o s u r e and m e t a b o l i t e e x c r e t i o n i n d i c a t e s shortcomings i n study design or e x e c u t i o n . The s t u d y d e s i g n c r i t e r i a d i s c u s s e d a b o v e a r e e m p l o y e d here t o examine f i v e s t u d i e s w h i c h have c o l l e c t e d p a t c h and m e t a b o l i t e d a t a s i m u l t a ­ neously. T h e s e s t u d i e s a r e summarized i n T a b l e I I . Table

II.

Patch Technique

Study P e s t i c i d e

18 19 20 21 22

parathion 2/4/5-T ethion phenoxy 2/4-D

N

r

11 21 17 17 8

.45 .36 .67

—

Validation

Studies

Extraneous Exposur

Interfering Exposure

Urine Sample

yes possible possible no

hand/resp hand hand/resp resp

5 days spot spot 7 days

Durham a n d W o l f e f i r s t a t t e m p t e d to v a l i d a t e the p a t c h t e c h n i q u e by m o n i t o r i n g a s i n g l e e x p o s u r e a n d d e ­ t e r m i n i n g t o t a l p a r a t h i o n m e t a b o l i t e e x c r e t i o n (17). E x t r a n e o u s e x p o s u r e a p p a r e n t l y was n o t c o n t r o l l e d : no pre-exposure u r i n e s a m p l e s were r e p o r t e d and t h e l e n g t h o f u r i n e s a m p l i n g was e x p r e s s e d s i m p l y a s u n t i l "levels were no l o n g e r o f s i g n i f i c a n c e " . Interfering exposures were n o t c o n t r o l l e d : r e s p i r a t o r y e x p o s u r e a n d hand e x p o ­ sure both c o n t r i b u t e d to m e t a b o l i t e l e v e l s . Parathion a p p e a r s t o h a v e b e e n a g o o d c h o i c e a s a s t u d y compound due t o i t s r e l a t i v e l y r a p i d a b s o r p t i o n and e x c r e t i o n . The s t u d y r e p o r t e d a "good c o r r e l a t i o n " b e t w e e n p a t c h e x p o s u r e and e x c r e t i o n / b u t no s t a t i s t i c a l a n a l y s i s was c o n d u c t e d . A c o r r e l a t i o n c o e f f i c i e n t o f r=.69 f o r t h e 11 s u b j e c t s c a n be c a l c u l a t e d / b u t t h i s v a l u e i s h e a v i l y i n f l u e n c e d by v e r y h i g h r e s u l t s f o r one s u b j e c t . When t h i s s u b j e c t i s removed from t h e d a t a s e t / t h e r e i s no s i g n i f i c a n t c o r r e l a t i o n ( r = . 1 9 ) . The i n f l u e n c e o f a s i n g l e h i g h v a l u e i s an i n h e r e n t weakness o f t h e P e a r s o n c o r r e l a t i o n c o e f f i c i e n t / a n d c o r r e l a t i o n s b a s e d on one extreme v a l u e a r e g e n e r a l l y n o t c o n s i d e r e d adequate e v i ­ dence o f a p o s i t i v e a s s o c i a t i o n . thus/ t h e s e d a t a do n o t d e m o n s t r a t e a c o r r e l a t i o n between t h e two measures. A s t u d y by L a v y a n d c o - w o r k e r s o f 2/4/5-T e x p o s u r e was b a s e d on a s i n g l e e x p o s u r e d e s i g n a n d was c o n d u c t e d t w i c e (JJB). The i m p o r t a n c e o f r e d u c i n g e x t r a n e o u s e x p o ­ s u r e was c l e a r l y r e c o g n i z e d / a s i n d i c a t e d by t h e r e c r u i t ­ ment o f p a r t i c i p a n t s who h a d n o t u s e d 2/4/5-T f o r two weeks/ a n d t h e c o l l e c t i o n o f 24 h o u r p r e - e x p o s u r e u r i n e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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samples. U n f o r t u n a t e l y , b a c k g r o u n d l e v e l s o f 2,4,5-T were v e r y h i g h f o r a number o f t h e w o r k e r s , p a r t i c u l a r i n the second study, c a l l i n g i n t o q u e s t i o n the u t i l i t y of the e x c r e t i o n data f o rc o m p a r a t i v e purposes. In regard to i n t e r f e r i n g exposures, r e s p i r a t o r y e x p o s u r e w a s meas u r e d a n d was d e t e r m i n e d t o be o n l y a s m a l l f r a c t i o n o f the dermal exposure. H o w e v e r , hand e x p o s u r e was n o t measured o r c o n t r o l l e d . Thus, hand e x p o s u r e r e p r e s e n t s an i m p o r t a n t c o n f o u n d i n g f a c t o r , p a r t i c u l a r l y s i n c e a p p l i c a t i o n s w i t h b a c k p a c k s y s t e m s a n d m i s t b l o w e r s comm o n l y r e s u l t i n s i g n i f i c a n t hand e x p o s u r e . T h i s s t u d y was a l s o hampered by t h e c h o i c e o f compounds. T h e h e r b i c i d e 2,4,5-T i s e x c r e t e d a l m o s t t o t a l l y i n urine, but e x c r e t i o n f o l l o w i n g dermal exposure extends beyond 5 days. S e v e r a l workers e x h i b i t e d t h e i highest e x c r e t i o n l e v e l s on On a v e r a g e t h e e x c r e t i o of t h e t o t a l 5 day e x c r e t i o n , s u g g e s t i n g t h a t a s i g n i f i c a n t f r a c t i o n o f t h e compound c o n t i n u e d t o be e x c r e t e d beyond t h i s p e r i o d . The s t u d y r e p o r t s a l o w c o r r e l a t i o n c o e f f i c i e n t ( r = . 4 5 ) f o r t h e 21 w o r k e r s s t u d i e d . In l i g h t of problems w i t h extraneous exposure, interfering exposure, and i n c o m p l e t e m e t a b o l i t e c o l l e c t i o n , however, t h i s r e s u l t i s not unexpected. Thus, the study does n o t p r o v i d e c l e a r evidence that patche exposure e s t i m a t e s and excretion are not correlated. Wojeck and co-workers examined the r e l a t i o n s h i p between e t h i o n exposure and d i a I k y l p h o s p h a t e e x c r e t i o n i n 17 F l o r i d a c i t r u s m i x e r s a n d a p p l i c a t o r s (1_9). Extran e o u s e x p o s u r e was m o n i t o r e d by a s i n g l e u r i n e s a m p l e one week b e f o r e t h e s t u d y . M e t a b o l i t e l e v e l s on t h e day p r i o r t o the s t u d y were n o t measured. Spot sampling o f u r i n e w a s c o n d u c t e d r a t h e r t h a n t o t a l 24 h o u r collections. These s a m p l i n g p r o c e d u r e s c l e a r l y l i m i t the u t i l i t y o f the u r i n e v a l u e s as r e l a t i v e i n d i c a t o r s o f exposure. R e s p i r a t o r y e x p o s u r e was l i m i t e d by u s e o f r e s p i rators. C o t t o n g l o v e s w e r e w o r n by a l l w o r k e r s t o meas u r e hand e x p o s u r e , b u t w o r k e r s d i d n o t wear g l o v e s a s p r o t e c t i v e d e v i c e s . The u s e o f c o t t o n g l o v e s r e p r e s e n t s a p a r t i c u l a r l y complex confounding factor, wherein the dermal sampling procedure i t s e l f i s l i k e l y to a l t e r the b i o l o g i c a l m o n i t o r i n g d a t a i n u n p r e d i c t a b l e ways. Regression a n a l y s i s produced a low c o r r e l a t i o n coeff i c i e n t ( r = . 3 6 ) f o r t h e g r o u p mean t o t a l d e r m a l exposure ( d e r m a l p a t c h e s + h a n d s ) a n d t h e g r o u p mean m e t a b o l i t e c o n c e n t r a t i o n when t h e s e v a l u e s w e r e c o m p a r e d on a d a i l y basis. C o n s i d e r i n g the l i m i t a t i o n s i n urine sampling and the p o t e n t i a l confounding e f f e c t o f the c o t t o n g l o v e s , t h i s study does n o t p r o v i d e an adequate test of patch technique v a l i d i t y .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

FENSKE

Validation of Environmental Monitoring

11

Manninen and c o - w o r k e r s c o n d u c t e d a s t u d y of 17 workers exposed t o p h e n o x y a c i d h e r b i c i d e s (20)» No e v a l u a t i o n o f p r i o r e x p o s u r e among w o r k e r s i s m e n t i o n e d / a n d no p r e - e x p o s u r e u r i n e s a m p l e s w e r e c o l l e c t e d . Two s p o t s a m p l e s w e r e t a k e n : one a f t e r t h e w o r k i n g day i n t h e e v e n i n g a n d one on t h e f o l l o w i n g m o r n i n g . Respiratory e x p o s u r e and hand e x p o s u r e were l e f t u n c o n t r o l l e d . Some w o r k e r s wore r e s p i r a t o r s or g l o v e s but o t h e r s d i d not, i n t r o d u c i n g an unknown amount of v a r i a b i l i t y i n t o the t o t a l e x p o s u r e r e f l e c t e d by t h e m e t a b o l i t e s . This study a n a l y z e d u r i n e f o r four d i f f e r e n t phenoxy h e r b i c i d e s : MCPA/ d i c h l o r p r o p / m e c o p r o p / a n d d i c a m b a . Workers used f o r m u l a t i o n s w i t h d i f f e r e n t c o m b i n a t i o n s o f t h e s e compounds. C l e a r l y the v a r i a b l e dermal p e n e t r a t i o n and p h a r m a c o k i n e t i c s o f t h e s e c o m p o u n d s make t h e s p o t u r i n a r y e x c r e t i o n v a l u e s d i f f i c u l t to i n t e r p r e t The a u t h o r s c o n d u c t e e x c r e t i o n a n d d e r m a l p a t c h e s t i m a t e s f o r t h e 17 w o r k e r s / r e s u l t i n g i n a p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t (r=.67). However/ v a r i a b i l i t y i n the h i g h e r e x p o s u r e v a l u e s were pronounced. H i g h l y exposed workers with s i m i l a r levels o f s k i n e x p o s u r e ( a p p r o x i m a t e l y 2 mg) v a r i e d b y m o r e t h a n an o r d e r of magnitude i n the e x c r e t i o n o f m e t a b o l i t e s ( 2 28 u m o l / L ) . The l a c k o f c o n t r o l o v e r e x t r a n e o u s a n d i n t e r f e r i n g exposures/ together with spot sampling of a v a r i e t y o f compounds r a i s e s q u e s t i o n s r e g a r d i n g the c o r r e l a t i o n of exposure and e x c r e t i o n . The f i n a l s t u d y t o be r e v i e w e d was conducted by C a n a d i a n r e s e a r c h e r s o n 8 f a r m e r s e x p o s e d t o 2,4-D amine (21). P r e - e x p o s u r e u r i n e samples were c o l l e c t e d to determine background l e v e l s . T o t a l 24 h o u r u r i n e samples w e r e c o l l e c t e d d u r i n g t h e s t u d y a n d f o r 4-7 d a y s t h e r e after. S o m e o f t h e p a r t i c i p a n t s s p r a y e d 2,4-D again w i t h i n a few days a f t e r the i n i t i a l s t u d y p e r i o d / p r e c l u d i n g use o f a s t r i c t s i n g l e e x p o s u r e d e s i g n . However/ a l l u r i n e was c o l l e c t e d f r o m t h e f i r s t e x p o s u r e t h r o u g h t h e l a s t a n d f o r 4-7 days a f t e r the l a s t / providing adequate data f o r d e v e l o p i n g a r e l a t i v e index of exposure. E a c h e x p o s u r e was m e a s u r e d by t h e p a t c h a n d h a n d wash t e c h n i q u e s . Workers d i d not use r e s p i r a t o r s or protective gloves. T h i s s t u d y was w e l l c o n d u c t e d / a v o i d ing problems r e g a r d i n g e x t r a n e o u s exposure seen i n the o t h e r s t u d i e s / and c o l l e c t i n g u r i n e f o r a p e r i o d a p p r o p r i a t e t o t h e e x c r e t i o n o f 2/4-D. C u m u l a t i v e u r i n a r y e x c r e t i o n was a s s o c i a t e d only w i t h hand e x p o s u r e (r=.67). P a t c h exposure e s t i m a t e s and t o t a l d e r m a l e x p o s u r e ( p a t c h e s t i m a t e s + hand e x p o s u r e ) did not c o r r e l a t e with u r i n a r y e x c r e t i o n . The a u t h o r s c o n c l u d e d t h a t the l a c k of c o r r e l a t i o n between total d e r m a l e x p o s u r e a n d u r i n a r y e x c r e t i o n was a t t r i b u t a b l e t o the v a r i a b l e n a t u r e of dermal d e p o s i t i o n and the i n a b i l i t y of the patch technique to e s t i m a t e such d e p o s i t i o n .

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In summary/ t h e s t u d i e s r e v i e w e d d e f i n i t i v e e v i d e n c e r e g a r d i n g the the patch technique. Confounding exposure/ i n t e r f e r i n g exposure and l e c t i o n i n t r o d u c e an unknown d e g r e e c a n n o t be s e p a r a t e d f r o m v a r i a b i l i t ment t e c h n i q u e . F l u o r e s c e n t Tracer Technique

h e r e do n o t p r o v i d e r e l a t i v e a c c u r a c y of f a c t o r s of extraneous inadequate urine colof v a r i a b i l i t y which y due t o t h e m e a s u r e -

Validation

Study

The f l u o r e s c e n t t r a c e r t e c h n i q u e was f i r s t e m p l o y e d i n f i e l d s t u d i e s i n 1 9 7 9 (j>). S u b s e q u e n t l y / a q u a n t i t a t i v e v i d e o i m a g i n g s y s t e m was d e v e l o p e d t o m e a s u r e dermal fluorescence. The r e l a t i o n s h i p b e t w e e n t h e t r a c e r a n d p e s t i c i d e s d u r i n g a p p l i c a t i o n was e x a m i n e d / a n d t h e t e c h n i q u e was f i e l d t e s t e d ( 2 2 - 2 4 ) A d e t a i l e d account of the v a l i d a t i o n stud recently (Fen ske press). The s t u d y i n c l u d e d s e v e n m i x e r s a n d 14 a p p l i c a t o r s . E a c h a p p l i c a t o r s p r a y e d f o u r t a n k s on a h i g h - v o l u m e a i r b l a s t spray s c h e d u l e t y p i c a l f o r c i t r u s i n the r e g i o n . The f l u o r e s c e n t w h i t e n i n g a g e n t / 4-methy1-7-diethylaminoc o u m a r i n / was e m p l o y e d a s a t r a c e r . D e p o s i t i o n of the t r a c e r compound a n d m a l a t h i o n on g a u z e p a t c h e s a t t a c h e d to t h e s p r a y r i g was highly correlated (r=.94). The i n s t r u m e n t e m p l o y e d t o q u a n t i f y d e r m a l f l u o r e s c e n c e was a computer-based image p r o c e s s i n g s y s t e m i n t e r f a c e d w i t h a t e l e v i s i o n camera. T h e t r a d i t i o n a l p a t c h t e c h n i q u e was a l s o e m p l o y e d (2_5). Hand w a s h e s w e r e n o t c o n d u c t e d due to use o f t h e t r a c e r compound. The p r i m a r y c o n c e r n i n d e s i g n i n g t h i s s t u d y was t h e s e l e c t i o n o f a p e s t i c i d e w h i c h w o u l d be r a p i d l y e x c r e t e d i n the u r i n e p r o p o r t i o n a l to absorbed dose. Several l a b o r a t o r y s t u d i e s p r o v i d e s u p p o r t f o r the use o f o r g a n o phosphorus p e s t i c i d e m e t a b o l i t e l e v e l s e x c r e t e d i n the u r i n e as r e l a t i v e i n d i c a t o r s of exposure. A controlled s t u d y i n humans w i t h m e t h y l and e t h y l p a r a t h i o n demons t r a t e d a s t r o n g a s s o c i a t i o n b e t w e e n two o r a l d o s e levels a n d e x c r e t i o n l e v e l s (^26^). More r e c e n t l y a study of azinphosmethyl a p p l i e d d e r m a l l y to rats demonstrated that d i m e t h y 1 t h i o p h o s p h a t e e x c r e t i o n was p r o p o r t i o n a l t o d o s e o v e r a f o u r - f o l d dose range. S t u d i e s w i t h human v o l u n teers a l s o suggested a proportional dose-excretion patt e r n (27). One o f t h e m o s t r a p i d l y e x c r e t e d o r g a n o p h o s p h o r u s compounds i s m a l a t h i o n / due t o i t s h y d r o l y s i s i n mammals to f o r m c a r b o x y l i c a c i d m e t a b o l i t e s (^8). Feldman and Maibach demonstrated t h a t e x c r e t i o n of m a l a t h i o n metabol i t e s f o l l o w i n g dermal a p p l i c a t i o n i n acetone peaked w i t h i n 12 h o u r s f o l l o w i n g e x p o s u r e / a n d t h a t o v e r 9 0 % o f the i n t r a v e n o u s a d m i n i s t r a t i o n was r e c o v e r e d a s m e t a b o -

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

FENSKE

79

Validation of Environmental Monitoring

l i t e s i n u r i n e (3^0). B r a d w a y a n d S h a f i k d e m o n s t r a t e d a s i m i l a r pharmacokinetic p a t t e r n f o r malathion i n mice (29). T h u s , m a l a t h i o n a p p e a r e d t o be a n e x c e l l e n t c a n d i date f o r t h i s v a l i d a t i o n study. The s e c o n d s t u d y d e s i g n i s s u e i n v o l v e d m i n i m i z i n g extraneous exposures. S u b j e c t s were asked to a v o i d a l l e x p o s u r e t o m a l a t h i o n o r any o t h e r d i m e t h y l organophosp h o r u s compounds f o r a t l e a s t 3 days p r i o r t o the study. Pre-exposure s a m p l e s were c o l l e c t e d a t the b e g i n n i n g of the study p e r i o d to determine p r i o r exposure. In one c a s e m e t a b o l i t e l e v e l s w e r e f o u n d t o be a b o v e b a c k g r o u n d l e v e l s , n e c e s s i t a t i n g e x c l u s i o n of t h i s s u b j e c t ' s data from a n a l y s i s . A l l u r i n e was c o l l e c t e d f o r 72 h o u r s f r o m t h e commencement o f the s t u d y / and s u b j e c t s were r e m o v e d from any f u r t h e r e x p o s u r e d u r i n g t h i s c o l l e c t i o n p e r i o d The r e l a t i v e l y s h o r subject cooperation r e c o v e r a p p r o x i m a t e l y 95% o f the m e t a b o l i t e s w h i c h w o u l d b e e x c r e t e d i n 5 d a y s (,10). Thus/ the t o t a l amount of m a l a t h i o n m e t a b o l i t e s r e c o v e r e d i n t h e 3 day u r i n e s a m p l e a p p e a r e d to a c c u r a t e l y r e f l e c t the r e l a t i v e exposure which workers received. I n t e r f e r i n g e x p o s u r e was minim i z e d by p r o v i d i n g a l l w o r k e r s w i t h r e s p i r a t o r s a n d c h e m ical resistant gloves. D e r m a l e x p o s u r e was m o n i t o r e d by the f l u o r e s c e n t t r a c e r t e c h n i q u e (body + hands) and by the p a t c h t e c h n i q u e (body o n l y ) d u r i n g a s i n g l e study p e r i o d f o r each worker. Table

III.

C o r r e l a t i o n of Exposure Measurements w i t h 24-Hr M a l a t h i o n M e t a b o l i t e E x c r e t i o n

Exposure Measurements Fluorescent P a t c h : Mean P a t c h : Head * ** + ++

Tracer Value Exposure

Corr. Coeff.*

RSquared

Rank Corr.**

.84+ .71 + .63 +

.71 .51 .40

.62 + .39++ .33

Pearson c o r r e l a t i o n c o e f f i c i e n t Spearman rank c o r r e l a t i o n c o e f f i c i e n t S t a t i s t i c a l l y s i g n i f i c a n t ; p<.001 S t a t i s t i c a l l y s i g n i f i c a n t ; p<.05

A p l o t of f l u o r e s c e n t t r a c e r e x p o s u r e and m e t a b o l i t e e x c r e t i o n f o r t h e 2 0 w o r k e r s i s p r e s e n t e d F i g u r e 1/ a n d r e s u l t s of r e g r e s s i o n a n a l y s i s are l i s t e d i n Table I I I . A l l w o r k e r s h a d t h e i r p e a k e x c r e t i o n w i t h i n t h e f i r s t 24 hours/ and the f l u o r e s c e n t t r a c e r measurements were h i g h l y c o r r e l a t e d w i t h e x c r e t i o n (r=.84). The correlation c o e f f i c i e n t and R - s q u a r e d v a l u e f o r the a s s o c i a t i o n of f l u o r e s c e n t t r a c e r measurements and 2 4 - h r m e t a b o l i t e e x c r e t i o n w e r e c o m p a r e d t o t h e same s t a t i s t i c s f o r two measurements d e r i v e d from patch data. T h e two p a t c h d a t a v a l u e s a r e 1) t h e m e a n o f t h e e i g h t o u t e r p a t c h e s a b o v e

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2500

MALATHION EXCRETION big) F i g u r e 1. R e g r e s s i o n a n a l y s i s o f f l u o r e s c e n t tracer e x p o s u r e a n d 24 h r m a l a t h i o n m e t a b o l i t e e x c r e t i o n .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

6.

FENSKE

81

Validation of Environmental Monitoring

t h e w a i s t o n e a c h w o r k e r , a n d 2) t h e e s t i m a t e d exposure to t h e head a n d neck b a s e d on e x t r a p o l a t i o n from four patches ( c h e s t , back a n d two s h o u l d e r ) . The head a n d neck were the o n l y exposed r e g i o n s . Since the study d i d not i n c l u d e a f u l l s e t of patches beneath the c l o t h i n g , no a t t e m p t w a s made t o e x t r a p o l a t e p a t c h d a t a t o d e t e r mine dermal exposure t o t h e w h o l e body. B o t h t h e mean patch value (r=.71) a n d t h e head e x p o s u r e estimate (r=.63) were s i g n i f i c a n t l y c o r r e l a t e d w i t h e x c r e t i o n . The R - s q u a r e d v a l u e i n d i c a t e s t h e p r o p o r t i o n o f t h e m e t a b o l i t e e x c r e t i o n v a r i a b l e t h a t c a n be e x p l a i n e d by the exposure v a r i a b l e , a n d i s t h e most u s e f u l statistic for comparison of the d i f f e r e n t exposure measurements. The t r a c e r d a t a e x p l a i n e d a p p r o x i m a t e l y 7 0 % o f t h e t o t a l v a r i a n c e , whereas the patch data e x p l a i n e d a t best about 50%. A l l of the c o r r e l a t i o n demonstrating that th c a t i o n of r e l a t i v e exposure potential. The S p e a r m a n R a n k C o r r e l a t i o n s t a t i s t i c ( r [ s ] ) was a l s o a p p l i e d to these data, s i n c e the Pearson Correlation C o e f f i c i e n t r e q u i r e s an assumption o f normal d i s t r i b u t i o n and i s h i g h l y i n f l u e n c e d by l a r g e v a l u e s . Here the c o n t r a s t i n the a s s o c i a t i o n of e x c r e t i o n and t r a c e r data a s c o m p a r e d t o p a t c h d a t a was e v e n more p r o n o u n c e d . The t r a c e r r a n k c o r r e l a t i o n was h i g h l y s i g n i f i c a n t ( r [ s ] = . 6 2 ; p < . 0 0 1 ) , w h e r e a s t h e mean p a t c h d a t a w e r e o n l y m a r g i n a l l y c o r r e l a t e d ( r [ s ] = . 3 9 ; p<.05), a n d p a t c h - e s t i m a t e d head exposure h a s no s i g n i f i c a n t c o r r e l a t i o n ( r [ s ] = . 3 3 ) . Current

S t a t u s o f Derma 1 E x p o s u r e A s s e s s m e n t

Methods

The p a t c h t e c h n i q u e c a n p r o v i d e a r e l a t i v e i n d i c a t i o n o f exposure, b u t may n o t b e a b l e t o r a n k e x p o s u r e s w i t h a high degree of accuracy. With l a r g e data sets the v a r i a b i l i t y r e f l e c t e d i n p a t c h d a t a may b e o v e r c o m e . The e s t a b l i s h m e n t o f a g e n e r i c data base o f a l l patch t e c h nique data i s a promising development i n t h i s regard (30). The p a t c h t e c h n i q u e i s i n h e r e n t l y l i m i t e d due t o i t s r e l i a n c e on t h e a s s u m p t i o n t h a t e x p o s u r e i s u n i f o r m o v e r v a r i o u s body p a r t s . Studies with the f l u o r e s c e n t t r a c e r and w i t h t h e w h o l e body t e c h n i q u e i n t h e U n i t e d Kingdom have i l l u s t r a t e d the i n a p p r o p r i a t e n e s s of t h i s assumption f o r a p p l i c a t o r exposures (31-32). The p a t c h m e t h o d may a l s o s u f f e r f r o m a s y s t e m a t i c e r r o r ; i . e . , f o r some m i x i n g a n d a p p l i c a t i o n p r o c e d u r e s t h e p a t c h e s t i mates a r e c o n s i s t e n t l y h i g h , w h i l e f o r o t h e r procedures the e s t i m a t e s a r e c o n s i s t e n t l y l o w (31). The v a l i d a t i o n s t u d y d i s c u s s e d h e r e p r o v i d e s e v i dence t h a t measurement o f f l u o r e s c e n t t r a c e r d e p o s i t i o n on t h e s k i n i s a v a l i d m e t h o d i n r e g a r d t o r e l a t i v e accuracy. As d i s c u s s e d e a r l i e r , the technique also showed a h i g h c o r r e l a t i o n between exposure and time

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w o r k e d among t i m b e r m i l l w o r k e r s , w h e r e t i m e w o r k e d i s a v a l i d parameter of exposure p o t e n t i a l . Additionally, t h i s approach has s u c c e s s f u l l y demonstrated quantitative d i f f e r e n c e s i n exposure as a f u n c t i o n of the type of p r o t e c t i v e c l o t h i n g w o r n (3_3). The f l u o r e s c e n t t r a c e r t e c h n i q u e p r o v i d e s new i n f o r m a t i o n r e g a r d i n g d e r m a l e x p o sure, as both the i n v e s t i g a t o r and worker can see patterns of d e p o s i t i o n and immediately a n a l y z e possible causes. The method r e q u i r e s r e l a t i v e l y few p e s t i c i d e r e s i d u e a n a l y s e s , s i n c e most o f t h e exposure data are c o l l e c t e d and a n a l y z e d a u t o m a t i c a l l y w i t h the imaging system. The m a j o r d i s a d v a n t a g e o f t h i s t e c h n i q u e when compared to the patch technique i s i t s r e l a t i v e complexity in the f i e l d . The s t a b i l i t f UV-A i l l u m i n a t i o d th instrument's performanc p e s t i c i d e and t r a c e be d e t e r m i n e d e m p i r i c a l l y i n e a c h s t u d y t h r o u g h field sampling. I n s o m e c a s e s c l o t h i n g p e n e t r a t i o n may b e d i f f e r e n t f o r t h e two compounds, r e q u i r i n g a c o r r e c t i o n factor. The quantitative accuracy of fluorescent tracer measurements has n o t been demonstrated and i s c u r r e n t l y under i n v e s t i g a t i o n . Laboratory experiments a r e aimed a t d e t e r m i n i n g the b e h a v i o r o f the f l u o r e s c e n t t r a c e r once i t reaches the skin. Recent s t u d i e s suggest that the t r a c e r p r o d u c e s a s t a b l e f l u o r e s c e n t e m i s s i o n on t h e s k i n f o r a t l e a s t s i x hours. F u r t h e r s t u d i e s w i l l examine the e f f e c t s o f s u n l i g h t a n d p e r s p i r a t i o n on t r a c e r s t a b i l i t y . Finally, the problem of quenching i s i n h e r e n t to any m e t h o d m e a s u r i n g d e r m a l f l u o r e s c e n c e . When e x c e s s f l u o r e s c e n t m a t e r i a l i s d e p o s i t e d on t h e s k i n t h e f l u o r e s c e n c e p r o d u c e d i s no l o n g e r p r o p o r t i o n a l t o d e p o s i t i o n . Thus, a r e a s o f v e r y h i g h e x p o s u r e may b e u n d e r e s t i m a t e d w i t h t h i s technique. Proper choice of tracer concentration a t t h e o u t s e t o f a s t u d y c a n do much t o m i t i g a t e t h i s problem. Conclusion The v a l i d a t i o n o f exposure methods i s an e s s e n t i a l step in developing a s c i e n t i f i c basis f o r the e v a l u a t i o n of human h e a l t h r i s k s . B i o l o g i c a l m o n i t o r i n g has enormous p o t e n t i a l f o rp r o v i d i n g e s t i m a t e s of i n t e r n a l dose and for i d e n t i f y i n g e a r l y indicators of c l i n i c a l e f f e c t s of exposure. Environmental monitoring plays a v i t a l role i n r e d u c i n g h e a l t h r i s k s by d e t e r m i n i n g t h e m a g n i t u d e o f exposure and documenting exposure pathways. The f l u o r e s cent t r a c e r technique and patch technique s e r v e as i n d e pendent and complementary approaches f o r e v a l u a t i n g and reducing dermal exposure i n a g r i c u l t u r e .

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Acknowledgments The c o o p e r a t i o n o f t h e a d m i n i s t r a t i o n a n d s t a f f o f t h e University of C a l i f o r n i a Lindcove Field Station i s acknowledged and g r e a t l y a p p r e c i a t e d . F i e l d w o r k was s u p p o r t e d by t h e N a t i o n a l I n s t i t u t e f o r O c c u p a t i o n a l S a f e t y a n d H e a l t h (1 R 0 1 0 1 2 3 4 - 0 1 A l ) . D a t a a n a l y s i s was s u p p o r t e d b y s t a t e f u n d s t h r o u g h t h e New J e r s e y Agricult u r a l E x p e r i m e n t S t a t i o n ( P u b l i c a t i o n No. F - 0 7 1 2 3 - 1 - 8 7 ) .

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8. 9. 10. 11. 12. 13. 14. 15.

Kay, K.; Monkman, L.; Windish, J.P.; Doherty, T.; Pare, J.; R a c i c o t , C. Ama Arch. Ind. Hyg. & Occ. Med. 1952, 6, 252-252. TLV - Threshold Limit Values for Chemical Substances i n the Work Environment ference of Governmenta c i n n a t i , Ohio, 1986. Lowry, L.K. J. Occup. Med. 1986 28, 578-582. Fenske, R.A.; Horstman, S.W.; B e n t l e y , R.K. Appl. Ind. Hyg. 1987, 2, 143-147 F r a n k l i n , C.A.; Fenske, R.A.; Greenhalgh, R.; Mathieu, L.; Denley, B.V.; L e f f i n g w e l l , J.T.; Spear, R.C. J. Toxicol. Environ. Health 1981, 7, 715-731 Hackathorn, D.R.; Eberhart, D.C. In Dermal Exposure Related to Pesticide Use Honeycutt, R.C.; Zweig, G.; Ragsdale, N.N., Eds.; ACS Symposium S e r i e s No. 273; American Chemical S o c i e t y : Washington, D.C., 1985; pp. 341-356. R e i n e r t , J.C.; Severn, D.J. In Dermal Exposure Rel a t e d to P e s t i c i d e Use Honeycutt, R.C.; Zweig, G.; Ragsdale, N.N., Eds.; ACS Symposium S e r i e s No. 273; American Chemical S o c i e t y : Washington, D.C., 1985; pp. 357-368. Grover, R.; Cessna, A.J.; Muir, N.I.; R i e d e l , D.; F r a n k l i n , C.A.; Yoshida, K. Arch. E n v i r o n . Contam. Toxicol. 1986, 15, 677-686. Bernard, A.; Lauwerys, R. J. Occup. Med. 1986, 28 558-562. Feldman, R.J.; Maibach, H.I. T o x i c o l . Appl. Pharma­ c o l . 1974, 28 126-132. Durham, W.F.; Wolfe, H.R. B u l l . World Health Organi­ zation 1962, 26, 75-91. World Health Organization Toxicol. Letters 1986, 33, 223-235. P e s t i c i d e Assessment G u i d e l i n e s , S u b d i v i s i o n U: Applicator Exposure, U.S. Environmental Protection Agency, NTIS, V i r g i n i a , 1986. B o n s a l l , J.L. In O c c u p a t i o n a l Hazards of P e s t i c i d e Use; T u r n b u l l , G.J.; Ed.; T a y l o r & F r a n c i s : London, 1985; pp. 13-34. Lavy, T.L.; M a t t i c e , J.D. T o x i c o l . L e t t e r s 1986, 33, 61-71.

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16. Lunchick, C.; Burin, G.; R e i n e r t , J.C.; In Chemical Basis of B i o l o g i c a l Monitoring ACS Symposium Series (this volume). 17. Durham, W.F. B u l l . World H e a l t h O r g a n i z a t i o n 1963, 29, 279-281. 18. Lavy, T.L.; Shepard, J.S.; M a t t i c e , J.D. J. A g r i c . Food Chem., 1980, 28 626-630. 19. Wojeck, G.A.; Nigg, H.N.; Stamper, J.H.; Bradway, D.E. Arch. E n v i r o n . Contam. T o x i c o l . 1981,10,725735 20. Manninen, A.; Kangas, J.; Klen, T.; S a v o l a i n e n , H. Arch. Environ. contam. Toxicol. 1986, 15, 107-111. 21. Grover, R.; F r a n k l i n , C.A.; Muir, N.I.; Cessna, A.J.; R i e d e l , D. T o x i c o l . L e t t e r s 1986, 33 73-83. 22. Fenske, R.A.; L e f f i n g w e l l , J.T.; Spear, R.C. In Dermal Exposure Related to Pesticide Use Honeycutt R.C.; Zweig, G. Series No. 273 ton, D.C., 1985; pp. 377-393. 23. Fenske, R.A.; L e f f i n g w e l l , J.T.; Spear, R.C. Am. Ind. Hyg. Assoc. J. 1986, 47 764-770 24. Fenske, R.A.; Wong, S.M.; L e f f i n g w e l l , J.T.; Spear, R.C. Am. Ind. Hyg. Assoc. J . 1986, 47, 771-775. 25. Davis, J.E. Residue Reviews 1980, 75 33-50. 26. Morgan, D.P.; H e t z l e r , H.L.; S l a c h , E.F.; L i n , L.I. Arch. Environ, contam. Toxicol. 1977, 6, 159-173. 27. F r a n k l i n , C.A.; Muir, N.I.; Moody, R.P. T o x i c o l . Letters 1986, 33, 127-136 28. Chen, R.R.; Tucker, W.P.; Dauterman, W.C. J . A g r i c . Food Chem. 1969,17,86. 29. Bradway, D.E.; Shafik, T.M. J. A g r i c . Food Chem. 1977, 25, 1342-1344. 30. Honeycutt, R.C. In Dermal Exposure Related to Pest­ i c i d e Use Honeycutt, R.C.; Zweig, G.; Ragsdale, N.N., Eds.; ACS Symposium S e r i e s No. 273; American Chemical S o c i e t y : Washington, D.C. 1985; pp. 369375. 31. Fenske, R.A. In P e s t i c i d e Science and B i o t e c h ­ nology; Greenhalgh, R.; Roberts, R.R.; Eds.; B l a c k w e l l S c i e n t i f i c Publications: London, 1987; pp. 579582. 32. Chester, G.; Ward, R.J. Human T o x i c o l o g y 1983, 2, 555-556. 33. Fenske, R.A. In Performance of Protective Clothing; American Society for the Testing of Materials: P h i l ­ adelphia, 1988. RECEIVED May 4, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 7

Biological Markers To Study Exposure in Animals and Bioavailability of Environmental Contaminants L. R. Shugart, S. M . Adams, B. D. Jiminez, S. S. Talmage, and J. F. McCarthy Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 Epidemiologic studie environment seek contribute to the causation of a specific toxic, clinical, or pathological endpoint. The multifactorial nature of disease etiology, long latency periods and the complexity of exposure, a l l contribute to the difficulty of establishing associations and causal relationships between a specific exposure and an adverse outcome. These barriers to studies of exposure and subsequent risk assessment cannot generally be changed. However, the appropriate use of biological markers in animal species living in a contaminated habitat can provide a measure of potential damage from that exposure and, in some instances, act as a surrogate for human environmental exposures. Quantitative predictivity of the effect of exposure to environmental pollutants is being approached by employing an appropriate array of biological end points. Human exposure to environmental pollutants is often exceedingly complex. Sources of exposure are multiple, and the importance of a particular source may depend on individual practices. Humans are highly mobile, and direct study, particularly when physiological testing is required, is generally expensive and logistically difficult. For these reasons, a simpler animal model which provides a parallel biological marker in humans would be useful. Small mammals have frequently been used to monitor the presence of metals and other contaminants in the environment. Such biomonitoring can be essential for determining bioavailability and resultant biological effects under natural conditions. Small mammals can be particularly effective biomonitors of soil contaminants i f the species used are abundant, easily caught, do not migrate long distances, and have well documented food habits This chapter not subject to U.S. copyright Published 1989 American Chemical Society

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(1). Good b i o m o n i t o r s a r e i n e q u i l i b r i u m w i t h the environment and s h o u l d show a graded response t o a range o f p o l l u t a n t s . Data a r e s p a r s e on the amounts o f hazardous s u b s t a n c e s r e l e a s e d to the environment. M e a s u r i n g c o n c e n t r a t i o n s o f t h e s e s u b s t a n c e s i n s o i l , p l a n t s , and a n i m a l s would r e q u i r e an e x h a u s t i v e m o n i t o r i n g program w h i c h would be c o s t l y , e s p e c i a l l y s i n c e many c h e m i c a l s w i l l n o t be p r e s e n t as the p a r e n t compound i n l i v i n g organisms due t o t h e i r r a p i d metabolism. The i m p o r t a n t q u e s t i o n i s n o t whether t h e s e s u b s t a n c e s a r e p r e s e n t , b u t r a t h e r whether t h e s e s u b s t a n c e s e n t e r the b i o t a i n c h e m i c a l forms and c o n c e n t r a t i o n s t h a t w i l l r e s u l t i n an a d v e r s e b i o l o g i c a l e f f e c t . Thus, m o n i t o r i n g the b i o l o g i c a l a v a i l a b i l i t y o f t h e s e s u b s t a n c e s i n b i o t a becomes a c r i t i c a l parameter f o r d e t e r m i n i n g whether t h e y have p o t e n t i a l f o r b e i n g hazardous and whether t h e y c o u l d r e s u l t i n s i g n i f i c a n t exposures t o humans ( 2 ) . The e s t i m a t i o n o f the r i s k r e s u l t i n g from exposure t o environmental p o l l u t a n t p h a r m a c o k i n e t i c s o f the i n an i n d i v i d u a l can be o b t a i n e d by q u a n t i t a t i o n o f the c h e m i c a l o r i t s m e t a b o l i t e s i n body f l u i d s such as b l o o d , u r i n e , f e c e s , sputum, or m i l k , however, a major drawback t o t h i s approach i s t h a t the a n a l y s i s must be performed s h o r t l y a f t e r exposure, t h a t i s , p r i o r to the c l e a r a n c e o f the m e t a b o l i t e s from the body. A l t e r n a t i v e p r o c e d u r e s f o r m o n i t o r i n g exposure t o hazardous s u b s t a n c e s a r e needed. I n p a r t i c u l a r , new methods a r e needed f o r measuring r e a c t i o n p r o d u c t s o f c h e m i c a l s , such as m e t a b o l i t e s and a d d u c t s , i n a n i m a l s and humans t h a t c o r r e l a t e w i t h p a t t e r n s o f a s s o c i a t e d b i o l o g i c a l change and p a t h o l o g i c a l c o n d i t i o n s Q , 4 ) . Our approach t o d e t e c t i n g exposure t o hazardous c h e m i c a l s i n the environment, and t o e s t i m a t i n g the p o t e n t i a l f o r subsequent adverse e f f e c t s , i s to monitor b i o l o g i c a l endpoints i n w i l d a n i m a l s . B i o l o g i c a l e n d p o i n t s i n c l u d e i n d i c a t o r s t h a t : (a) q u a n t i f y exposure by measuring c h e m i c a l i n t e r a c t i o n w i t h c r i t i c a l m o l e c u l a r t a r g e t s (adducts i n n u c l e i c a c i d s and p r o t e i n s ) ; (b) measure a l t e r a t i o n s i n s p e c i f i c , s e n s i t i v e , and c r i t i c a l p h y s i o l o g i c a l and b i o c h e m i c a l p r o c e s s e s ( d e t o x i f y i n g enzyme a c t i v i t i e s and m e t a l - b i n d i n g p r o t e i n s ) ; and, ( c ) m o n i t o r e a r l y e x p r e s s i o n o f c e l l u l a r d y s f u n c t i o n ( n e o p l a s i a o r l o s s o f immune competence). U s i n g t h i s approach the organism i s seen t o f u n c t i o n as an i n t e g r a t o r o f exposure, a c c o u n t i n g f o r a b i o t i c and p h y s i o l o g i c a l f a c t o r s t h a t modulate the dose o f t o x i c a n t t a k e n up from the environment. The b i o l o g i c a l marker i s used t o d e t e c t the response o f the organism t o the e n v i r o n m e n t a l i n s u l t . Because o f the o f t e n l o n g l a t e n t p e r i o d between exposure and subsequent i r r e v e r s i b l e d e l e t e r i o u s impact on h e a l t h , i t would be d e s i r a b l e t o have s e n s i t i v e b i o l o g i c a l i n d i c a t o r s t h a t c o u l d d e t e c t exposure e a r l y enough so t h a t p r e v e n t i v e o r r e m e d i a l measures c o u l d be t a k e n . Three elements a r e c r i t i c a l t o our approach: (a) marker s e l e c t i o n based on t o x i c o l o g i c a l mechanisms; (b) f i e l d s t u d i e s t o e s t a b l i s h c o r r e l a t i o n s between e n v i r o n m e n t a l c o n t a m i n a t i o n and markers; and, ( c ) l a b o r a t o r y c o n f i r m a t i o n o f c a u s a l r e l a t i o n s h i p s between exposure, b i o l o g i c a l markers, and adverse e f f e c t s . B i o l o g i c a l m o n i t o r i n g can be used as a c o s t - e f f e c t i v e s u r v e y o f c o n t a m i n a t e d s i t e s , f o r r o u t i n e m o n i t o r i n g o f uncontaminated s i t e s ,

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SHUGART ET AL.

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t o p r o v i d e e v i d e n c e f o r contaminant impact on b i o t a , and t o e s t a b l i s h p r i o r i t i e s f o r s i t e cleanup. A s u i t e o f b i o m a r k e r s t h a t we c u r r e n t l y u s e t o m o n i t o r f o r exposure t o s e l e c t e d e n v i r o n m e n t a l p o l l u t a n t s , i s shown i n T a b l e I . I n f o r m a t i o n o b t a i n e d upon m o n i t o r i n g t h e response o f t h e s e b i o m a r k e r s i s compared t o l o n g - t e r m adverse e f f e c t s i n t h e organism (reduced f e c u n d i t y , d e c r e a s e d d i s e a s e r e s i s t a n c e , and tumor formation). T a b l e I . B i o l o g i c a l Markers o f Exposure and B i o a v a i l a b i l i t y o f E n v i r o n m e n t a l Contaminants

Environmental Pollutant

A.

B.

Toxic Metals: Cu, Hg, Ag, Zn, Cd, Pb,

O r g a n i c Cmpds: 1. PAH's

Reliability Index*

Biological Marke

DNA i n t e g r i t y / m d C y d metal-binding proteins porphyrin biosynthesis immune response x e n o b i o t i c metabolism

s s,d s,d,p s s

DNA/hemoglobin adducts x e n o b i o t i c metabolism immune response DNA i n t e g r i t y / m d C y d

s, d,p s,d s s

x e n o b i o t i c metabolism porphyrin p r o f i l e immune response DNA i n t e g r i t y / m d C y d DNA/hemoglobin adducts

s s s s s,d

5

5

2.

PCB's & TCDD

5

*

s: s i g n a l o f p o t e n t i a l problem d: d e f i n i t i v e i n d i c a t o r o f type o r c l a s s o f p o l l u t a n t p: p r e d i c t i v e i n d i c a t o r o f l o n g - t e r m adverse e f f e c t

S t u d i e s w i t h C e l l u l a r Biomarkers DNA M o d i f i c a t i o n . As a r e s u l t o f exposure t o hazardous s u b s t a n c e s i n t h e environment, i t i s n o t u n r e a s o n a b l e t o e x p e c t t h a t damage t o DNA may o c c u r t h a t goes u n c o r r e c t e d , w i t h subsequent a d v e r s e e f f e c t s t o t h e organism. The r a t i o n a l e u n d e r l y i n g t h e s t r a t e g y o f c h e m i c a l d o s i m e t r y by d e t e r m i n i n g l e v e l s o f compounds which become c o v a l e n t l y bound t o c e l l u l a r macromolecules i s based on o u r u n d e r s t a n d i n g o f t h e mode o f a c t i o n o f g e n o t o x i c compounds (5-7) . The m e t a b o l i s m o f c h e m i c a l s i s a r e l a t i v e l y complex phenomenon and d e t o x i f i c a t i o n by c e l l u l a r mechanisms i s n o t always complete, sometimes r e s u l t i n g i n h i g h l y r e a c t i v e e l e c t r o p h i l i c m e t a b o l i t e s . These m e t a b o l i t e s c a n

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undergo a t t a c k by n u c l e o p h i l i c c e n t e r s i n macromolecules such as l i p i d s , p r o t e i n s , DNA and RNA which o f t e n r e s u l t s i n c e l l u l a r toxicity. B i n d i n g w i t h DNA, however, can cause f o r m a t i o n o f a l t e r e d bases t h a t can be r e p a i r e d , be innocuous, or r e s u l t i n a l t e r a t i o n s w h i c h become f i x e d and are t r a n s m i t t e d t o daughter cells. C u r r e n t r e s e a r c h suggests t h a t r e a c t i o n o f c h e m i c a l s w i t h DNA and the e n s u i n g changes w h i c h r e s u l t can cause c a n c e r . G i v e n t h a t g e n o t o x i c agents e x e r t t h e i r a c t i v i t y t h r o u g h i r r e v e r s i b l e r e a c t i o n s w i t h n u c l e o p h i l i c atoms ( a d d u c t s ) , the amount o f such r e a c t i o n p r o d u c t s w i l l be p r o p o r t i o n a l not o n l y t o the i n v i v o c o n c e n t r a t i o n o f the e l e c t r o p h i l e , b u t a l s o t o the time t h i s c o n c e n t r a t i o n i s m a i n t a i n e d . T h e r e f o r e , the amount o f m e t a b o l i t e bound t o c e l l u l a r DNA ( i n v i v o d o s e ) , p r o v i d e s a r e l i a b l e d o s i m e t r i c b a s i s upon w h i c h t o a s s e s s the r i s k c o n n e c t e d w i t h exposure t o a g e n o t o x i c compound (3,7,8). I t s h o u l d be n o t e d t h a t the d e t e c t i o n and q u a n t i t a t i o n o f DNA adducts i s not a s i m p l e techniques c u r r e n t l y a v a i l a b l e a s s a y o f t e n has to accommodate the unique c h e m i c a l and/or p h y s i c a l p r o p e r t i e s o f the i n d i v i d u a l compound or i t s adduct; (b) the p e r c e n t a g e o f t o t a l c h e m i c a l t h a t becomes a t t a c h e d to the DNA i n the t a r g e t t i s s u e i s q u i t e s m a l l because most o f i t i s u s u a l l y d e t o x i f i e d and e x c r e t e d ; (c) not a l l adducts t h a t form between the g e n o t o x i c agent and DNA are s t a b l e o r are i n v o l v e d i n the development o f subsequent d e l e t e r i o u s events i n the organism; and, (d) the amount o f DNA a v a i l a b l e f o r a n a l y s i s from the t a r g e t t i s s u e o r organ i s o f t e n q u i t e l i m i t e d . R e c e n t l y our l a b o r a t o r y demonstrated an a l t e r n a t i v e method t o r a d i o m e t r i c , immuno, or p o s t d e r i v a t i z a t i o n d e t e c t i o n o f the p o l y c y c l i c a r o m a t i c h y d r o c a r b o n , b e n z o [ a j p y r e n e (BaP) b i n d i n g t o DNA ( 9 ) . E s s e n t i a l l y , the t e c h n i q u e c o n s i s t s o f the a c i d - i n d u c e d removal o f the d i o l e p o x i d e adduct o f BaP from the macromolecule as the s t r o n g l y f l u o r e s c e n t f r e e t e t r o l s w h i c h are t h e n s e p a r a t e d and q u a n t i t a t e d by f l u o r e s c e n c e / H P L C a n a l y s i s . The r e s u l t i n g c h r o m a t o g r a p h i c p r o f i l e can be used t o e s t a b l i s h the s t e r e o c h e m i c a l o r i g i n o f the d i o l e p o x i d e i n v o l v e d i n adduct f o r m a t i o n ( 1 0 ) . Benzofalpyrene-DNA Adduct D e t e c t i o n . Our i n i t i a l r e s e a r c h w i t h the f l u o r e s c e n c e / H P L C t e c h n i q u e has demonstrated t h a t we can d e t e c t , i d e n t i f y , and q u a n t i t a t e , subsequent t o s e v e r a l r o u t e s o f exposure, the b i n d i n g o f BaP w i t h c e l l u l a r DNA o f mice (10-12) and f i s h (13.) . The d e t e c t i o n and q u a n t i t a t i o n o f adduct f o r m a t i o n i n f i s h exposed t o BaP demonstrate t h a t a q u a t i c s p e c i e s are s i m i l a r to o t h e r organisms i n t h a t they p o s s e s s c e l l u l a r enzymes c a p a b l e o f m e t a b o l i z i n g BaP to the u l t i m a t e c a r c i n o g e n i c form o f the c h e m i c a l , the d i o l e p o x i d e . A comparison o f BaP-adducts formed w i t h the DNAs o f mice and f i s h demonstrate s i m i l a r i t i e s (Table I I ) , and s u g g e s t the f e a s i b i l i t y o f e x t r a p o l a t i o n o f exposure d a t a , b a s e d on the d e t e c t i o n and q u a n t i t a t i o n o f DNA adduct l e v e l s , between s p e c i e s . F i e l d S t u d i e s w i t h B e l u g a Whale. Large q u a n t i t i e s o f PAHs, i n c l u d i n g the s t r o n g l y c a r c i n o g e n i c BaP, have been r e p o r t e d i n the main h a b i t a t o f the S t . Lawrence b e l u g a whale p o p u l a t i o n . These compounds are the p r o b a b l e cause o f the i n c r e a s e d f r e q u e n c y o f

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

7. SHUGARTETAL.

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Biological Markers in Animals

T a b l e I I . Comparison o f DNA Adduct F o r m a t i o n i n t h e Mouse and B l u e g i l l S u n f i s h a f t e r an A c u t e Exposure t o Benzo|[ajpyrene

Species

Macromolecule

DNA Adduct F o r m a t i o n * 1-1 II-2 %I-1

Mice

S k i n DNA**

588

220

73

Fish

L i v e r DNA***

928

127

88

* DNA adduct f o r m a t i o n d e t e r m i n e d 72 hours subsequent t o BaP exposure and e x p r e s s e d as nanograms o f t e t r o l 1-1 o r I I - 2 p e r gram o f DNA ** Topical applicatio body w e i g h t . *** I n t r a p e r i t o n e a l i n j e c t i o n o f BaP a t 5 micrograms p e r gram body w e i g h t . F i s h m a i n t a i n e d a t 20 C. o

b l a d d e r tumors seen among aluminum workers from t h e same a r e a ( 1 4 ) , and s i n c e t h e compounds contaminate t h e b e l u g a whale f o o d c h a i n , t h e y a r e c o n s i d e r e d t o be t h e e t i o l o g i c a l f a c t o r f o r t h e h i g h f r e q u e n c y o f tumors seen i n t h i s p o p u l a t i o n ( 1 5 ) . D e t e c t a b l e l e v e l s o f BaP-DNA adducts were found i n t h e b r a i n s o f b e l u g a whales from t h e S t . Lawrence e s t u a r y , w h i l e t h e o c c u r r e n c e o f BaP-DNA adducts o f t h e b r a i n and l i v e r o f b e l u g a s t a k e n from t h e Mackenzie e s t u a r y was n o t o b s e r v e d ( T a b l e I I I ) . Table I I I .

D e t e c t i o n o f Benzo[a]pyrene Adducts i n DNA o f B e l u g a whales ( D e l p h i n a p t e r u s l e u c a s )

Sample

Tissue

BaP Adduct F o r m a t i o n binding* level**

S t . Lawrence E s t u a r y #1 #2 #3

Brain Brain Brain

206 94 69

Mackenzie E s t u a r y #1 - #4

Brain

none d e t e c t e d

#1 - #4

Liver

none d e t e c t e d

* per ** 10 7

2. 15 0. 98 0. 73

BaP adducts t o DNA e x p r e s s e d as nanograms o f t e t r o l 1-1 gram o f DNA. Data t a k e n from ( 1 5 ) . L e v e l o f adduct f o r m a t i o n e x p r e s s e d as BaP adducts p e r DNA n u c l e o t i d e s .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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The d a t a o b t a i n e d a r e i m p o r t a n t f o r s e v e r a l r e a s o n s . F i r s t , they p r o v i d e e v i d e n c e t h a t t h e whales from t h e S t . Lawrence e s t u a r y had been exposed t o BaP, and had m e t a b o l i z e d i t t o t h e d i o l e p o x i d e , w h i c h s u b s e q u e n t l y became c o v a l e n t l y bound t o t h e DNA o f t h e b r a i n t i s s u e . Over t h e p a s t s e v e r a l y e a r s a s t r o n g and c o n v i n c i n g argument has d e v e l o p e d f o r a c a u s a l r e l a t i o n s h i p between t h e c a r c i n o g e n i c potency o f BaP and t h e amount bound t o an organism's DNA as a r e s u l t o f c e l l u l a r metabolism ( 6 , 1 6 ) . T h i s i s r e i n f o r c e d by t h e premise t h a t t h e i n t e g r i t y o f DNA i s e s s e n t i a l f o r s u r v i v a l . A l t e r a t i o n s , i f l e f t u n c o r r e c t e d , c o u l d t r i g g e r a sequence o f e v e n t s t h a t c u l m i n a t e s i n t h e appearance o f an o v e r t m a l i g n a n c y . Second, t h e l e v e l o f adducts d e t e c t e d approaches t h a t found i n a n i m a l s , b o t h t e r r e s t r i a l and a q u a t i c , exposed under c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s t o a c a r c i n o g e n i c dose o f BaP (12,13,17,18). T h i r d , c e l l s i n b r a i n t i s s u e a r e known t o have slow t u r n o v e r r a t e s and l a c k o r have v e r y low c a p a c i t y f o r e x c i s i o n r e p a i r o f DNA damage. T h e r e f o r e , s i g n i f i c a n o c c u r i n t h i s t i s s u e fro e n v i r o n m e n t a l l e v e l s o f BaP t o o low t o i n d u c e n e o p l a s i a ( 1 9 ) . I t s h o u l d be n o t e d t h a t t h e h e a l t h o f an a n i m a l may i n f l u e n c e t h e f o r m a t i o n o f a d d u c t s . The whales from t h e Mackenzie e s t u a r y were h u n t e d a n i m a l s and n o t known t o be d i s e a s e d a t t h e time o f t h e i r c a p t u r e , w h i l e those from t h e S t . Lawrence were beached a n i m a l s . Non-adductive DNA Damage. C e r t a i n g e n o t o x i c compounds o r agents such as m e t a l s , r a d i o n u c l i d e s , some c h e m i c a l s , e t c . , do n o t c o v a l e n t l y b i n d t o DNA, b u t n e v e r t h e l e s s i n d u c e damage. I f t h i s damage i s e x p r e s s e d as s t r a n d breakage ( o r t h e p o t e n t i a l f o r s t r a n d b r e a k a g e ) , i t c a n be d e t e c t e d by measuring t h e r a t e o f a l k a l i n e i n d u c e d s t r a n d s e p a r a t i o n (20) . A n a l y s i s by a m o d i f i e d a l k a l i n e u n w i n d i n g a s s a y (21) demonstrated t h a t c h e m i c a l l y i n d u c e d DNA s t r a n d breakage c o u l d be d e t e c t e d i n b l u e g i l l s u n f i s h and f a t h e a d minnows exposed t o c h r o n i c , low l e v e l s o f BaP. T h i s t e c h n i q u e has been u s e d t o determine t h e r e l a t i v e DNA damage, as measured by s t r a n d breakage, i n b l u e g i l l s u n f i s h t a k e n from s e v e r a l s i t e s i n White Oak Creek, w h i c h i s a s m a l l stream t h a t f l o w s t h r o u g h t h e Department o f Energy r e s e r v a t i o n a t t h e Oak Ridge N a t i o n a l L a b o r a t o r y and t e r m i n a t e s i n White Oak Lake. B i o m o n i t o r i n g a c t i v i t i e s a r e b e i n g c o n d u c t e d i n t h i s system ( 2 7 ) . The d a t a i n T a b l e IV show t h a t t h e i n t e g r i t y o f t h e DNA o f t h e f i s h i n t h e White Oak Creek System, e x c e p t f o r those a t t h e 3.0-3.4 km s i t e , i s s i m i l a r t o t h a t o f f i s h t a k e n from a c o n t r o l s i t e (Brushy F o r k Creek, which i s l o c a t e d some d i s t a n c e from t h e ORNL r e s e r v a t i o n ) f o r b o t h t h e S p r i n g and F a l l c o l l e c t i o n t i m e s . A d d i t o n a l s t u d i e s w i t h f i s h c o l l e c t e d a t t h e 3.0-3.4 km s i t e a r e i n p r o g r e s s . These d a t a , a l t h o u g h p r e l i m i n a r y , i n d i c a t e t h e p o t e n t i a l f o r measuring DNA damage as a g e n e r a l b i o l o g i c a l marker o f exposure o f i n d i g e n o u s a n i m a l s t o contaminants t h a t o c c u r i n t h e i r environment. Hemoglobin M o d i f i c a t i o n . Because i t meets a number o f e s s e n t i a l r e q u i r e m e n t s , hemoglobin has been proposed as an a l t e r n a t i v e c e l l u l a r macromolecule t o DNA f o r e s t i m a t i n g t h e i n v i v o dose o f

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

7. SHUGARTETAL.

Biological Markers in Animals

T a b l e IV.

R e l a t i v e DNA S t r a n d Breaks i n B l u e g i l l from the White Oak Creek Watershed

Site

n* Spring

W.O.C.(3.0-3.4 km) W.0.C.(2.6 km) White Oak Lake Brushy F o r k Creek

1987

4.77 0.68 0.75 0.91

F a l l 1987

2..01 0..03 1..13 1..15

* n - ( I n F / l n F ) - 1 - number o f b r e a k s p e r a l k a l i n e u n w i n d i n u n i t f DNA (se r e f 20.21) F represents form from f i s s e n t s f r a c t i o n o f DNA i n double s t r a n d e d form from f i s h a t M e l t o n Branch, a s m a l l stream t h a t e n t e r s the White Oak Creek (W.O.C.) a t 2.5 km. A minimum o f f i v e f i s h were a n a l y z e d a t each s i t e . s

c

s

c h e m i c a l s subsequent t o exposure (22.-25.). F i r s t , i t has r e a c t i v e n u c l e o p h i l i c s i t e s and the r e a c t i o n p r o d u c t s w i t h e l e c t r o p h i l i c agents a r e s t a b l e . Over f i f t y compounds t o date have been shown t o y i e l d c o v a l e n t r e a c t i o n p r o d u c t s w i t h hemoglobin i n a n i m a l e x p e r i m e n t s . These compounds i n c l u d e r e p r e s e n t a t i v e s from most o f the i m p o r t a n t c l a s s e s o f g e n o t o x i c c h e m i c a l s c u r r e n t l y known. No t e s t e d mutagenic o r c a n c e r - i n i t i a t i n g compound has f a i l e d t o produce c o v a l e n t r e a c t i o n p r o d u c t s w i t h hemoglobin. Secondly, hemoglobin has a w e l l e s t a b l i s h e d l i f e - s p a n , i s r e a d i l y a v a i l a b l e i n humans and a n i m a l s , and i t s c o n c e n t r a t i o n i n an i n d i v i d u a l i s not subject to large v a r i a t i o n . T h i r d l y , m o d i f i c a t i o n of hemoglobin has been shown t o g i v e an i n d i r e c t measure o f the dose o f r e a c t i o n p r o d u c t t h a t b i n d s t o the DNA i n the t a r g e t c e l l s . B i o m o n i t o r i n g o f hemoglobin-bound m e t a b o l i t e s r e p r e s e n t s a n o v e l approach t o the e s t i m a t i o n o f exposure t o p o t e n t i a l l y h a r m f u l c h e m i c a l s ( 2 6 ) . A l t h o u g h these adducts have no p u t a t i v e m e c h a n i s t i c r o l e i n c a r c i n o g e n e s i s , t h e y do r e l a t e q u a n t i t a t i v e l y t o exposure and t o a c t i v a t i o n s i n c e t h e y may approximate the s y s t e m i c dose ( i n v i v o dose) o f a c h e m i c a l . They can be a measure o f the c h e m i c a l ' s g e n o t o x i c potency as w e l l . Furthermore, s i n c e the adducts w h i c h form w i t h hemoglobin a r e s t a b l e and have l i f e spans e q u a l t o t h a t o f the c i r c u l a t i n g e r y t h r o c y t e ( 2 3 ) , q u a n t i t a t i o n o f these adducts can be used t o i n t e g r a t e the dose o b t a i n e d from c h r o n i c l o w - l e v e l types o f exposure w h i c h most l i k e l y o c c u r i n c h e m i c a l l y - p o l l u t e d environments. F i e l d S t u d i e s w i t h T e r r e s t r i a l A n i m a l s . A f l o o d p l a i n on E a s t F o r k P o p l a r Creek (EFPC) was s e l e c t e d as a s t u d y s i t e . EFPC o r i g i n a t e s a t New Hope Pond, a p o i n t source o f i n d u s t r i a l p o l l u t i o n , and the s t u d y s i t e i s about 4 k i l o m e t e r s downstream. The f l o o d p l a i n i s low and the c r e e k p e r i o d i c a l l y o v e r f l o w s , d e p o s i t i n g sediment. There

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

91

92

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

i s abundant v e g e t a t i o n . Measured BaP c o n c e n t r a t i o n s i n the s o i l was 70 nanograms/gram, a l t h o u g h 10 times t h i s amount have been r e p o r t e d . C o n c e n t r a t i o n s i n p l a n t s was l e s s than l / 1 0 t h o f t h a t i n the s o i l . S m a l l animals were t r a p p e d a t t h i s s i t e t o determine the p r a c t i c a l i t y o f u s i n g hemoglobin as a s u i t a b l e b i o l o g i c a l marker o f BaP p o l l u t i o n . P r e l i m i n a r y d a t a (Table V) i n d i c a t e d e t e c t a b l e c o n c e n t r a t i o n s of BaP adducts w i t h hemoglobin were p r e s e n t i n some i n d i v i d u a l s o f s p e c i e s which have i n t i m a t e c o n t a c t w i t h the sediment o r s o i l . The s h o r t t a i l shrew burrows i n t o the ground and e a t s earthworms and i n s e c t s , w h i l e muskrats f e e d on i n v e r t e b r a t e s found i n the c r e e k bed and browse on muddy v e g e t a t i o n a l o n g the c r e e k bank. BaP adducts were n o t d e t e c t a b l e i n h e r b i v o r e s such as the w h i t e - f o o t e d mouse. T a b l e V.

Concentratio Hemoglobin o

Species

f Benzo[a]pyren

Number Analyzed

W h i t e - f o o t e d mouse (Peromvscus leucopus) House mouse (Mus muscuius) S h o r t t a i l shrew ( B l a r i n a brevicauda) B l a c k r a t snake (Elaphe o b s o l e t a o b s o l e t a ) Snapping t u r t l e (Chelvdra serpentina) Norway r a t (Rattus norvepicus) Muskrat (Ondatra z i b e t h i c a )

Adduct

i

th

BaP-hemoglobin Adducts*

15

0,.00

1

0..00

2

0,.35

1

0..00

1

0,.16

1

0,.15

6

0,.23

* BaP adducts t o hemoglobin e x p r e s s e d as nanograms o f t e t r o l 1-1 p e r gram o f hemoglobin. Data t a k e n from ( 2 7 ) . BaP-hemoglobin Adducts i n Humans. Human d a i l y exposure t o BaP i n c r e a s e s d r a m a t i c a l l y i n smokers, as one c i g a r e t t e c o n t a i n s between 10 and 50 nanograms o f BaP. S i n c e the l i f e - s p a n o f a human red b l o o d c e l l i s s e v e r a l months, c o n t i n u a l exposure t o BaP a t one or more packs o f c i g a r e t t e s p e r day s h o u l d r e s u l t i n a measurable s t e a d y s t a t e c o n c e n t r a t i o n o f BaP-hemoglobin a d d u c t s . A p r e l i m i n a r y s t u d y was conducted i n a l i m i t e d p o p u l a t i o n o f humans to determine whether BaP-hemoglobin adducts c o u l d be d e t e c t e d . The d a t a i n T a b l e VI (methodology a c c o r d i n g t o (25,26)) show t h a t adducts were p r e s e n t i n f i v e out o f s i x i n d i v i d u a l s who smoked w h i l e s i m i l a r a n a l y s e s o f non-smokers gave lower l e v e l s r e l a t i v e t o smokers. These d a t a demonstrate the f e a s i b i l i t y o f the t e c h n i q u e ,

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

7. SHUGARTETAL.

Biological Markers in Animals

93

however, i n t r i n s i c f a c t o r s such as age, sex, h e a l t h , and n u t r i t i o n a l s t a t u s , as w e l l as e x t r i n s i c f a c t o r s such as t h e p r e s e n c e o f o t h e r c h e m i c a l s , may i n f l u e n c e t h e p h a r m a c o k i n e t i c s o f BaP metabolism, and t h e i n c i d e n c e o f adducts w i t h hemoglobin. Table VI.

B e n z o [ a j p y r e n e Adducts i n Hemoglobin o f Smokers and Non-smokers

Sample No. Sex Age Race

1 2 3 4 5 6 7 8 9 10 11 12 13 14

m m f f m m f m m m f m f m

23 44 54 56 36 40 28 42 49 27 56 49 50 57

C B C C 0 C C C 0 B C C C C

Smoking Habit*

none none

none f o r 8 y r s none f o r 15 y r s none 20 40 none none f o r 1 y r 20 40 40+

BaP-hemoglobin Adducts**

64 24

73 nd nd 24 159 129 1402*** 468 272 229

C i g a r e t t e s p e r day. ** BaP adducts t o hemoglobin e x p r e s s e d as picograms o f t e t r o l 1-1 p e r gram o f hemoglobin; nd: none d e t e c t e d . *** Numerous f l u o r e s c e n t peaks p r e s e n t i n chromatogram i n a d d i t i o n t o t e t r o l 1-1 peak. X e n o b i o t i c M e t a b o l i s m . X e n o b i o t i c s i n t h e environment, i f t a k e n up by an organisms, may induce microsomal enzymes (mixed f u n c t i o n o x i d a s e s , MFO's) t h a t c a n d e t o x i f y many o f t h e s e compounds (28.). The MFO's c a t a l y z e a v a r i e t y o f r e a c t i o n s , i n c l u d i n g a r o m a t i c and a l i p h a t i c N - h y d r o x y l a t i o n , N - d e a l k y l a t i o n , and O - d e a l k y l a t i o n . P a r a d o x i c a l l y , t h e MFO's may a c t i v a t e c e r t a i n c h e m i c a l s t o t h e i r c a r c i n o g e n i c m e t a b o l i t e ( s ) ( 2 9 ) . The b i o c h e m i c a l systems i n f i s h r e s p o n s i b l e f o r t h e s e types o f r e a c t i o n s a r e s i m i l a r t o t h o s e found i n mammalian s p e c i e s (30,31). The use o f enzyme a s s a y s i n b i o l o g i c a l m o n i t o r i n g programs was r e v i e w e d by Payne (32) f o r d i f f e r e n t p h y l a o f a q u a t i c a n i m a l s and was found t o be u s e f u l i n d e t e c t i n g known p o i n t s o u r c e s o f p o l l u t i o n . F i e l d S t u d i e s i n A q u a t i c Organisms. A c t i v i t i e s o f s e v e r a l MFO parameters have been measured i n f i s h c o l l e c t e d from s t a t i o n s a l o n g E a s t F o r k P o p l a r Creek ( 3 3 ) . A m u l t i v a r i a t e d i s c r i m i n a n t a n a l y s i s was used t o examine t h e p a t t e r n o f s e v e r a l measures o f t h e MFO system ( e t h o x y r e s o r u f i n d e e t h y l a s e , NAD r e d u c t a s e , NADP r e d u c t a s e ,

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cytochrome P-450, and cytochrome D5 l e v e l s ) i n f i s h from the d i f f e r e n t s t a t i o n s . The s t a t i s t i c a l a n a l y s i s ( F i g u r e 1) c o n f i r m t h a t female b l u e g i l l s u n f i s h a t s t a t i o n n o . l (EFPC a t New Hope Pond), a s o u r c e o f i n d u s t r i a l p o l l u t i o n , e x h i b i t e d a s i g n i f i c a n t l y d i f f e r e n t p a t t e r n o f MFO responses t h a n f i s h c o l l e c t e d 20 k i l o m e t e r s down stream ( s t a t i o n no. 5) o r from a p r i s t i n e stream (control). In addition, histopathological analysis of various organs from f i s h a l o n g EFPC showed h i g h e r l e v e l s o f l e s i o n s i n the f i s h closest to s t a t i o n n o . l . These r e s u l t s ( F i g u r e 1) suggest t h a t the MFO enzyme system can be u s e f u l as a b i o l o g i c a l marker i n a m o n i t o r i n g program i f the response o f the system t o e n v i r o n m e n t a l and p h y s i o l o g i c a l i n f l u e n c e s such as temperature, season, f o o d , hormonal ( s e x ) , e t c . , are taken i n t o c o n s i d e r a t i o n . Summary and C o n c l u s i o n s E f f e c t i v e environmenta t r a n s p o r t and f a t e o f contaminants i n n a t u r a l systems ( 3 4 ) . S m a l l mammals can s e r v e as i n d i c a t o r s o f the presence and b i o a v a i l a b i l i t y o f contaminants and s e l e c t e d m o n i t o r i n g s p e c i e s can be used t o measure the a c c u m u l a t i o n o f c o n t a m i n a n t s . Furthermore, knowledge o f f o o d h a b i t s and h a b i t a t may i n d i c a t e pathways o f c o n t a m i n a n t s t o organisms M o n i t o r i n g b i o l o g i c a l e n d p o i n t s i n w i l d a n i m a l s can be used as an a l t e r n a t i v e t o e x t e n s i v e s a m p l i n g and c h e m i c a l a n a l y s e s , p a r t i c u l a r l y where a l a r g e a r e a i s i n v o l v e d and the o r i g r n o r e x t e n t o f c o n t a m i n a t i o n i s unknown o r p o o r l y d e f i n e d . E v i d e n c e o f exposure o f b i o t a as d e t e r m i n e d by b i o l o g i c a l m o n i t o r i n g c o u l d h e l p t o e s t a b l i s h the need f o r c l e a n u p . Our l a b o r a t o r y has been i n t e r e s t e d i n d e v i s i n g methods f o r d e t e c t i n g the exposure o f l i v i n g organisms t o hazardous c h e m i c a l s and p h y s i c a l agents i n the environment. I n i t i a l studies with c h e m i c a l d o s i m e t r y a n a l y s i s began w i t h the development o f a f l u o r e s c e n c e / H P L C a s s a y f o r the d e t e c t i o n and q u a n t i t a t i o n o f BaPDNA adducts w i t h a s e n s i t i v i t y s u f f i c i e n t f o r d e t e c t i n g e n v i r o n m e n t a l exposures. Subsequent r e s e a r c h demonstrated the a p p l i c a b i l i t y o f t h i s t e c h n i q u e t o a n i m a l models, b o t h a q u a t i c and t e r r e s t r i a l , under v a r i o u s e x p e r i m e n t a l c o n d i t i o n s . Furthermore, hemoglobin m o d i f i c a t i o n by c h e m i c a l s was shown t o be e q u i v a l e n t t o DNA b i n d i n g as a d o s i m e t e r . S t u d i e s i n a q u a t i c organisms, b o t h i n the l a b o r a t o r y and i n the f i e l d , have demonstrated the f e a s i b i l i t y o f measuring MFO enzyme a c t i v i t i e s a s s o c i a t e d w i t h the metabolism o f x e n o b i o t i c s , such as BaP, i n c o n j u n c t i o n w i t h n o n - a d d u c t i v e DNA damage as p o t e n t i a l l y u s e f u l b i o m a r k e r s f o r exposure and b i o a v a i l a b i l i t y o f e n v i r o n m e n t a l pollutants. Other b i o l o g i c a l markers a r e c u r r e n t l y b e i n g i n v e s t i g a t e d f o r t h e i r c a p a c i t y t o d e t e c t and q u a n t i t a t e exposure t o c o n t a m i n a n t s . These i n c l u d e : (a) parameters o f the immune response ( i n t e r f e r o n i n d u c t i o n , a n t i b o d y - f o r m i n g c e l l s , and lymphocyte response t o m i t o g e n s ) ; (b) i n d u c t i o n o f m e t a l - b i n d i n g p r o t e i n s ; and, (c) minor n u c l e o s i d e c o n t e n t o f DNA.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

SHUGART ET AL.

Biological Markers in Animals

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FEMALE BLUEGILLS (WINTER)

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F i g u r e 1. S e g r e g a t i o n o f i n t e g r a t e d v a r i a b l e groups by d i s c r i m i n a n t a n a l y s i s u s i n g s e v e r a l measures o f t h e MFO system. C i r c l e s r e p r e s e n t 95% c o n f i d e n c e r a d i i o f c o n t r o l and r e s p e c t i v e t e s t s t a t i o n s . S t a t i o n no. 1 (New Hope Pond) i s t h e headwaters o f E a s t Fork P o p l a r Creek; i n c r e a s i n g s t a t i o n numbers a r e f u r t h e r down stream. Data t a k e n from ( 3 3 ) .

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Acknowledgments Portions of this work were sponsored by the Department of Environmental Management in the Health, Safety, Environment and Accountability Division of the Oak Ridge Y-12 Plant; and the ORNL Director's R&D Program. The Oak Ridge Y-12 Plant and the Oak Ridge National Laboratory are operated by Martin Marietta Energy Systems, Inc., under contract DE-AC05-84OR21400. Environmental Sciences Division, Oak Ridge National Laboratory Publication No. 3088. Literatute Cited 1. Beardsley, A., Vogg, J., Beckett, P.H.T., and Sanso, B.F. Environ. Pollut. 1978, 16, 65-71. 2. Maugh, T.H. Science 1984, 219, 1032-1033. 3. Perera, F.P., Poirer M.C. Yuspa S.H. Nakayama J. Jaretski, A., Curnern I.B. Carcinogenesi 4. Montesano, R., Rajewksy, M.F., Pegg, A.F., and Miller, E. Cancer Res. 1982, 42, 5236-5239. 5. Gelboin, H., and T'so, P.O.P. Polycyclic Hydrocarbons and Cancer: Molecular Biology and Environment: Academic: New York, 1979. 6. Harvey, R.G. Am. Scientist 1982, 70, 386-393. 7. Wogan, G.N., and Gorelick, N.J. Environ. Health Perspec. 1985, 62, 5-18. 8. Shamsuddin, A.K.M., Sinopoli, N.J., Hemminki, K., Boesch, R.R., and Harris, C.C. Cancer Res. 1985, 45, 66-68. 9. Rahn, R., Chang, S., Holland, J.M., and Shugart, L.R. Biochem. Biophys. Res. Commun. 1982, 109, 262-269. 10. Shugart, L.R., Holland, J.M., and Rahn, R. Carcinogenesis 1983, 4, 195-199. 11. Shugart, L.R., J. Toxicol. Environ. Health 1985, 15, 255-263. 12. Shugart, L.R., and Kao, J. Environ. Health Perspec. 1985, 62, 223-226. 13. Shugart, L.R., McCarthy, J.F., Jimenez, B.D., and Daniel, J. Aquatic Toxicol. 1987, 9, 319-325. 14. Theriault, G., and Tremblay, C. The Lancet 1984, 1, 947-950. 15. Martineau, D., Legace, A., Beland, P., Higgins, R., Armstrong, D., and Shugart, L.R. Proc. 7th Biennial Conf. on Biology of Marine Mammals 1987, p 45. 16. Phillip, D.H. Nature 1983, 303, 468-472. 17. Varanasi, U., Stein, J.E., and Hom, T. Biochem.Biophys.Res. Commun. 1981, 103, 780-787. 18. Stowers, S.J., and Anderson, M.W. Chem.-Biol. Interact. 1984, 51, 151-166. 19. Stowers, S.J., and Anderson, M.W. Environ. Health Perspec. 1985, 62, 31-39. 20. Rydberg, B. Rad. Res. 1975, 61, 274-287. 21. Shugart, L.R. Mar. Environ. 1988, 24, 321-325. 22. Osterman-Golkar, S., Ehrenberg, L., Segebrack, D., and Halstrom, I. Mut. Res. 1976, 34, 1-20. 23. Calleman, C.J. Ph.D. Thesis Department of Radiobiology, University of Stockholm, Sweden, 1984.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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24. 25. 26. 27.

28. 29. 30. 31. 32. 33. 34.

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Shugart, L.R. Toxicol. 1985, 34, 211-220. Shugart, L.R. Anal. Biochem. 1986, 152, 365-369. Pereira, M.A., and Chang, L. In: Banbury Report #13; Bridges, B.A., Butterworth, B.E., and Weinstein, I.B., Eds.: Cold Spring Harbor Laboratory, New York, 1982; pp 177-187. Walton, B.T., Talmage, S.s., and Meyers, L.J. In: First Annual Report on the ORNL Biological Monitoring and Abatement Program: Loar, J.M., Ed.; ORNL-TM-10399, 1987, Oak Ridge National Laboratory, Oak Ridge, TN; pp 234-244. Parke, V.D. J. Aquatic Toxicol. 1981, 1, 367-376. Cummings, S.W., and Prough, R.A. In: Biological Basis of Detoxification: Caldwell, J., and Jakoby, W.B., Eds.: Academic; New York, 1983; pp 1-30. Bend, J.R., and James, M.O. Biochem. Biophys. Perspec. Mar. Biol. 1979, 4, 125-188. Chambers, J.E., and Yarbrough J.D Comp Biochem Physiol 1976, 55c, 77-84 Payne, J.F., In: Ecologica Environment: Personne, E., Jasper, E., and Clares, C., Eds.; State University of Ghant, Belgium, 1984; pp 625-655. Loar, J.M., (Ed.) 1988. First Annual Report on the Y-12 Plant Biological Monitoring and Abatement Program. Draft ORNL/TM. Oak Ridge National Laboratory, Oak Ridge, TN. Wren, C.D. Environ. Monit. Assess. 1986, 6, 127-144.

RECEIVED June 1, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 8

Use of Urine for Monitoring Human Exposure to Genotoxic Agents 1

1,2

1

Richard H. C. San , Miriam P. Rosin , Raymond H. See , Bruce P. Dunn , and Hans F. Stich 1

1

1

Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada School of Kinesiology, Simon Fraser University, Burnaby, BC V5A 1S6, Canada We have recently assessed the feasibility of using two approaches for biologica individuals: (a) components in urine specimens, and (b) the analysis of urothelial cells for the presence of chromosomal damage, as measured by the frequency of micronuclei. Urine specimens were collected from non-smoking orchardists in the Okanagan Valley, B.C. during the pre-spraying and pesticide spraying periods of the fruit growing season. The urine was concentrated by preparative reversed-phase high-pressure liquid chromatography, and assayed for chromosome-damaging activity using Chinese hamster ovary cell cultures. A significant elevation in chromosome-damaging activity was observed only in the urine collected during the spraying period. Samples from the same individuals obtained during the pre-spraying period did not differ from non-smoking controls. Urothelial cells collected by centrifugation of urine from a single void proved to be insufficient in number for analysis of micronuclei. This approach may be applicable i f multiple urine samples are obtained. A brief description of our experience with micronuclei analysis in urothelial cells from individuals in other populations is also presented.

2

Short-term in vitro assays of pure chemicals have aided in the dis­ covery of the genotoxic action of many chemicals. However, such tests do not permit the assessment of human exposure to environmental agents. The tests also do not simulate the number of complex ways in which chemical agents may interact in biological systems. At present, two methods are widely applicable for monitoring the effects of genotoxic chemicals on exposed populations. One method involves the analysis of in vitro target indicators such as microbial or mammalian cells for genotoxic damage after exposure to body fluids (e.g., urine) from exposed individuals. The other approach involves the analysis of cells and tissues of the exposed individual for evidence of cytogenetic damage such as micronuclei or chromosomal aberrations. c

0097-6156/89/0382-0098$06.00/0 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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The r a t i o n a l e b e h i n d the a n a l y s i s o f u r i n e i s based on the knowledge t h a t i n g e s t i o n o r m e t a b o l i c t r a n s f o r m a t i o n o f endogenous and exogenous s u b s t a n c e s may r e s u l t i n the e x c r e t i o n o f m e t a b o l i t e s i n t o t h e u r i n e (_1). The d e t e c t i o n o f the m e t a b o l i t e s i n u r i n e would i n d i c a t e t h a t a b s o r p t i o n o f the p r e c u r s o r compound has o c c u r r e d , and t h a t c e l l s i n v a r i o u s organs a r e a t r i s k o f exposure d u r i n g s y s t e m i c d i s t r i b u t i o n o f the p a r e n t compound. U n f o r t u n a t e l y , t h e concent r a t i o n o f g e n o t o x i c m e t a b o l i t e s i n the u r i n e may be r e l a t i v e l y low, r e q u i r i n g e x t r a c t i o n and c o n c e n t r a t i o n p r o c e d u r e s f o r adequate d e t e c t i o n . T h i s d i f f i c u l t y was a l l e v i a t e d by Yamasaki and Ames (2^) , who used n o n - p o l a r A m b e r l i t e XAD-2 r e s i n t o c o n c e n t r a t e compounds from the u r i n e o f c i g a r e t t e smokers. S i n c e t h e n , t h i s approach has been used t o examine many t y p e s o f exposure t h r o u g h l i f e s t y l e (_3) , d i e t ( 4 - 6 ) , o c c u p a t i o n (7-11), and m e d i c a l t r e a t m e n t (12,13). R e c e n t l y , p r e p a r a t i v e r e v e r s e d - p h a s e h i g h - p r e s s u r e l i q u i d chromat o g r a p h y was shown t o be s u p e r i o r t o XAD-2 r e s i n s i n terms o f p e r formance and r e c o v e r y i t o x i c c o n s t i t u e n t s (14) The m i c r o n u c l e u s t e s t has been s u c c e s s f u l l y a p p l i e d t o e x f o l i a t e d u r i n a r y b l a d d e r c e l l s o f smokers and c o f f e e d r i n k e r s ( 1 5 ) , as w e l l as o f b i l h a r z i a l p a t i e n t s (16). M i c r o n u c l e i are a c e n t r i c chromosome fragments d e r i v e d from a b e r r a n t chromosomes (17). Their p r e s e n c e i n the c y t o p l a s m o f e x f o l i a t e d c e l l s as D N A - c o n t a i n i n g bodies i n d i c a t e s the c a p a c i t y of genotoxins t o i n f l i c t genetic damage on the d i v i d i n g b a s a l c e l l s o f the e p i t h e l i u m (15) . An i n c r e a s e i n the m i c r o n u c l e u s f r e q u e n c y o f the b l a d d e r c e l l s , t o g e t h e r w i t h a d e m o n s t r a t i o n o f g e n o t o x i c a c t i v i t y i n the u r i n e , would p r o v i d e s t r o n g e v i d e n c e o f exposure t o e n v i r o n m e n t a l c o n t a m i n a n t s . I n t h i s paper we r e p o r t on our s t u d y o f u r i n e g e n o t o x i c i t y and micronucleus frequency i n e x f o l i a t e d u r i n a r y bladder c e l l s of i n d i v i d u a l s o c c u p a t i o n a l l y exposed t o p e s t i c i d e s . Methodology Study Groups. In t h i s s t u d y , u r i n e samples were c o l l e c t e d from 21 o r c h a r d i s t s ( a l l non-smokers) i n the Okanagan V a l l e y , B.C., when they were engaged i n t h e a p p l i c a t i o n o f p e s t i c i d e s d u r i n g the f r u i t growing season. As c o n t r o l s , u r i n e was c o l l e c t e d from t h e s e same i n d i v i d u a l s d u r i n g the p r e - s p r a y i n g as w e l l as the p o s t - s p r a y i n g seasons. I n a d d i t i o n , 16 i n d i v i d u a l s from an a g r i c u l t u r a l r e s e a r c h s t a t i o n i n the Okanagan r e g i o n were r e c r u i t e d t o p r o v i d e u r i n e samples d u r i n g the same t i m e p e r i o d as t h e o r c h a r d i s t s . As c o n t r o l s o u t s i d e t h e f r u i t g r o w i n g r e g i o n , non-smoking i n d i v i d u a l s from Vancouver and Grand F o r k s , B.C. were r e c r u i t e d t o p r o v i d e one u r i n e specimen. C o l l e c t i o n of U r i n e Samples. I t was d e c i d e d t h a t a l o n g i t u d i n a l study would be most i d e a l f o r t h i s p r o j e c t s i n c e v a r i a b l e s such as m e t a b o l i c and l i f e s t y l e d i f f e r e n c e s would be e s s e n t i a l l y e l i m i n a t e d . A t l e a s t one s i n g l e u r i n e sample was c o l l e c t e d from each o f the o r c h a r d i s t s d u r i n g the peak p e s t i c i d e s p r a y i n g season. Initially, the specimen c o n s i s t e d o f a l l v o i d s c o l l e c t e d between 16 and 24 hours a f t e r p e s t i c i d e a p p l i c a t i o n . Based on the o b s e r v a t i o n s made on t h e s e samples, i t was s u s p e c t e d t h a t t h e c o l l e c t i o n o f u r i n e a t 16 t o 24 hours p o s t - s p r a y i n g may have passed the t i m e a t w h i c h p e s t i c i d e s

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are m e t a b o l i z e d and e x c r e t e d . C o n s e q u e n t l y , a l l u r i n e specimens were c o l l e c t e d from the o r c h a r d i s t s w i t h i n t h e e n t i r e f i r s t 8 h o u r s a f t e r p e s t i c i d e a p p l i c a t i o n . A l a t e a f t e r n o o n sample was a l s o c o l l e c t e d from the same i n d i v i d u a l s d u r i n g t h e p r e - s p r a y i n g season t o s e r v e as a c o n t r o l . U r i n e samples from a l l c o n t r o l groups were c o l l e c t e d l a t e i n t h e a f t e r n o o n (4 t o 8 p.m.). A l l u r i n e specimens were c o l l e c t e d i n s t e r i l e 500-ml p o l y ­ e t h y l e n e b o t t l e s (Nalgene, R o c h e s t e r , NY) w i t h o u t any p r e s e r v a t i v e s . B o t t l e s were r e f r i g e r a t e d by t h e p a r t i c i p a n t s d u r i n g the c o l l e c t i o n period u n t i l f u l l . B e f o r e f r e e z i n g , each sample was c e n t r i f u g e d t o obtain e x f o l i a t e d urinary bladder c e l l s . The s u p e r n a t a n t was s t o r e d a t -20°C u n t i l a n a l y s i s . The volume, pH, and c r e a t i n i n e c o n t e n t o f each u r i n e sample were measured. The c r e a t i n i n e d e t e r m i n a t i o n , o b t a i n e d u s i n g a m o d i f i c a t i o n o f t h e p r o c e d u r e d e s c r i b e d by I o s e f s o h n (18), s e r v e d t o a d j u s t f o r v a r y i n g u r i n e c o n c e n t r a t i o n s and d i f f e r e n c e s i n body w e i g h t s between i n d i v i d u a l s Preparation of Concentrate c o n c e n t r a t e d by r e v e r s e d - p h a s e h i g h - p r e s s u r e l i q u i d chromatography u s i n g a Waters Prep LC/System 500A p r e p a r a t i v e l i q u i d chromatograph (Waters S c i e n t i f i c , M i l f o r d , MA) and a Waters Prep Pak 500/C18 r e v e r s e d - p h a s e column. A l l t h e o r g a n i c m a t e r i a l on the column was e l u t e d w i t h 50% a c e t o n e / 5 0 % 2.5 niM p h o s p h o r i c a c i d (v/v) (4_) . The e l u a t e was r o t a r y e v a p o r a t e d a t 40°C t o remove t h e a c e t o n e , n e u t r a l ­ i z e d w i t h 1 M sodium h y d r o x i d e , and f r e e z e - d r i e d . A f t e r d r y i n g , the powdered u r i n e e x t r a c t was s t o r e d a t -20°C and r e d i s s o l v e d i n d o u b l e d i s t i l l e d water i m m e d i a t e l y b e f o r e use. Assay f o r C l a s t o g e n i c (Chromosome-Damaging) A c t i v i t y i n U r i n e . Urine e x t r a c t s were a s s a y e d f o r t h e i r c a p a c i t y t o induce chromosome and c h r o m a t i d damage i n c u l t u r e d C h i n e s e hamster o v a r y (CHO) fibroblasts. D i l u t i o n s o f t h e u r i n e e x t r a c t s were p r e p a r e d t o o b t a i n v a r i o u s c o n c e n t r a t i o n s o f c r e a t i n i n e e q u i v a l e n c e (14). Following addition o f t h e u r i n e e x t r a c t f o r 3 h o u r s , t h e CHO c e l l s were i n c u b a t e d f o r a f u r t h e r 16 h o u r s , t h e n a r r e s t e d a t metaphase w i t h c o l c h i c i n e f o r 3 h o u r s , h a r v e s t e d , s t a i n e d , and a n a l y z e d f o r the p r e s e n c e o f chromo­ some as w e l l as c h r o m a t i d b r e a k s and exchanges (19). Using t h i s e x p e r i m e n t a l p r o t o c o l , t h e c y t o g e n e t i c damage o b s e r v e d was p r e ­ d o m i n a n t l y (^96%) o f the c h r o m a t i d t y p e . Analysis of Micronuclei i n E x f o l i a t e d U r o t h e l i a l C e l l s . E x f o l i a t e d u r o t h e l i a l c e l l s were o b t a i n e d from f r e s h l y c o l l e c t e d u r i n e samples by c e n t r i f u g a t i o n , f i x e d i n e t h a n o l / g l a c i a l a c e t i c a c i d (3:1, v / v ) , dropped onto p r e c l e a n e d s l i d e s , a i r - d r i e d o v e r n i g h t , and s t a i n e d a c c o r d i n g t o t h e p r o c e d u r e s d e s c r i b e d by S t i c h e t a l . (20). A minimum o f 300 i n t a c t u r o t h e l i a l c e l l s were a n a l y z e d t o d e t e r m i n e the f r e q u e n c y o f m i c r o n u c l e a t e d c e l l s (20-22). S t a t i s t i c a l A n a l y s i s . A l l s t a t i s t i c a l comparisons were p e r f o r m e d u s i n g n o n - p a r a m e t r i c methods. The c l a s t o g e n i c a c t i v i t y o f u r i n e samples from t h e a g r i c u l t u r a l r e s e a r c h s t a t i o n workers b e f o r e and d u r i n g p e s t i c i d e exposure was e v a l u a t e d f o r s i g n i f i c a n c e u s i n g the Wilcoxon paired-sample t e s t . Tukey's m u l t i p l e comparisons t e s t was used t o e v a l u a t e d i f f e r e n c e s i n the c l a s t o g e n i c a c t i v i t y o f u r i n e from o r c h a r d i s t s d u r i n g d i f f e r e n t p e s t i c i d e exposure p e r i o d s . This t e s t was a l s o used t o examine d i f f e r e n c e s i n the mean c l a s t o g e n i c

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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a c t i v i t y among t h e t h r e e s t u d y groups ( o r c h a r d i s t s , a g r i c u l t u r a l r e s e a r c h s t a t i o n w o r k e r s , and r e f e r e n c e c o n t r o l s ) . The Mann-Whitney U t e s t was used t o e v a l u a t e u r i n a r y c r e a t i n i n e v a l u e s and u r i n a r y pH. Results C l a s t o g e n i c A c t i v i t y i n U r i n e from R e f e r e n c e C o n t r o l Group. The u r i n e o f t h e r e f e r e n c e c o n t r o l group e x h i b i t e d low b u t s i g n i f i c a n t l e v e l s o f c l a s t o g e n i c a c t i v i t y compared t o those found f o r c o n t r o l d i s t i l l e d water c o n c e n t r a t e s (P<0.001; Mann-Whitney U t e s t ) (Table I). D o s e - r e l a t e d i n c r e a s e s i n c l a s t o g e n i c a c t i v i t y were o b s e r v e d f o r many u r i n e e x t r a c t s (data n o t shown). C l a s t o g e n i c r e s p o n s e s were g e n e r a l l y found a t c o n c e n t r a t i o n s between 2.0 and 5.0 mg/ml c r e a t i n i n e equivalence. Beyond t h i s c o n c e n t r a t i o n r a n g e , CHO c e l l s showed evidence o f m i t o t i c i n h i b i t i o n i n a d d i t i o n t o t o x i c i t y , t h e l a t t e r i n d i c a t e d by a d e c r e a s e i n c e l l number and t h e presence o f pycnotic nuclei. T a b l e I . E f f e c t o f P e s t i c i d e Exposure on U r i n e C l a s t o g e n i c i t y P e r c e n t Metaphases w i t h Chromatid A b e r r a t i o n s (Mean±S.D.) 5..2±2.3*> a

Group R e f e r e n c e c o n t r o l s (n=22) Research s t a t i o n personnel ( n = l l ) : Before spraying 5..4±2.0 During spraying 6..3±3.1 (P>0.05) O r c h a r d i s t s (n=22): 4..9±3.5 Before spraying During spraying: c d 19..9±10.2 (P<0.001) ' Afternoon v o i d Next morning v o i d 7..1±3.9 (P>0.05) D i s t i l l e d water c o n c e n t r a t e c o n t r o l s 1.,0±1.0 ^Average o f t h e maximum v a l u e o b t a i n e d f o r each i n d i v i d u a l o v e r t h e dose range t e s t e d (1.0 t o 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e ) . ^ S i g n i f i c a n t i n c r e a s e (P<0.001,- Mann-Whitney U t e s t ) compared t o value f o r d i s t i l l e d water concentrates. S t a t i s t i c a l s i g n i f i c a n c e o f d i f f e r e n c e between p r e - s p r a y i n g and s p r a y i n g u r i n e samples, as d e t e r m i n e d by t h e W i l c o x o n p a i r e d - s a m p l e test. ^ H i g h l y s i g n i f i c a n t (P<0.001) r e l a t i v e t o v a l u e o b t a i n e d f o r t h e r e f e r e n c e c o n t r o l group, as d e t e r m i n e d by t h e Mann-Whitney U t e s t . c

c

F i g u r e 1-F d e s c r i b e s t h e d i s t r i b u t i o n o f u r i n e c l a s t o g e n i c i t y among t h e r e f e r e n c e c o n t r o l i n d i v i d u a l s a t a c r e a t i n i n e e q u i v a l e n c e o f 4.0 mg/ml. A t t h i s c o n c e n t r a t i o n , t h e u r i n e e x t r a c t s o f 76% o f the s u b j e c t s d i d n o t induce more t h a n 5% metaphases w i t h c h r o m a t i d a b e r r a t i o n s . The u r i n e e x t r a c t s o f 19% o f t h e i n d i v i d u a l s demonstrat e d c l a s t o g e n i c a c t i v i t y between 6 and 10% metaphases w i t h c h r o m a t i d a b e r r a t i o n s , w h i l e o n l y 5% o f t h e c o n t r o l group s u b j e c t s showed 11 t o 15% metaphases w i t h c h r o m a t i d a b e r r a t i o n s . The maximum c l a s t o g e n i c a c t i v i t y found o v e r t h e dose range t e s t e d (1.0 t o 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e ) i s shown f o r each i n d i v i d u a l i n F i g u r e 2-C. F o r t h e group, a mean p e r c e n t a g e o f 5.2±2.3 metaphases c o n t a i n e d a t l e a s t one c h r o m a t i d a b e r r a t i o n . These f i g u r e s s t r o n g l y suggest (P<0.001; Mann-Whitney U t e s t ) t h a t

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

102

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

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F i g u r e 1. Frequency d i s t r i b u t i o n o f the c l a s t o g e n i c a c t i v i t y o f u r i n e e x t r a c t s from o r c h a r d i s t s (A-C) and c o n t r o l s (D-F), measured as t h e c a p a c i t y t o i n d u c e c h r o m a t i d a b e r r a t i o n s i n C h i n e s e hamster o v a r y (CHO) c e l l c u l t u r e s . U r i n e samples were o b t a i n e d from o r c h a r d i s t s p r i o r t o s p r a y i n g (A) and on two o c c a s i o n s d u r i n g the s p r a y i n g season: 6-8 hours p o s t - s p r a y i n g ( B ) , and 16-24 h o u r s p o s t s p r a y i n g (C). V a l u e s shown i n D a r e from r e s e a r c h s t a t i o n p e r s o n n e l sampled d u r i n g the p r e - s p r a y i n g p e r i o d , w i t h E d i s p l a y i n g t h e s e same i n d i v i d u a l s d u r i n g the s p r a y i n g p e r i o d . V a l u e s shown i n F a r e from u r i n e samples t a k e n from i n d i v i d u a l s i n a n o n - a g r i c u l t u r a l a r e a . A l l samples were measured a t 4.0 mg/ml c r e a t i n i n e e q u i v a l e n c e .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8. SAN ET AL.

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F i g u r e 2. I n d i v i d u a l v a r i a t i o n s i n t h e c l a s t o g e n i c a c t i v i t y o f u r i n e e x t r a c t s from t h e o r c h a r d i s t s (A) and c o n t r o l groups (B and C), Each b a r r e p r e s e n t s t h e maximum p e r c e n t a g e o f metaphases w i t h c h r o m a t i d a b e r r a t i o n s i n CHO c e l l c u l t u r e s o b t a i n e d o v e r t h e dose range t e s t e d (1.0 t o 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e ) . Values shown i n B were o b t a i n e d from u r i n e samples o f i n d i v i d u a l s a t t h e a g r i c u l t u r a l r e s e a r c h s t a t i o n , w h i l e C g i v e s c o n t r o l v a l u e s from u r i n e o f i n d i v i d u a l s from a n o n - a g r i c u l t u r a l a r e a .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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r e l a t i v e t o t h e b a s e l i n e l e v e l s o f c l a s t o g e n i c a c t i v i t y observed w i t h d i s t i l l e d water c o n c e n t r a t e s , u r i n e c o n t a i n s m a t e r i a l s capable o f i n d u c i n g g e n e t i c damage (Table I ) . C l a s t o g e n i c A c t i v i t y i n U r i n e from A g r i c u l t u r a l Research S t a t i o n P e r s o n n e l . U n l i k e t h e r e f e r e n c e c o n t r o l group, where p e s t i c i d e exposure was known t o be s m a l l o r n o n - e x i s t e n t , i n d i v i d u a l s from t h e a g r i c u l t u r a l r e s e a r c h s t a t i o n may have e x p e r i e n c e d some exposure t o p e s t i c i d e s d u r i n g t h e s p r a y i n g p e r i o d due t o t h e p o s s i b l e d r i f t o f p e s t i c i d e s from t h e s i t e o f a p p l i c a t i o n . U r i n e samples o b t a i n e d from 11 non-smoking a g r i c u l t u r a l r e s e a r c h s t a t i o n p e r s o n n e l d u r i n g t h e p r e - s p r a y i n g and s p r a y i n g p e r i o d s were c o n c e n t r a t e d as d e s c r i b e d p r e v i o u s l y , and assayed f o r t h e p r e s e n c e o f c l a s t o g e n i c m a t e r i a l s . For t h e m a j o r i t y o f u r i n e e x t r a c t s , d o s e - r e l a t e d r e s p o n s e s were observed (two examples a r e shown i n F i g u r e s 3-C and 3-D). C l a s t o g e n i c a c t i v i t y was g e n e r a l l y d e t e c t a b l e a t 3.0 mg/ml c r e a t i n i n e equivalence, with signs mg/ml c o n c e n t r a t i o n . The frequency d i s t r i b u t i o n o f u r i n a r y c l a s t o g e n i c a c t i v i t y f o r the p r e - s p r a y i n g and s p r a y i n g p e r i o d s a t 4.0 mg/ml c r e a t i n i n e e q u i v a l e n c e i s shown i n F i g u r e s 1-D and 1-E. L i t t l e d i f f e r e n c e was apparent i n t h e f r e q u e n c i e s d u r i n g t h e p r e - s p r a y i n g and s p r a y i n g p e r i o d s . D u r i n g b o t h these p e r i o d s , o v e r 80% o f t h e i n d i v i d u a l s e x h i b i t e d u r i n e c l a s t o g e n i c i t y w i t h i n t h e range o f 0 t o 5% metaphases w i t h c h r o m a t i d a b e r r a t i o n s . Over b o t h p e r i o d s , no i n d i v i d u a l s showed u r i n e c l a s t o g e n i c i t y o f more t h a n 10% metaphases w i t h c h r o m a t i d aberrations. I l l u s t r a t e d i n F i g u r e 2-B i s t h e observed maximum c l a s t o g e n i c a c t i v i t y over t h e dose range t e s t e d (1.0 t o 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e ) . F o r t h e p r e - s p r a y i n g p e r i o d , an average o f 5.4±2.0 a b e r r a n t metaphases were found f o r t h e u r i n e specimens t e s t e d . D u r i n g t h e s p r a y i n g p e r i o d , t h e u r i n e s demonstrated an average o f 6.3±3.1% metaphases w i t h c h r o m a t i d a b e r r a t i o n s . Comparison o f t h e s e mean v a l u e s by t h e W i l c o x o n p a i r e d - s a m p l e t e s t (23) showed no s i g n i f i c a n t d i f f e r e n c e (P>0.50). I t t h e r e f o r e appears t h a t i n d i r e c t exposure t o p e s t i c i d e s may be i n s u f f i c i e n t t o induce any f u r t h e r chromosome-damaging a c t i v i t y i n t h e u r i n e above t h a t o f n o r m a l / b a s e l i n e l e v e l s . C l a s t o g e n i c A c t i v i t y i n U r i n e from O r c h a r d i s t s ( U r i n e C o l l e c t e d W i t h i n 16 t o 24 Hours o f P e s t i c i d e A p p l i c a t i o n ) . P e s t i c i d e exposure d i d n o t make t h e u r i n e more t o x i c towards t h e CHO c e l l s , s i n c e t h e r e f e r e n c e c o n t r o l u r i n e samples demonstrated t h e same e x t e n t o f toxicity. The frequency d i s t r i b u t i o n o f u r i n a r y c l a s t o g e n i c a c t i v i t y a t 4.0 mg/ml c r e a t i n i n e e q u i v a l e n c e d u r i n g b o t h t h e p r e - s p r a y i n g ( F i g u r e 1-A) and s p r a y i n g ( F i g u r e 1-C) p e r i o d s appears t o be s i m i l a r . D u r i n g these two time p e r i o d s , t h e u r i n e s o f about 90% o f t h e s u b j e c t s were unable t o i n d u c e more t h a n 5% a b e r r a n t metaphases. T h e r e f o r e p e s t i c i d e s p r a y i n g d i d n o t appear t o a f f e c t t h e u r i n a r y c l a s t o g e n i c a c t i v i t y o f t h e o r c h a r d i s t s . The maximum c l a s t o g e n i c a c t i v i t y o b t a i n e d o v e r t h e dose range o f 1.0 t o 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e i s p r e s e n t e d i n F i g u r e 2-A ( b e f o r e s p r a y i n g , and a t 16 t o 24 hours p o s t - s p r a y i n g ) . D e s p i t e i n t r a - and i n t e r - i n d i v i d u a l v a r i a t i o n s , t h e group mean v a l u e s o f c l a s t o g e n i c a c t i v i t y f o r t h e s e two s e t s o f samples were n o t s i g n i f i c a n t l y d i f f e r e n t (Table I ) .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8. SAN ET AL.

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Equivalence

8 (mg/ml)

F i g u r e 3. Dose-response c u r v e s f o r t h e c l a s t o g e n i c a c t i v i t y o f e x t r a c t s p r e p a r e d from u r i n e c o l l e c t e d d u r i n g t h e p r e - s p r a y i n g and s p r a y i n g p e r i o d s from two o r c h a r d i s t s (A and B ) , and two c o n t r o l s from t h e a g r i c u l t u r a l r e s e a r c h s t a t i o n (C and D). The p e r c e n t a g e o f metaphases w i t h c h r o m a t i d a b e r r a t i o n s i n CHO c e l l c u l t u r e s i n d u c e d by u r i n e e x t r a c t s from t h e p r e - s p r a y i n g (o) and s p r a y i n g (A) p e r i o d s a r e shown f o r each s u b j e c t . A l l s u b j e c t s a r e non-smokers.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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T a b l e I I l i s t s t h e d i f f e r e n t t y p e s o f p e s t i c i d e s used by t h e o r c h a r d i s t s d u r i n g t h e days o f u r i n e c o l l e c t i o n . I n many i n s t a n c e s , more t h a n one p e s t i c i d e was a p p l i e d by an i n d i v i d u a l s p r a y e r d u r i n g the course o f t h e day. Many o f t h e s e p e s t i c i d e s p o s s e s s g e n o t o x i c p r o p e r t i e s . W i t h t h e e x c e p t i o n o f one o r two i n d i v i d u a l s , a l l o f t h e o r c h a r d i s t s a p p l i e d a t l e a s t one g e n o t o x i c p e s t i c i d e . However, even though g e n o t o x i c p e s t i c i d e s were used by many o r c h a r d i s t s , no o v e r a l l i n c r e a s e i n u r i n a r y c l a s t o g e n i c a c t i v i t y beyond normal l i m i t s was observed. Table I I .

P e s t i c i d e s Used by t h e O r c h a r d i s t s

Usea Reference Pesticides Genotoxicity 24 Az inpho s-methyl + I + 25 Benomyl F Captan + 26 F Carbaryl I Cyhexatin M + 29 Diazinon I + 30 Dimethoate I Dinitrocresol I ND 31 Dodine F 32 Endosulfan I 28 Ethephon PG 28 Ethion I Nab am F ND + Paraquat 33 H Phosalone I ND 34 + Simazine H 28 Ziram F I , i n s e c t i c i d e ; F, f u n g i c i d e ; M, m i t i c i d e ; PG, p l a n t growth r e g u l a t o r ; H, h e r b i c i d e . k+, p o s i t i v e r e s u l t ; -, n e g a t i v e r e s u l t ; ND, no i n f o r m a t i o n a v a i l a b l e f o r the chemical. b

a

C l a s t o g e n i c A c t i v i t y i n U r i n e from O r c h a r d i s t s (Urine C o l l e c t e d W i t h i n 6 t o 8 Hours o f P e s t i c i d e A p p l i c a t i o n ) . Owing t o t h e l a c k o f an i n c r e a s e i n g e n o t o x i c a c t i v i t y i n u r i n e c o l l e c t e d w i t h i n 16 t o 24 h o u r s o f p e s t i c i d e s p r a y i n g , t h e t i m i n g o f u r i n e c o l l e c t i o n was changed t o w i t h i n 8 hours o f p e s t i c i d e a p p l i c a t i o n . T h i s d e c i s i o n was i n f l u e n c e d by a r e c e n t l y p u b l i s h e d r e p o r t t h a t changes i n u r i n e g e n o t o x i c i t y may be time-dependent (35). Dose-response c u r v e s o f u r i n e samples from two r e p r e s e n t a t i v e p e s t i c i d e s p r a y e r s a r e shown i n F i g u r e s 3-A and 3-B. Compared t o u r i n e from t h e p r e - s p r a y i n g p e r i o d , u r i n e specimens c o l l e c t e d d u r i n g the s p r a y i n g p e r i o d e x h i b i t e d p o t e n t c l a s t o g e n i c a c t i v i t y , as i n d i c a t e d by t h e s h a r p l y i n c r e a s e d frequency o f c h r o m a t i d a b e r r a t i o n s . The c l a s t o g e n i c a c t i v i t y was dose-dependent b u t n o t l i n e a r , and o c c u r r e d o v e r a narrow c o n c e n t r a t i o n range. The predominant t y p e o f damage was c h r o m a t i d exchange, and t h e i r p r e v a l e n c e d i d n o t appear t o change w i t h dose. The p e r c e n t a g e o f metaphases w i t h c h r o m a t i d aberr a t i o n s g e n e r a l l y i n c r e a s e d w i t h u r i n e dose t o a maximum, t h e n dec r e a s e d w i t h a f u r t h e r i n c r e a s e i n dose. I n many c a s e s , t h e h i g h e s t a c t i v i t i e s were found a t near t o x i c u r i n e c o n c e n t r a t i o n s . T o x i c i t y was u s u a l l y noted between 5.0 and 8.0 mg/ml c r e a t i n i n e e q u i v a l e n c e .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8.

SAN ET AL.

Exposure to Genotoxic Agents

107

A t t h e s e c o n c e n t r a t i o n s , a d e c r e a s e i n c l a s t o g e n i c a c t i v i t y was t y p i c a l l y o b s e r v e d s i n c e s e v e r e l y a b e r r a n t c e l l s l y s e r a p i d l y and are not d e t e c t e d . T o x i c i t y was prominent a t h i g h c r e a t i n i n e doses i n u r i n e s c o l l e c t e d d u r i n g b o t h the p r e - s p r a y i n g and s p r a y i n g p e r i o d s . F i g u r e s 1-A and 1-B c o n t r a s t the d i s t r i b u t i o n o f u r i n a r y c l a s t o g e n i c a c t i v i t y f o r the two p e r i o d s a t 4.0 mg/ml c r e a t i n i n e e q u i v a l e n c e . I n the p r e - s p r a y i n g p e r i o d , no i n d i v i d u a l s demonstrated any a c t i v i t y beyond 10% c e l l s w i t h c h r o m a t i d a b e r r a t i o n s . E i g h t y p e r c e n t o f the s u b j e c t s showed 0 t o 5% metaphases w i t h c h r o m a t i d a b e r r a t i o n s . However, a change i n d i s t r i b u t i o n was o b s e r v e d a f t e r exposure t o p e s t i c i d e s . A t 4.0 mg/ml c r e a t i n i n e c o n c e n t r a t i o n , the u r i n e s o f a l a r g e r p r o p o r t i o n o f i n d i v i d u a l s showed the a b i l i t y t o induce h i g h f r e q u e n c i e s o f c h r o m a t i d a b e r r a t i o n s . For example, the u r i n e s o f 38% o f t h e s p r a y e r s , a f t e r p e s t i c i d e usage, demonstrated more t h a n t w i c e the t y p i c a l b a s e l i n e l e v e l s o f a b e r r a t i o n s ( i . e . , 0 t o 5% metaphases w i t h chromatid a b e r r a t i o n s ) The maximum c l a s t o g e n i (1.0 t o 8.0 mg/ml c r e a t i n i n d e p i c t e d i n F i g u r e 2-A. D u r i n g the p r e - s p r a y i n g p e r i o d , an average o f 4.9±3.5% o f the metaphases e x h i b i t e d c h r o m a t i d a b e r r a t i o n s . W i t h exposure t o p e s t i c i d e s , the a b e r r a t i o n f r e q u e n c y was s i g n i f i c a n t l y e l e v a t e d t o 19.9±10.2% c e l l s w i t h c h r o m a t i d a b e r r a t i o n s (P<0.001; W i l c o x o n p a i r e d - s a m p l e t e s t ) . M u l t i p l e exchanges and fragmentation o f the chromosomal complement were f r e q u e n t l y observed. The most commonly used p e s t i c i d e s on the day o f u r i n e c o l l e c t i o n were the i n s e c t i c i d e s a z i n p h o s - m e t h y l and p h o s a l o n e . On a v e r a g e , each s u b j e c t s p r a y e d a t o t a l o f 4.2 h o u r s per day. The Spearman rank c o r r e l a t i o n t e s t i n d i c a t e d no c o r r e l a t i o n between the t o t a l number o f hours o f s p r a y i n g and the o b s e r v e d maximum c l a s t o g e n i c a c t i v i t y (r =0.317; P>0.20). S i n c e many o r c h a r d i s t s used a c o m b i n a t i o n o f p e s t i c i d e s d u r i n g the c o u r s e o f one day, a c o r r e l a t i o n o f a s p e c i f i c p e s t i c i d e w i t h the u r i n a r y c l a s t o g e n i c a c t i v i t y was not f e a s i b l e . However, i t i s w o r t h p o i n t i n g out t h a t f o u r o r c h a r d i s t s , who used o n l y a s i n g l e p e s t i c i d e on t h a t day, demonstrated h i g h u r i n a r y c l a s t o g e n i c a c t i v i t y when s p r a y i n g e i t h e r p h o s a l o n e , s i m a z i n e , o r paraquat. Of the 17 p e s t i c i d e s s p r a y e d on the day o f u r i n e s a m p l i n g , 8 (47%) had been r e p o r t e d t o e x h i b i t g e n o t o x i c a c t i v i t y in vitro. The use o f such g e n o t o x i c p e s t i c i d e s may e x p l a i n the i n c r e a s e i n u r i n a r y c l a s t o g e n i c a c t i v i t y a s s o c i a t e d w i t h p e s t i c i d e exposure. I t s h o u l d be noted t h a t a l l o f t h e above e f f e c t s were o b t a i n e d w i t h o u t m e t a b o l i c a c t i v a t i o n in vitro, s u g g e s t i n g t h a t the c l a s t o g e n i c agents i n the u r i n e were d i r e c t - a c t i n g . The e f f e c t o f i n c l u d i n g an S9 m i c r o s o m a l a c t i v a t i o n system i n the a s s a y on the c l a s t o g e n i c a c t i v i t y has n o t been examined. I n a l a r g e p r o p o r t i o n o f the u r i n e samples t e s t e d , low b u t s i g n i f i c a n t ( r e l a t i v e t o s o l v e n t c o n t r o l s ) l e v e l s o f c l a s t o g e n i c a c t i v i t y were o b s e r v e d i n the u r i n e o f unexposed non-smokers, i n d i c a t i n g the r o l e o f o t h e r f a c t o r s i n t h e appearance o f u r i n e c l a s t o g e n i c i t y . U r i n a r y pH and c r e a t i n i n e d i d n o t d i f f e r among the study groups. s

A n a l y s i s o f M i c r o n u c l e i i n E x f o l i a t e d B l a d d e r C e l l s . An attempt was made t o a n a l y z e the e x f o l i a t e d u r o t h e l i a l c e l l s o f p e s t i c i d e s p r a y e r s f o r t h e presence o f m i c r o n u c l e i , an in vivo i n d i c a t i o n o f g e n o t o x i c damage. U n f o r t u n a t e l y , e x f o l i a t e d u r o t h e l i a l c e l l s i n the u r i n e o f

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male s u b j e c t s a r e n o t abundant, and l a r g e volumes o f u r i n e a r e g e n e r a l l y r e q u i r e d t o o b t a i n an adequate number o f c e l l s . Because of the s c a r c i t y of c e l l s i n the s l i d e preparations, only a l i m i t e d number o f specimens p r o v i d e d s u f f i c i e n t c e l l s f o r a n a l y s i s . As shown i n T a b l e I I I , 3.36% o f t h e e x f o l i a t e d u r o t h e l i a l c e l l s c o l l e c t e d from t h e o r c h a r d i s t s w i t h i n 6 t o 8 h o u r s o f p e s t i c i d e a p p l i c a t i o n were m i c r o n u c l e a t e d . This represents a 5 . 6 - f o l d increase i n micron u c l e a t e d c e l l s compared t o specimens c o l l e c t e d d u r i n g t h e p r e s p r a y i n g p e r i o d . I t s h o u l d be n o t e d t h a t these samples were o b t a i n e d from i n d i v i d u a l s 10 t o 12 days i n t o t h e s p r a y i n g season. Genotoxic damage b y e n v i r o n m e n t a l agents o c c u r s i n d i v i d i n g e p i t h e l i a l c e l l s . I t r e q u i r e s one t o two weeks f o r t h e d a u g h t e r c e l l s t o m i g r a t e up t h r o u g h t h e e p i t h e l i u m and e x f o l i a t e . T a b l e I I I . Frequency o f M i c r o n u c l e a t e d C e l l s from P o p u l a t i o n Groups a t E l e v a t e d R i s k f o r Cancer

Controls Egypt: Schistosoma haematobium i n f e c t i o n

a

0.57 (0.2-0.9)

(n=ll) Vancouver: smokers

cigarette

0.56 (0.0-0.8)

(n=20) Okanagan V a l l e y : p e s t i c i d e sprayers

0.60 (0.2-0.8)

(n=10)

Group

Increase

7.56 (4.7-12.5)

13.3

(n=19) b

3.34 (1.6-6.5)

5.96

(n=20) 3.36 (1.1-5.3)

5.6

(n=5)

F i g u r e s r e p r e s e n t average p e r c e n t a g e o f m i c r o n u c l e a t e d c e l l s , w i t h t h e range g i v e n i n p a r e n t h e s e s . ^40-65 c i g a r e t t e s p e r day.

exfoliated

Discussion U r i n e C l a s t o g e n i c i t y A s s o c i a t e d w i t h P e s t i c i d e Exposure. The c u r r e n t i n v e s t i g a t i o n i n d i c a t e s t h a t i n d i v i d u a l s exposed t o h i g h c o n c e n t r a t i o n s o f p e s t i c i d e s show s u b s t a n t i a l g e n o t o x i c a c t i v i t y i n t h e i r urine. I n c o n t r a s t , p r e - s p r a y i n g u r i n e samples c o l l e c t e d from t h e same s u b j e c t s demonstrated o n l y low " b a s e l i n e " l e v e l s o f c l a s t o g e n i c a c t i v i t y . The mean p e r c e n t a g e o f CHO c e l l s w i t h c h r o m a t i d a b e r r a t i o n s i n c r e a s e d f i v e f o l d f o r t h e group d u r i n g t h e peak s p r a y i n g s e a s o n , s t r o n g l y i m p l y i n g t h e a s s o c i a t i o n o f p e s t i c i d e exposure w i t h the h i g h u r i n a r y c l a s t o g e n i c a c t i v i t y . G e n o t o x i c i t y o f u r i n e from p e s t i c i d e s p r a y e r s has n o t been previously reported. However, e v i d e n c e o f t h e a b i l i t y o f p e s t i c i d e s t o induce c y t o g e n e t i c damage i n humans i s known. Yoder e t a l . (36) n o t e d a marked i n c r e a s e i n t h e c h r o m a t i d l e s i o n s o f lymphocyte c u l t u r e s p r e p a r e d from i n d i v i d u a l s exposed d u r i n g heavy s p r a y i n g p e r i o d s . The a b e r r a t i o n s were p a r t i c u l a r l y s t r i k i n g among w o r k e r s exposed p r i m a r i l y t o h e r b i c i d e s . C r o s s e n e t a l . (37) and D u l o u t e t a l . (38) b o t h found a s i g n i f i c a n t l y e l e v a t e d i n c i d e n c e o f s i s t e r c h r o m a t i d exchanges (another e n d p o i n t f o r g e n o t o x i c damage) i n p e r i p h e r a l lymphocyte chromosomes o f s u b j e c t s o c c u p a t i o n a l l y exposed t o p e s t i c i d e s . However, some o f these f i n d i n g s have n o t been

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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s u b s t a n t i a t e d by o t h e r i n v e s t i g a t o r s (39-42). D i f f e r e n c e s i n t h e degree o f exposure o r t h e u s e o f d i f f e r e n t p e s t i c i d e s may have contributed t o the negative findings. The h i g h u r i n e c l a s t o g e n i c i t y a s s o c i a t e d w i t h p e s t i c i d e exposure was o n l y observed i n u r i n e specimens c o l l e c t e d w i t h i n 6 t o 8 h o u r s o f p e s t i c i d e s p r a y i n g . U r i n e c o l l e c t e d w i t h i n 16 t o 24 hours o f s p r a y i n g demonstrated no s i g n i f i c a n t i n c r e a s e i n c l a s t o g e n i c a c t i v i t y o v e r c o n t r o l b a s e l i n e v a l u e s . I t can be s p e c u l a t e d t h a t the maximum e x c r e t i o n r a t e o f p e s t i c i d e s g e n e r a l l y o c c u r s w i t h i n hours o f exposure. T h i s i n d i c a t e s t h e importance o f c o l l e c t i n g u r i n e a t a p e r i o d when t h e m e t a b o l i t e e x c r e t i o n r a t e i s h i g h o r o p t i m a l . I n t e r i n d i v i d u a l v a r i a t i o n s were f o u n d i n t h e l e v e l s o f c l a s t o g e n i c a c t i v i t y i n u r i n e c o l l e c t e d d u r i n g t h e s p r a y i n g p e r i o d . Some i n d i v i d u a l s had e x t r e m e l y h i g h a c t i v i t y , whereas a few showed moderate t o low l e v e l s o f a c t i v i t y . These v a r i a t i o n s may r e f l e c t d i f f e r e n c e s i n a b s o r p t i o n d i s t r i b u t i o n m e t a b o l i s m and e x c r e t i o n o f p e s t i c i d e s between i n d i v i d u a l s i n d i c a t i o n of the v a r i a t i o n The i n c l u s i o n o f a c o n v e n t i o n a l p a t c h t e s t t o determine p e s t i c i d e exposure may h e l p t o e x p l a i n some o f t h e v a r i a b i l i t y observed i n c l a s t o g e n i c a c t i v i t y (43). H i g h u r i n a r y c l a s t o g e n i c a c t i v i t y may be i n t e r p r e t e d i n one o f two ways. The r a p i d c l e a r a n c e o f c l a s t o g e n s from t h e body may be t a k e n as evidence o f d i m i n i s h e d r i s k . On t h e o t h e r hand, such f a c i l e e x c r e t i o n may mean t h a t organs such as t h e b l a d d e r w i l l be exposed t o h i g h c o n c e n t r a t i o n s o f g e n o t o x i c m a t e r i a l (44).

I t s h o u l d be emphasized t h a t t h e t e s t e d doses o f o r g a n i c m a t e r i a l i n t h e u r i n e e x t r a c t s a r e n o t v e r y d i f f e r e n t from t h o s e present i n urine i n the bladder. C r e a t i n i n e concentrations i n the u r i n e t y p i c a l l y ranged from 0.5 t o 2.5 mg/ml. Many o f t h e u r i n e e x t r a c t s o b t a i n e d from t h e o r c h a r d i s t s d u r i n g t h e s p r a y i n g p e r i o d were a c t i v e in vitro a t 4.0 mg c r e a t i n i n e e q u i v a l e n c e p e r ml o f t i s s u e c u l t u r e medium o r l e s s . Thus t h e c o n c e n t r a t i o n s o f g e n o t o x i c m a t e r i a l s used i n t h e assay a r e r e a s o n a b l y comparable t o t h e concent r a t i o n s t o w h i c h b l a d d e r mucosal c e l l s may be exposed. M o n i t o r i n g U r i n e G e n o t o x i c i t y as a Means o f D e t e c t i n g Exposure t o E n v i r o n m e n t a l Carcinogens and Mutagens: L i m i t a t i o n s and A p p l i c a t i o n s . The p r e s e n t s t u d y demonstrates t h e v a l u e o f u s i n g u r i n e a n a l y s i s as a means o f a s s e s s i n g human exposure t o e n v i r o n m e n t a l contaminants. I t i s n e c e s s a r y t o emphasize here t h a t t h e study i s o n l y an attempt t o make a q u a l i t a t i v e a s s o c i a t i o n between exposure t o a p o t e n t i a l c a r c i n o g e n o r mutagen and a b n o r m a l i t i e s observed i n t h e proposed genetic endpoints. S e v e r a l advantages o f t h e b i o a s s a y o f u r i n e a r e i l l u s t r a t e d i n t h i s investigation. Unlike epidemiological studies, u r i n e a n a l y s i s f o r g e n o t o x i c i t y takes i n t o account the v a r i a t i o n s i n exposure regimes, and thus t h e s c r e e n i n g p r o c e d u r e c a n p i n p o i n t i n d i v i d u a l r i s k (45). W i t h t h e i m p l e m e n t a t i o n o f such s c r e e n i n g t e c h n i q u e s , exposure t o h i g h r i s k environments may be m i n i m i z e d l o n g b e f o r e t h e o c c u r r e n c e o f any i r r e v e r s i b l e p a t h o l o g i c a l changes. The assay o f u r i n e f o r g e n o t o x i c i t y does n o t measure t h e a c t i v i t y o f j u s t one c h e m i c a l component, b u t t h e a c t i v i t y o f a c o m b i n a t i o n o f t h e p e s t i c i d e s per se and t h e m e t a b o l i t e s p r e s e n t i n the u r i n e as a r e s u l t o f complex exposures. Within the urine are thousands o f c h e m i c a l s , some o f which may be i n h i b i t o r s o r enhancers

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o f g e n o t o x i c a c t i v i t y . The assay o f u r i n e samples measures the net e f f e c t o f the c h e m i c a l s p r e s e n t t o g e t h e r , t a k i n g i n t o c o n s i d e r a t i o n the s y n e r g i s t i c o r a n t a g o n i s t i c i n t e r a c t i o n s t h a t may be s i g n i f i c a n t . Another advantage o f a n a l y z i n g human u r i n e i s t h e a c c e s s i b i l i t y o f t h e samples. U r i n e c o l l e c t i o n p r o c e d u r e s are n o n - i n v a s i v e and can be conducted on a r e p e a t e d b a s i s . I n a d d i t i o n , the a n a l y s i s o f u r i n e f o r g e n o t o x i c i t y may be c o u p l e d w i t h q u a n t i t a t i v e c h e m i c a l a n a l y s i s . O v e r a l l , t h e a n a l y s i s o f u r i n e samples i s s i m p l e and r a p i d . U r i n e m o n i t o r i n g i s most u s e f u l i n e v a l u a t i n g human exposure t o m u l t i p l e agents. R a r e l y i s an i n d i v i d u a l ever exposed t o a s i n g l e agent. I n t h e m a j o r i t y o f c a s e s , t h e e x t e n t o f exposure o r the r o u t e (e.g., dermal, r e s p i r a t o r y , i n g e s t i o n ) w i l l not be known. F o r example, i n our i n v e s t i g a t i o n , most s p r a y e r s were exposed t o a v a r i e t y o f p e s t i c i d e s , and i t was unknown whether t h i s m i x t u r e would be g e n o t o x i c in vivo. T h i s q u e s t i o n can be answered by u r i n e genot o x i c i t y assays. The a c t u a l p r e s e n c e o f g e n o t o x i c a c t i v i t y c o i n c i d i n g w i t h t h e time o f exposur e v i d e n c e t h a t exposure ha were t h u s a t r i s k f o r g e n e t i c damage w h i l e the c h e m i c a l s were b e i n g s y s t e m a t i c a l l y d i s t r i b u t e d t h r o u g h o u t t h e body. Thus the b i o a s s a y o f u r i n e i s u s e f u l f o r i d e n t i f y i n g c a r c i n o g e n i c and mutagenic r i s k s a s s o c i a t e d w i t h v a r i o u s work environments. However, the a n a l y s i s o f u r i n e f o r g e n o t o x i c i t y i s not w i t h o u t i t s l i m i t a t i o n s . U n l i k e chemical a n a l y s i s , there i s a lack of s e n s i t i v i t y f o r s p e c i f i c c h e m i c a l s (46). As demonstrated i n t h i s s t u d y , a l t h o u g h c l a s t o g e n i c a c t i v i t y was observed w i t h p e s t i c i d e usage, t h e i d e n t i t y o f t h e c l a s t o g e n s remains unknown. Because o f the unknown n a t u r e o f t h e compounds, t h e assay r e s u l t s can o n l y be i n t e r p r e t e d i n a q u a l i t a t i v e manner i n terms o f exposure assessment. To g a i n more u s e f u l i n f o r m a t i o n ( q u a n t i t a t i v e assessment), c h e m i c a l t e c h n i q u e s (e.g., a n a l y t i c a l h i g h - p r e s s u r e l i q u i d chromatography) must be employed i n c o n j u n c t i o n w i t h t h e e x a m i n a t i o n f o r u r i n e genotoxic i t y . Moreover, our s t u d i e s and t h o s e o f o t h e r s (2,47,48) show t h a t u r i n e a n a l y s i s may be u s e f u l o n l y f o r m o n i t o r i n g r e c e n t exposures. Cumulative exposures cannot be d e t e c t e d (49). Depending on the x e n o b i o t i c , m e t a b o l i t e s may be e l i m i n a t e d i n t o the u r i n e w i t h i n one t o perhaps two days. Beyond t h i s p e r i o d , the l e v e l s o f m e t a b o l i t e s i n the u r i n e may be too low f o r d e t e c t i o n . Thus i t i s n e c e s s a r y t o e i t h e r determine t h e e x c r e t i o n k i n e t i c s o f the m e t a b o l i t e s o r t o o b t a i n m u l t i p l e u r i n e samples a f t e r exposure t o an agent. S i n c e b o t h c h o i c e s are l i k e l y t o be i m p r a c t i c a l , t h e c o l l e c t i o n o f u r i n e a few h o u r s a f t e r exposure o r l a t e i n the e v e n i n g may be a r e a s o n a b l e a l t e r n a t i v e . K r i e b e l e t a l . (_50) found t h a t an e v e n i n g u r i n e sample p r o v i d e d a good e s t i m a t i o n o f t h e mutagen c o n c e n t r a t i o n s o f a 24-hour u r i n e sample. N e v e r t h e l e s s , r e g a r d l e s s o f the sampling schedule u s e d , t h e c o l l e c t i o n o f u r i n e samples f a r beyond t h e o p t i m a l metab o l i t e e x c r e t i o n p e r i o d w i l l r e s u l t i n l e v e l s of genotoxic a c t i v i t y u n d i s t i n g u i s h a b l e from normal background v a l u e s . D e c o m p o s i t i o n o f u r i n a r y c l a s t o g e n s may p r e s e n t a problem i n the a n a l y s i s o f u r i n e . Some m e t a b o l i t e s a r e s h o r t - l i v e d and may never be d e t e c t e d i n the u r i n e . Only t h o s e m e t a b o l i t e s w i t h l o n g h a l f - l i v e s can be assayed. Even s t o r a g e a t -20°C does not ensure the s t a b i l i t y o f the e x t r a c t components. A few i n d i v i d u a l s have i n v e s t i g a t e d the s t a b i l i t y of u r i n e concentrates. P u t z r a t h e t a l . (51) observed t h e

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l o s s o f g e n o t o x i c a c t i v i t y o f a few u r i n e c o n c e n t r a t e s w i t h s t o r a g e . Beek e t a l . (52) a l s o noted decreased g e n o t o x i c a c t i v i t y w i t h r e - u s e d u r i n e e x t r a c t s . I n b o t h c a s e s , t h e a u t h o r s a t t r i b u t e d the l o s s o f a c t i v i t y t o r e p e a t e d f r e e z i n g and thawing o f the u r i n e e x t r a c t s . Genotoxins may a l s o be l o s t through the e x t r a c t i o n and concent r a t i o n procedures. F o r example, w i t h our methods, v o l a t i l e compounds (e.g., n i t r o s a m i n e s ) may be l o s t through the r o t a r y evaporat i o n or f r e e z e - d r y i n g p r o c e s s . Loss o f a c t i v i t y may a l s o o c c u r through l i m i t a t i o n s i n the e x t r a c t i o n p r o c e d u r e s . Because o f the presence o f h i g h s a l t c o n c e n t r a t i o n s i n the u r i n e , h y d r o p h i l i c m a t e r i a l s a r e d i f f i c u l t t o i s o l a t e . T o x i c i t y of the s a l t s t o mammalian c e l l s a l s o makes i t i m p o s s i b l e t o t e s t h y d r o p h i l i c components i n u r i n e . T h i s t o x i c i t y problem was e v i d e n t when f r e e z e - d r i e d u n c o n c e n t r a t e d u r i n e was assayed f o r g e n o t o x i c m a t e r i a l . New p r o c e d u r e s must be developed t o e x t r a c t p o l a r o r g a n i c compounds i n urine. F i n a l l y , c a u t i o n mus t o x i c i t y d a t a . The absenc always n e c e s s a r i l y i n d i c a t e a l a c k o f exposure. M e t a b o l i t e s may be p r e s e n t i n c o n c e n t r a t i o n s below t h e d e t e c t a b l e l i m i t s o f the assay system. I n a d d i t i o n , compounds e x c r e t e d t h r o u g h r o u t e s o t h e r than u r i n e (e.g., l u n g s , f e c e s , sweat) may never be d e t e c t e d by u r i n e a s s a y s . Some c h e m i c a l s may a l s o be b i o t r a n s f o r m e d i n t o p r o d u c t s t h a t cannot be r e a c t i v a t e d in vitro (53). G l u c u r o n i d e and s u l f a t e conj u g a t e s may be r e a d i l y c l e a v e d by the i n c l u s i o n o f 3 - g l u c u r o n i d a s e and s u l f a t a s e enzymes i n t h e assay system (1,2) . I n c o n t r a s t , c h e m i c a l s c o n j u g a t e d t o g l u t a t h i o n e cannot be e a s i l y c l e a v e d t o y i e l d t h e compound i n i t s o r i g i n a l form. Instead, g l u t a t h i o n e conjugates a r e l i k e l y t o be m e t a b o l i z e d such t h a t the g l u t a t h i o n e - m e t a b o l i t e l i n k a g e i s r e t a i n e d (54). T h i s c l a s s o f m e t a b o l i t e s i s not d e t e c t a b l e i n u r i n e c l a s t o g e n i c i t y o r m u t a g e n i c i t y t e s t s (55). Other methods such as those t h a t assay f o r m e r c a p t u r i c a c i d s and o t h e r t h i o e t h e r s must be used t o d e t e c t g l u t a t h i o n e c o n j u g a t e s (56). Another f a c t o r t o c o n s i d e r i s t h a t a l t h o u g h a b s o r p t i o n o f the compound by t h e body may have o c c u r r e d , the s u s p e c t e d agent may not have y e t been m e t a b o l i z e d . T h i s p e r t a i n s , i n p a r t i c u l a r , t o l i p o p h i l i c substances t h a t p e r s i s t i n the body f o r a l o n g p e r i o d o f t i m e . The slow r e l e a s e o f t h e s e compounds from t i s s u e s t o r e s w i l l make t h e i r detection quite d i f f i c u l t . Confounding F a c t o r s A f f e c t i n g the U r i n e C l a s t o g e n i c i t y Assay. The presence o f g e n o t o x i c a c t i v i t y i n u r i n e from c i g a r e t t e smokers has been demonstrated i n s e v e r a l s t u d i e s (2,4,45,51,57,58). F i n d i n g s o f i n c r e a s e d u r i n e m u t a g e n i c i t y r e l a t e d t o p a s s i v e smoking have been r e p o r t e d (2,3,59) . C e r t a i n l y , t h e s e r e s u l t s i l l u s t r a t e the importance o f r e s t r i c t i n g u r i n e s t u d i e s t o non-smokers when a t t e m p t i n g t o demonstrate o c c u p a t i o n a l exposure t o e n v i r o n m e n t a l c h e m i c a l s o t h e r than c i g a r e t t e smoke. Smokers s h o u l d o n l y be used as a p o s i t i v e control population. Low l e v e l s o f u r i n a r y c l a s t o g e n i c a c t i v i t y were d e t e c t e d i n many s u b j e c t s i n t h e p r e s e n t s t u d y , d e s p i t e the absence o f exposure t o p e s t i c i d e s o r c i g a r e t t e smoke. S i m i l a r f i n d i n g s o f low l e v e l s o f u r i n e m u t a g e n i c i t y u n r e l a t e d t o o c c u p a t i o n o r smoking have been r e p o r t e d (45,52,60,61). R e c e n t l y , v a r i o u s d i e t a r y f a c t o r s a f f e c t i n g t h e u r i n e m u t a g e n i c i t y assay have been d i s c o v e r e d , such as i n g e s t i o n

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o f a f r i e d b e e f meal (48) o r a f r i e d pork o r bacon meal (_5) , b u t not microwaved meat (48). U r i n e m u t a g e n i c i t y was a l s o i n c r e a s e d i n another study where s u b j e c t s were r e s t r i c t e d t o a v e g e t a r i a n d i e t c o n s i s t i n g o f soy p r o d u c t s , n u t s , f r u i t s and v e g e t a b l e s (6). These s t u d i e s demonstrate t h a t consumption o f m u t a g e n - c o n t a i n i n g foods may be p a r t l y r e s p o n s i b l e f o r t h e presence o f u r i n e g e n o t o x i c i t y . The use o f m e d i c a t i o n may a l s o be a c o n t r i b u t i n g f a c t o r . P a t i e n t s t r e a t e d w i t h the a n t i t u m o u r d r u g , cyclophosphamide (62), and w i t h the a n t i s c h i s t o s o m a l d r u g s , n i r i d a z o l e and m e t r o n i d a z o l e (63), and p s o r i a t i c p a t i e n t s r e c e i v i n g crude c o a l t a r t h e r a p y have e x h i b i t e d mutagenic a c t i v i t y i n t h e u r i n e (12). Some drugs may s y n e r g i z e t h e mutagenic a c t i v i t y o f u r i n e i n c o m b i n a t i o n w i t h o t h e r agents. F o r example, R e c i o e t a l . (64) noted t h a t t h e use o f an a r t h r i t i c drug ( A s c r i p t i o n A.D.) i n c r e a s e d the mutagenic a c t i v i t y o f the u r i n e o f a c i g a r e t t e smoker f i v e f o l d above t h a t o f u r i n e o f o t h e r smokers. However, th e p o t e n t i a l c o n f o u n d i n e f f e c t f drugs i l l u s t r a t e d by t he abov c a u t i o n i n the i n t e r p r e t a t i o o f low l e v e l s o f c l a s t o g e n i c a c t i v i t y may be t h e r e s u l t o f unknown exposure t o e n v i r o n m e n t a l contaminants such as automobile exhaust (65). I n any u r i n e s t u d y , i t i s v i r t u a l l y i m p o s s i b l e t o c o n t r o l f o r exposure t o such e n v i r o n m e n t a l s u b s t a n c e s . The o n l y s o l u t i o n would be t o a n a l y z e r e p e a t e d u r i n e samplings t o e s t a b l i s h a b a s e l i n e t o account f o r i n t e r f e r i n g exposures. The M i c r o n u c l e u s T e s t on E x f o l i a t e d Human C e l l s . A l t h o u g h t h i s study demonstrates the p r e s e n c e o f c l a s t o g e n i c a c t i v i t y i n u r i n e e x t r a c t s from o r c h a r d i s t s d u r i n g t h e s p r a y i n g season, the q u e s t i o n a r i s e s as t o whether t h e compounds c a u s i n g t h i s c l a s t o g e n i c a c t i v i t y w i t h in vitro c u l t u r e s would a c t in vivo t o i n d u c e g e n o t o x i c damage. A whole organism may p o s s e s s a c t i v a t i o n / i n a c t i v a t i o n mechanisms which would p r o v i d e p r o t e c t i o n from such g e n o t o x i c components. I n an e f f o r t t o answer t h i s q u e s t i o n , e x f o l i a t e d e p i t h e l i a l c e l l s were i s o l a t e d from t h e u r i n e o f p e s t i c i d e s p r a y e r s and examined f o r t h e f r e q u e n c y o f m i c r o n u c l e i . U n f o r t u n a t e l y , few o f the examined u r i n e samples c o n t a i n e d s u f f i c i e n t c e l l s t o p e r m i t an a c c u r a t e assessment o f damage. However, samples w i t h s u f f i c i e n t c e l l s d i d show a s i g n i f i c a n t increase i n micronucleated c e l l s . Future studies with t h i s procedure s h o u l d i n v o l v e r e p e a t e d s a m p l i n g o f each examined i n d i v i d u a l ( e . g . , 2-3 s u c c e s s i v e d a y s ) . I s o l a t e d c e l l s would be s t o r e d i n e t h a n o l and p o o l e d j u s t p r i o r t o d r o p p i n g them onto s l i d e s . The c o m b i n a t i o n o f a n a l y s i s o f b i o l o g i c a l f l u i d s f o r g e n o t o x i c a c t i v i t y and t h e c o n c u r r e n t assessment o f exposed e p i t h e l i a l c e l l s f o r in vivo damage i s h i g h l y p r o m i s i n g . T h i s approach has been used p r e v i o u s l y t o e l u c i d a t e t h e r e l a t i o n s h i p between exposure t o genot o x i n s and t h e i n d u c t i o n o f g e n e t i c damage i n t a r g e t t i s s u e s . One such group assayed were t o b a c c o / b e t e l q u i d chewers i n the P h i l i p p i n e s and I n d i a , a group a t e l e v a t e d r i s k f o r o r a l c a n c e r . S a l i v a samples t a k e n from i n d i v i d u a l s d u r i n g chewing were found t o c o n t a i n g e n o t o x i c a g e n t s , a p o r t i o n o f which have been i d e n t i f i e d as t o b a c c o - s p e c i f i c n i t r o s a m i n e s (66-68). O r a l smears o f e x f o l i a t e d c e l l s from t h e s e chewers showed a s i g n i f i c a n t e l e v a t i o n i n m i c r o n u c l e u s f r e q u e n c i e s . S u b s e q u e n t l y , a c o m b i n a t i o n o f t h e two approaches was used t o i d e n t i f y components which would a c t i n t h e s e p o p u l a t i o n s t o i n c r e a s e (e.g., c i g a r e t t e o r a l c o h o l usage) o r reduce (chemopreventive agents

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such as b e t a - c a r o t e n e ) g e n o t o x i c damage (69). A s i m i l a r approach c o u l d be used t o study g e n o t o x i n s i n t h e u r i n e o f c a r c i n o g e n - e x p o s e d individuals. We would be r e m i s s a t t h i s p o i n t i f we d i d n o t i n s e r t a note o f c a u t i o n i n t h e u s e o f t h e m i c r o n u c l e u s t e s t on e x f o l i a t e d c e l l s t o m o n i t o r p o p u l a t i o n s such as p e s t i c i d e s p r a y e r s . P r i o r t o t h i s s t u d y , a l l o f t h e examined p o p u l a t i o n groups had been more o r l e s s c h r o n i c a l l y exposed t o c a r c i n o g e n i c a g e n t s . A l l o f t h e p o p u l a t i o n s a t r i s k f o r o r a l c a n c e r c o n s i s t e d o f tobacco u s e r s w i t h a c h r o n i c usage of e i t h e r chewing tobacco o r c i g a r e t t e s . B i l h a r z i a s i s p a t i e n t s a t r i s k f o r u r i n a r y b l a d d e r c a n c e r may be exposed f o r s e v e r a l y e a r s t o c a r c i n o g e n i c agents i n t h e u r i n e (16). A s i m i l a r s i t u a t i o n e x i s t s f o r t h e second p o p u l a t i o n i n which an e l e v a t e d m i c r o n u c l e u s f r e q u e n c y was observed i n u r i n a r y sediments ( c i g a r e t t e smokers i n B r i t i s h Columbia) (15). I n t h e case o f acute exposure t o a g e n o t o x i c agent, such a s i n t h i s s t u d y , a temporary e l e v a t i o n i n m i c r o n u c l e u s f r e q u e n c i e s c o u l d be m i s s e experience w i t h p e r i o d i r a d i a t i o n t o t h e p e l v i c r e g i o n encompassing t h e u r i n a r y b l a d d e r i s t h a t t h e f r e q u e n c y o f m i c r o n u c l e a t e d c e l l s i n u r i n e samples i n c r e a s e s 10-12 days a f t e r t r e a t m e n t b e g i n s , and d e c l i n e s w i t h i n a week a f t e r t r e a t m e n t i s t e r m i n a t e d , r e f l e c t i n g t h e time r e q u i r e d f o r damage i n t h e d i v i d i n g c e l l s o f t h e b a s a l l a y e r s o f t h e e p i t h e l i u m t o produce m i c r o n u c l e i i n daughter c e l l s , and f o r t h e s e c e l l s t o r e a c h t h e s u r f a c e o f t h e e p i t h e l i u m and be e x f o l i a t e d . These k i n e t i c s s h o u l d be k e p t i n mind when examining i n d i v i d u a l s r e c e i v i n g a c u t e exposure t o an agent, and a r e p e a t e d s a m p l i n g p r o c e d u r e over t h e s e time i n t e r v a l s s h o u l d be employed. In c o n c l u s i o n , t h i s s t u d y i n d i c a t e s t h a t t h e e x a m i n a t i o n o f e x t r a c t e d u r i n e samples f o r c l a s t o g e n i c a c t i v i t y i n in vitro cell c u l t u r e s may be a v a l u a b l e i n d i c a t o r o f exposure o f p e s t i c i d e workers t o g e n o t o x i c a g e n t s . More s t u d i e s a r e r e q u i r e d p r i o r t o j u d g i n g t h e s u i t a b i l i t y o f employing t h e e x f o l i a t e d c e l l m i c r o n u c l e u s t e s t as a r o u t i n e m o n i t o r o f in vivo g e n o t o x i c damage i n such workers.

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38. Dulout, F.N.; Pastori, M.C.; Olivero, O.A.; Gonzalez Cid, M.; Loria, D.; Matos, E.; Sobel, N.; de Bujan, E.C.; Albiano, N. Mutation Res. 1985, 143, 237-44. 39. Rabello, M.N.; Becak, W.; De Almeida, W.F.; Pigati, P.; Ungaro, M.T.; Murata, T.; Pereira, C.A.B. Mutation Res. 1975, 28, 449-54. 40. Hogstedt, B.; Kolnig, A-M.; Mitelman, F.; Skerfving, S. Hereditas 1980, 92, 177-8. 41. de Cassia Stocco, R.; Becak, W.; Gaeta, R.; Rabello-Gay, M.N. Mutation Res. 1982, 103, 71-6. 42. Steenland, K.; Carrano, A.; Clapp, D.; Ratcliffe, J.; Ashworth, L.; Meinhardt, T. J. Occup. Med. 1985, 27, 729-32. 43. Franklin, C.A.; Fenske, R.A.; Greenhalgh, R.; Mathieu, L.; Denley, H.V.; Leffingwell, J.T.; Spear, R.C. J. Toxicol. Environ. Health 1981, 7, 715-31. 44. Barnes, W.S.; Weisburger, J.H. Mutation Res. 1984, 156, 83-91. 45. Guerrero, R.R.; Rounds D.E.; Hall T.C J Natl Cancer Inst 1979, 62, 805-9. 46. Brusick, D.; de Serres Neel, J.V.; Shelby, M.D.; Waters, M.D. In Guidelines for Studies of Human Populations Exposed to Mutagenic and Reproductive Hazards; Bloom, A.D., Ed.; March of Dimes Birth Defects Foundation: White Plains, N.Y., 1981; pp 111-40. 47. Kado, N.Y.; Manson, C.; Eisenstadt, E.; Hsieh, D.P.H. Mutation Res. 1985, 157, 227-33. 48. Sousa, J.; Nath, J.; Tucker, J.D.; Ong, T. Mutation Res. 1985, 149, 365-74. 49. Vainio, H.; Sorsa, M.; Falck, K. In Monitoring Human Exposure to Carcinogenic and Mutagenic Agents; Berlin, A.; Draper, M.; Hemminki, K.; Vainio, H., Eds.; International Agency for Research on Cancer: Lyon, 1984; IARC Sci. Publ. No. 59, pp 247-58. 50. Kriebel, D.; Gold, H.J.; Bronsdon, A.; Commoner, B. J. Environ. Pathol. Toxicol. 1985, 6, 157-69. 51. Putzrath, R.M.; Langley, D.; Eisenstadt, E. Mutation Res. 1981, 85, 97-108. 52. Beek, B.; Aranda, I.; Thompson, E. Mutation Res. 1982, 92, 333-60. 53. Falck, K. In Mutagens in Our Environment; Sorsa, M.; Vainio, H., Eds.; Alan R. Liss: New York, 1982; pp 387-400. 54. Dorough, H.W. J. Toxicol. Clin. Toxicol. 1983, 19, 637-59. 55. Ramel, C. In Individual Susceptibility to Genotoxic Agents in the Human Population; de Serres, F.J.; Pera, R.W., Eds.; Research Triangle Park, N.C., 1984; pp 293-303. 56. van Doorn, R.; Leijdekkers,C.M.;Bos, R.P.; Brouns, R.M.E.; Henderson, P.T. Ann. Occup. Hyg. 1981, 24, 77-92. 57. Aeschbacher, H.U.; Chappuis, C. Mutation Res. 1981, 89, 161-77. 58. Caderni, G.; Dolara, P. Pharmacol. Res. Commun. 1983, 5, 775-82. 59. Bos, R.P.; Theuws, J.L.G.; Henderson, P.T. Cancer Lett. 1983, 19, 85-90. 60. Barale, R.; Sozzi, G.; Toniolo, P.; Borghi, O.; Reali, D.; Loprieno, N.; Delia Porta, G. Mutation Res. 1985, 157, 235-40. 61. Everson, R.B.; Ratcliffe, J.M.; Flack, P.M.; Hoffman, D.M.; Watanabe, A.S. Cancer Res. 1985, 45, 6487-97. 62. Siebert, D.; Simon, U. Mutation Res. 1973, 21, 257-62.

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In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 9

Neuropathy Target Esterase in Blood Lymphocytes Monitoring the Interaction of Organophosphates with a Primary Target Marcello Lotti Istituto di Medicina del Lavoro, Universitá degli Studi di Padova, Via Facciolati 71 35127 Padova Ital Selective toxicit interactions of the toxin with primary targets. The i d e n t i f i c a t i o n of such targets and their accessibility in human fluids w i l l allow an evaluation of dose-effect relationships at a molecular level. Inter- and intra-individual variations of dose response relationships may then be rationalized and acceptable thresholds properly set. However, the assumption is made that the ultimate toxin is equally delivered to the accessible target and to where toxicity takes place. Some organophosphorus (OP) pesticides cause a rare selective toxicity called organophosphate-induced delayed polyneuropathy (OPIDP). The initiation of this toxicity involves specific interactions of a protein in the nervous system called Neuropathy Target Esterase (NTE). This protein is present in blood lymphocytes and its measurement after exposure to certain OP pesticides has been suggested as a biomonitor for OPIDP. NTE inhibition in blood lymphocytes predicts the development of OPIDP in man. However dose-response relationships are not available for man and a threshold is not yet established. Human lymphocytic NTE shows large interindividual variation, suggesting the need for an individual baseline to evaluate occupational exposures. An attempt to monitor occupational exposures showed a substantial effect on lymphocytic NTE not followed by toxicity. A likely explanation is that the assumption on the delivery previously made was incorrect. Furthermore the turnover of NTE in the target organ might be faster than in the monitored one and thus the effect of exposure overestimated. c

0097-6156/89/0382-0117$06.00/0 1989 American Chemical Society

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B i o l o g i c a l m o n i t o r i n g of o c c u p a t i o n a l and e n v i r o n m e n t a l exposures t o c h e m i c a l s w i l l become more m e a n i n g f u l f o r r i s k assessment when the mode of a c t i o n of the c h e m i c a l i s understood ( 1 ) . When t h e m o l e c u l a r t a r g e t of t o x i c i t y f o r a g i v e n c h e m i c a l i s i d e n t i f i e d , dose-response s t u d i e s are p o s s i b l e ( 2 ) . I f the t a r g e t i s a c c e s s i b l e , such s t u d i e s occupy a unique p o s i t i o n i n b i o l o g i c a l monitoring because they d e a l directly with adverse effects produced by c h e m i c a l s . I n a d d i t i o n , they s u p p l y i n t e g r a t e d i n f o r m a t i o n on e x p o s u r e , a b s o r p t i o n , d i s t r i b u t i o n , and response (3). A good example of an a c c e s s i b l e t a r g e t t h a t p r e d i c t s t o x i c e f f e c t s a t an i n a c c e s s i b l e s i t e i s the a c e t y l c h o l i n e s t e r a s e enzyme i n the red blood c e l l s which p r e d i c t s neuronal c h o l i n e s t e r a s e a c t i v i t y ( 4 ) . The purpose of t h i s paper i s t o r e p o r t another type of test to monitor a d i f f e r e n t toxicity c a u s e d by some organophosphorus p e s t i c i d e s , t h e organophosphorus induced d e l a y e d p o l y n e u r o p a t h y (OPIDP) OPIDP i s a syndrom and i s caused by some s i g n s of a p e r i p h e r a l p o l y n e u r o p a t h y , a x o n a l i n t y p e , a r e d e l a y e d 2-5 weeks a f t e r a s i n g l e exposure and l e s s p r e d i c t a b l y a f t e r r e p e a t e d exposures ( 5 ) . The c e n t r a l nervous system might a l s o be i n v o l v e d ( 6 ) . S e v e r a l commercial p e s t i c i d e s have produced OPIDP i n man, including trichlorphon, trichlornate, methamidophos, and chlorpyrifos (7). The mechanism of i n i t i a t i o n of OPIDP was e l u c i d a t e d by M.K. Johnson and r e v i e w e d s e v e r a l times (5,8,9,10,11), as w e l l as the consequent practical g a i n s f o r human OPIDP biomonitoring (12,13,14). T h i s paper g i v e s a b r i e f account of these s t u d i e s and d i s c u s s e s some l i m i t a t i o n s of the b i o m o n i t o r i n g t e s t which i s now available. The m o l e c u l a r t a r g e t f o r OPIDP i s a p r o t e i n w i t h e s t e r a t i c a c t i v i t y c a l l e d Neuropathy T a r g e t E s t e r a s e (NTE). OPs which cause OPIDP p h o s p h o r y l a t e more than 70% of NTE of the t a r g e t axons soon a f t e r d o s i n g i n the hen ( 1 0 , 1 5 ) . A f u r t h e r s t e p , c a l l e d a g i n g of the p h o s p h o r y l enzyme complex, l e a d s t o a n e g a t i v e l y charged complex and b i n d i n g of the a l k y l group l o s t d u r i n g t h i s p r o c e s s t o membranes. T h i s two s t e p r e a c t i o n u s u a l l y t a k e s p l a c e w i t h i n hours a f t e r d o s i n g and c o r r e l a t e s w i t h t h e development of OPIDP t h r e e weeks l a t e r . R e c e n t l y another aspect of the p a t h o g e n e s i s has been identified: a marked r e d u c t i o n of r e t r o g r a d e a x o n a l t r a n s p o r t o c c u r s 3-5 days a f t e r d o s i n g i n p e r i p h e r a l nerves which e v e n t u a l l y degenerate ( 1 6 ) . OPs and r e l a t e d i n h i b i t o r s w h i c h produce a non-ageable NTE complex do not cause OPIDP and a l s o p r o t e c t from a subsequent c h a l l e n g i n g dose of an e f f e c t i v e OP. Dudek and R i c h a r d s o n r e p o r t e d the presence of NTE a c t i v i t y i n the hen l y m p h a t i c system ( 1 7 ) , and t h i s f i n d i n g was c o n f i r m e d i n man ( 1 8 ) . The q u e s t i o n was r a i s e d whether measurements of NTE a c t i v i t y i n b l o o d lymphocytes a f t e r exposure t o OPs c a u s i n g OPIDP can be used t o monitor t h i s t o x i c e f f e c t i n humans. V a l i d a t i o n of t h i s t e s t i n humans p r o c e e d s , f o r obvious r e a s o n s , r a t h e r s l o w l y . N e v e r t h e l e s s , some d a t a have been accumulated so f a r and t h e i r s i g n i f i c a n c e r e c e n t l y d i s c u s s e d ( 1 4 ) . B r i e f l y , a case of death due to the organophosphate dimethoate (phosphorodithioic acid, 0 , 0 - d i m e t h y l S- ( 2 - m e t h y l a m i n o ) - 2 - o x o e t h y l e s t e r ) v a l i d a t e s the

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a n i m a l d a t a on the n e g l i g i b l e p o t e n t i a l of t h i s OP to cause OPIDP, i . e . dimethoate does not i n h i b i t NTE i n e i t h e r a n i m a l s or man at doses f a r above the l e t h a l ones ( 1 9 ) . I n a s u i c i d e case w i t h c h l o r p y r i f o s (0,0 - d i e t h y l - 0 - 3 , 5 , 6 , - t r i c h l o r o - 2- p y r i d y l p h o s p h o r o t h i o a t e ) , i n h i b i t i o n of l y m p h o c y t i c NTE correlates with t h a t i n p e r i p h e r a l nerve ( 2 0 ) . A f t e r a l a r g e dose of the same chemical to man, substantial inhibition of lymphocytic NTE predicted the development of OPIDP when measured s e v e r a l days b e f o r e the onset of symptoms ( 2 1 ) . A l l the e v i d e n c e c o l l e c t e d so f a r , however, l e a v e s the key q u e s t i o n open about the t h r e s h o l d of NTE i n h i b i t i o n / a g i n g r e q u i r e d to t r i g g e r the t o x i c response i n man. As more d a t a become a v a i l a b l e , a l s o w i t h a l a r g e r use of the l y m p h o c y t i c NTE t e s t , the q u e s t i o n w i l l p r o b a b l y be answered. NTE activity as s t u d i e d i n p e r i p h e r a l lymphocytes of an unexposed population reveals a large i n t e r i n d i v i d u a l variation (22). To a s c e r t a i n t h a t lo lymphocyti NTE a c t i v i t represent effect of occupationa baseline values i s necessary It i s p o s s i b l e that " f a l s e p o s i t i v e " or " f a l s e negative" r e s u l t s maybe o c c u r when l y m p h o c y t i c NTE i s used to monitor OPIDP. T a b l e 1 l i s t s some s i t u a t i o n s where i n h i b i t i o n of NTE i n p e r i p h e r a l lymphocytes might not be r e l a t e d t o OPIDP ( f a l s e p o s i t i v e s ) . As clarified by mechanistic s t u d i e s , NTE inhibitors like p h o s p h i n a t e s , carbamates and s u l p h o n a t e s do not cause OPIDP. To my knowledge however, none of these p r o t e c t i v e NTE inhibitors are commercial p e s t i c i d e s . The second s i t u a t i o n where " f a l s e p o s i t i v e " r e s u l t s might a r i s e i s when i n h i b i t e d NTE ages s l o w l y . Usually, whenever r o u t i n e t e s t s u s i n g NTE assay are performed, i t i s assumed that the aging process, i f formally possible, i s very rapid (23,24). T h i s assumption i g n o r e s the p o s s i b i l i t y of slow a g i n g of i n h i b i t e d NTE, and r e c e n t e v i d e n c e has m o d i f i e d t h i s view. Several OPs e x i s t i n s t e r o i s o m e r i c forms and s t u d i e s w i t h c h i r a l isomers have revealed a high degree of stereoselectivity in enzyme reactions with inhibitors and substrates and in biological a c t i v i t i e s (25). It i s known that racemic EPN (0-ethyl 0-(4-nitrophenyl) phenylphosphonothioate) and i t s L(-)-EPN isomer cause OPIDP i n the hen ( 2 6 ) , but not the D(+)-EPN isomer (27). In repeated dosing e x p e r i m e n t s L ( - ) - E P N caused OPIDP i n the hen, whereas D(+)-EPN had very marginal e f f e c t s (28). Some y e a r s ago we were p u z z l e d by r e s u l t s o b t a i n e d i n v i t r o w i t h NTE and AChE when c h a l l e n g e d w i t h the r e s o l v e d o p t i c a l isomers of EPNO (EPN-oxon), because i t had been s u g g e s t e d t h a t the ratio AChE I ^ / N T E I in vitro is p r e d i c t i v e of the l i k e l i h o o d f o r a g i v e n OP to cause OPIDP i n v i v o (29). Both EPNO isomers showed the same r a t i o of 0.01, s u g g e s t i n g an i d e n t i c a l potency i n c a u s i n g OPIDP ( L o t t i M.; Johnson M.K. U n p u b l i s h e d r e s u l t s , 1978). The s o l u t i o n came y e a r s l a t e r when i t was demonstrated t h a t the phosphony1-NTE complex, when formed by L ( - ) - E P N 0 , ages q u i c k l y , w h i l e t h a t formed by D(+)-EPN0 ages v e r y s l o w l y ( 3 0 ) . The same e f f e c t s on NTE occur i n v i v o e i t h e r w i t h EPA or EPNO isomers and the development of OPIDP i s c o r r e l a t e d w i t h the r a p i d a g i n g of p h o s p h o n y l a t e d NTE ( 3 1 ) . The unaged p h o s p h o n y l a t e d NTE a l s o p r o t e c t s the animal from a subsequent c h a l l e n g i n g dose of 5 Q

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an e f f e c t i v e OP. I n such cases of slow a g i n g OP i s o m e r s , we might therefore observe i n h i b i t i o n o f NTE w i t h o u t t h e c l i n i c a l correlate.

TABLE 1:

" F a l s e P o s i t i v e " R e s u l t s When Measuring Lymphocyte NTE A c t i v i t y f o r OPIDP B i o m o n i t o r i n g

a.

non-aging inhibited NTE (example: c a r b a m y l a t e d and s u l p h o n y l a t e d NTE) ( 8 ) .

b.

slow-aging i n h i b i t e d NTE) ( 3 2 ) .

c.

d i f f e r e n t a c c e s s of the i n h i b i t o r system (example: highl metabolites) (33).

d.

d i f f e r e n t s e n s i t i v i t y t o OPIDP v a r i a t i o n s of the t h r e s h o l d ( ? ) .

e.

t h e t u r n o v e r of i n h i b i t e d NTE i s d i f f e r e n t i n the nervous system (permanent c e l l s ) and i n b l o o d lymphocytes ( v a r i a b l e pool) ( ? ) .

NTE

(example:

Number i n b r a c k e t s i n d i c a t e r e f e r e n c e s .

phosphinylated,

D-(+)-EPN p h o s p h i n y l a t e d

t o lymphocytes and nervous

because

of

interindividual

( ? ) d a t a not a v a i l a b l e

A n o t h e r p o s s i b i l i t y i s when NTE i n h i b i t i o n i n the p r e d i c t i v e organ does not c o r r e l a t e w i t h t h a t i n the t a r g e t organ. T h i s may have been the case when a s u b s t a n t i a l i n h i b i t i o n of l y m p h o c y t i c NTE was observed i n an o c c u p a t i o n a l exposure t o t h e c o t t o n d e f o l i a n t s DEF (S,S,S - t r i b u t y l p h o s p h o r o t r i t h i o a t e ) and Merphos ( t r i b u t y l phosphorotrithioite) (32). Parallel t o enzyme measurements, e l e c t r o p h y s i o l o g i c a l t e s t s performed on exposed w o r k e r s , i n d i c a t e d no e x p o s u r e - r e l a t e d e f f e c t s on p e r i p h e r a l nervous system f u n c t i o n s . The h y p o t h e s i s i s t h a t t h e a c t i v e m e t a b o l i t e ( s ) of these c h e m i c a l s as formed i n t h e l i v e r ( ? ) , i s h i g h l y r e a c t i v e and p r e f e r e n t i a l l y i n h i b i t s NTE i n lymphocytes r a t h e r than i n t h e nervous system. However, the p o s s i b i l i t y t h a t NTE was i n h i b i t e d below t h e t h r e s h o l d cannot be r u l e d o u t . Once the t h r e s h o l d o f NTE i n h i b i t i o n / a g i n g w i t h o u t t o x i c r e s p o n s e i s known, p o s s i b l e i n t e r i n d i v i d u a l v a r i a t i o n s i n s e n s i t i v i t y might a l s o be d e t e c t a b l e . I n r e p e a t e d exposures to OPs c a p a b l e of c a u s i n g OPIDP, another p o s s i b i l i t y of m i s l e a d i n g r e s u l t s s h o u l d be t a k e n i n t o account: t h a t the t u r n o v e r of i n h i b i t e d NTE i s d i f f e r e n t i n lymphocytes and i n t h e b r a i n l e a d i n g t o e i t h e r h i g h e r or lower i n h i b i t i o n i n one organ as compared t o the o t h e r . T h i s i s because the nervous c e l l p o p u l a t i o n i s s t a b l e , whereas the p o o l of c i r c u l a t i n g lymphocytes is highly variable.

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TABLE I I :

" F a l s e N e g a t i v e " R e s u l t s When Measuring A c t i v i t y f o r OPIDP B i o m o n i t o r i n g

Lymphocyte NTE

a.

d e l a y e d i n h i b i t i o n / a g i n g of NTE (example: s e v e r a l days a f t e r c h l o r p y r i f o s ) ( 3 3 ) .

peak NTE i n h i b i t i o n

b.

d i f f e r e n t s e n s i t i v i t y t o OPIDP v a r i a t i o n s of the t h r e s h o l d ( ? ) .

of

c.

the t u r n o v e r of i n h i b i t e d NTE i s d i f f e r e n t i n t h e nervous system (permanent cells) and i n blood lymphocytes ( v a r i a b l e pool) ( ? ) .

because

interindividual

Numbers i n b r a c k e t s i n d i c a t

Measurements of l y m p h o c y t i c NTE a c t i v i t y might a l s o g i v e " f a l s e n e g a t i v e " r e s u l t s (Table I I ) . T h i s might occur when the t i m i n g of measurement i s not c a r e f u l l y s e l e c t e d . We suspected such " f a l s e n e g a t i v e " r e s u l t s i n a p a t i e n t who attempted s u i c i d e by i n g e s t i n g a l a r g e amount o f c h l o r p y r i f o s ( 2 1 ) . Surprisingly, l y m p h o c y t i c NTE was s t i l l i n h i b i t e d more than 20 days a f t e r t h e p o i s o n i n g ; however, the u s u a l d e l a y between h i g h NTE i n h i b i t i o n and the c l i n i c a l onset o f OPIDP was m a i n t a i n e d (2-3 weeks). As a result of t h i s o b s e r v a t i o n some experiments w i t h hens were performed t o c l a r i f y t h i s and o t h e r d i s c r e p a n c i e s observed i n t h e p o i s o n i n g case. C h l o r p h y r i f o s caused OPIDP i n hens but the e f f e c t d i d not c o r r e l a t e w i t h the i n h i b i t i o n of NTE i n the nervous system 24 hours a f t e r d o s i n g , as i t u s u a l l y o c c u r s . A t t h a t t i m e , the i n h i b i t i o n of nervous system NTE was below t h e t h r e s h o l d (about 50%) and the peak o c c u r r e d 4-5 days l a t e r ( 3 3 ) . I t i s concluded t h e r e f o r e t h a t f o r c e r t a i n OPs, t h e u s u a l 24 hour i n t e r v a l a f t e r exposure might not be a p p r o p r i a t e t o observe the maximal e f f e c t on NTE. I n c o n c l u s i o n , the u n d e r s t a n d i n g of the mechanism of a c t i o n of c e r t a i n OPs i n c a u s i n g OPIDP and t h e i d e n t i f i c a t i o n o f t h e m o l e c u l a r t a r g e t l e d t o the development of a s p e c i f i c t e s t t o be used i n human b i o m o n i t o r i n g . F u r t h e r m e c h a n i s t i c s t u d i e s and c l i n i c a l o b s e r v a t i o n s a r e l e a d i n g t o a b e t t e r a p p r e c i a t i o n of the l i m i t s of t h i s t e s t . Acknowledgment s I w i s h t o thank Mrs. C. D r a c e - V a l e n t i n i f o r her h e l p i n p r e p a r i n g the manuscript. Supported i n part by G r a n t s from CNR (85.00581.56,SP5), I t a l i a n M i n i s t r y of Education and Regions Veneto.

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Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Aldridge W.N. Ann. Rev. Pharmacol. Toxicol. 1986, 26, 39. World Health Organization; Report of Planning Meeting. International Program on Chemical Safety. ICS/87.17, 1987. Foa' V.; Emmett E.A.; Maroni M.; Colombi A. (Eds.) Occupational and Environmental Chemical Hazards, Cellular and Biochemical Indices for Monitoring Toxicity. Ellis Horwood Publ., Chichester, U.K., 1987. Derache R. (Ed.) Organophosphorus Pesticides, Criteria (dose/effect relationships) for Organophosphorus Pesticides. CEE. Pergamon Press, Oxford, New York, 1977. Lotti M.; Becker C.E.; Aminoff M.J. Neurology 1984, 34, 658. Vasilescu C. J. Neurol, Neurosurg. Psychiat. 1982, 45, 942. World Health Organization; Organophosphorus Insecticides: A General Introduction Environmental Health Criteria 63 Geneva, 1986. Johnson M.K. CRC Crit Davis C.S.; Richardson R.J. In: Experimental and Clinical Neurotoxicology; Spencer P.S.; Schaumburg H.H., Eds.; Williams & Wilkins, Baltimore, 1980; pp. 527-544. Johnson M.K. In: Rev. Biochem. Toxicol, vol. 4; Hodgson E.; Bend J.R.; Philpot R.M., Eds.; Elsevier, 1982; pp. 141-212. Johnson M.K. Trends Pharmacol. Sci. 1987, 8, 174. Lotti, M. Advances in Biosciences vol. 46; Gilioli R.; Cassitto M.G.; Foa' V., Eds; Pergamon Press, Oxford and New York, 1983; pp. 101-108. Lotti M. Toxicol. Lett. 1986, 33, 167. Lotti M. Trends Pharmacol. Sci. 1987, 8, 176. Lotti M.; Caroldi E.; Moretto A.; Johnson M.K.; Fish C.; Gopinath G.; Roberts N.L. Toxicol. Appl. Pharmacol. 1987, 88, 87. Moretto A.; Lotti M.; Sabri M.I.; Spencer P.S. J. Neurochem. 1987, 49, 1515. Dudek B.R.; Richardson R.J. Biochem. Pharmacol. 1982, 31, 1117. Moretto A; Fassina A; Lotti M. Proc. 2nd International Meeting on Cholinesterases, 1983, p. 171. Lotti M.; Ferrara S.D.; Caroldi S.; Sinigaglia F. Arch. Toxicol. 1981, 48, 265. Osterloh J.; Lotti M.; Pond S. J. Analyt. Toxicol. 1983, 7, 125. Lotti M.; Moretto A.; Zoppellari R.; Dainese R.; Rizzuto N.; Barusco G. Arch. Toxicol. 1986, 59, 176. Bertoncin D.; Russolo A.; Caroldi S.; Lotti M. Arch. Environ. Health 1985, 40, 139. Clothier B.; Johnson M.K. Biochem. J. 1979, 177, 549. Clothier B.; Johnson M.K. Biochem. J. 1980, 185, 739. Ohkawa H. In: Insecticide Mode of Action; Coats J.R.; Ed.; Academic Press, NY, London, 1982; pp. 163-185. Aldridge W.N.; Barnes J.M. Biochem. Pharmacol. 1966, 15, 541 and 549. Ohkawa H.; Mikami N.; Okuno Y.; Miyamoto J. Bull. Envir. Contam. Toxicol. 1977, 18, 534.

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32.

Lotti M.; Becker C.E.; Aminoff M.J.; Woodrow J.E.; Seiber J.N.; Talcott R.E.; Richardson R.J. J. Occup. Med. 1983, 25,

33.

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517.

RECEIVED March 15, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 10

Percutaneous Absorption and Inherent Toxicity Ronald C. Wester and Howard I. Maibach Department of Dermatology, University of California School of Medicine, San Francisco, CA 94143-0989 The aim of a toxicological study is to determine the inherent activity of some chemical relative to a target tissue. This process requires delivery to the target tissue -- usually described as bioavailability. With topical products, bioavailabilit cutaneous absorptio rent activity of the chemical. Failure of a patch test system to deliver chemical into skin will produce false negative results. In the skin, the amount of chemical present can correlate with erythema response and epidermal tumors. The skin also has metabolic potential and can alter chemical to more reactive metabolites. Relationship: Percutaneous Absorption and Inherent Toxicity The skin is recognized both as a barrier to absorption and as a primary route to the systemic circulation. The skin's barrier properties are often, but not always, impressive. Fluids and elec­ trolytes are reasonably well retained within the body, while at the same time many foreign chemicals are partially restricted from entering the systemic circulation. Despite these barrier proper­ ties, the skin is the route by which many chemicals enter the body. In most instances, the toxicology of the chemical is slight, and/or the bioavailability (rate and amount of absorption) of the chemical is too low to cause an immediate response. However, some chemicals applied to the skin have the potential to produce toxicity. It is now recognized that local and systemic toxicity depend on a chemical penetrating the skin. Table I shows the relationship of percutaneous absorption to toxicologic activity. A local or systemic effect cannot occur unless the chemical has inherent toxicity and the chemical is able to overcome the barrier properties of skin and enter a biologic system (local skin and/or systemic circulation (1). This chapter explores this concept of percutaneous absorption and inherent toxicity. 0097-6156/89/0382-0131$06.00/0 ° 1989 American Chemical Society

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T a b l e I . R e l a t i o n s h i p o f Percutaneous Absorption t o Toxicologic A c t i v i t y

Property o f Chemical

Absorption through s k i n

Local o r systemic effect

Inherent toxicity None None None

+ +

+

P a t c h T e s t Systems The development o f t o p i c a l drug p r o d u c t s r e q u i r e s t e s t i n g f o r s k i n t o x i c o l o g y r e a c t i o n s . A v a r i e t y o f p a t c h t e s t systems a r e a v a i l a b l e w i t h which c h e m i c a l s a r e a p p l i e d t o s k i n . The purpose o f t h i s s t u d y was t o determine t h e s k i n a b s o r p t i o n o f t h e a l l e r g e n p a r a p h e n y l enediamine (PPDA) from a v a r i e t y o f p a t c h t e s t i n g systems. [14C]PPDA ( 1 % i n petrolatum,USP) was p l a c e d i n a v a r i e t y o f p a t c h t e s t systems a t a c o n c e n t r a t i o n n o r m a l i z e d t o e q u a l s u r f a c e a r e a (2 mg/mm2). S k i n a b s o r p t i o n was d e t e r m i n e d i n t h e g u i n e a p i g by u r i n a r y e x e r t i o n o f 14C. There was a s i x - f o l d d i f f e r e n c e i n t h e range o f s k i n a b s o r p t i o n (p N 0.02) ( F i g u r e 1 ) . In decreasing o r d e r , p e r c e n t s k i n a b s o r p t i o n s from v a r i o u s p a t c h t e s t systems were H i l l Top Chamber (53.4 + 20.6), T e f l o n C o n t r o l P a t c h (48.6 + 9 . 3 ) , S m a l l F i n n Chamber w i t h paper d i s c i n s e r t (34.1 + 19.8), Small Finn Chamber (29.8 + 9.0), L a r g e F i n n Chamber (23.1 + 7.3), AL-Test Chamber (8.0 + 0.8). Thus, t h e c h o i c e o f p a t c h system c o u l d produce a f a l s e n e g a t i v e e r r o r i f t h e system i n h i b i t s s k i n a b s o r p t i o n , and subsequent s k i n t o x i c o l o g y r e a c t i o n ( 2 ) . The h i g h e s t e f f i c i e n c y o f s k i n a b s o r p t i o n was w i t h t h e H i l l - T o p Chamber. P o l i k a n d r i t o u and Conine (3) performed comparative s t u d i e s u s i n g t h e H i l l - T o p chamber system and W e b r i l p a t c h system t o compare d e l a y e d c o n t a c t h y p e r s e n s i t i v i t y . R e a c t i o n s were induced a t s i g n i f i c a n t l y lower c o n c e n t r a t i o n s f o r samples t e s t e d w i t h t h e H i l l - T o p Chamber. The r e a s o n f o r t h i s may have been h i g h e r s k i n absorption. M e t a b o l i c P r o d u c t i o n o f Cutaneous

Carcinogens

Much a t t e n t i o n has been f o c u s e d on p o l y c y c l i c a r o m a t i c hydrocarbons because they produce s k i n carcinomas. Metabolic a c t i v a t i o n o f these compounds i s u s u a l l y t h e f i r s t s t e p toward t h e i n d u c t i o n o f s k i n c a n c e r s . The m e t a b o l i t e s o f benz[a]pyrene (BP) g e n e r a l l y f a l l i n t o t h r e e c l a s s e s : p h e n o l s , quinones, and d i h y d r o d i o l s . I t i s t h e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

10.

WESTER AND MAIBACH

Inherent Toxicity

Midpoint (hours) Figure 1. Skin absorption of /7-phenylenediamine, measured by using various patch-testing systems.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

134

d i h y d r o d i o l s t h a t , when m e t a b o l i z e d t o epoxide d i o l s , become t h e more p o t e n t c a r c i n o g e n s . The e p o x i d e s r e a c t w i t h c e l l u l a r n u c l e o p h i l e s such as DNA, RNA, o r p r o t e i n s . The same i s t r u e f o r o t h e r p o l y c y c l i c a r o m a t i c hydrocarbons such as 3-methylcholanthrene and b e n z [ a ] a n t h r a c e n e d e r i v a t i v e s . These compounds induce s k i n tumors and t h i s p r o b a b l y i s caused by a r e a c t i v e m e t a b o l i t e (an e p o x i d e ) . Knowledge o f the b i n d i n g o f BP m e t a b o l i t e s t o macromolecules has reached a h i g h e r l e v e l o f s o p h i s t i c a t i o n . B i n d i n g o f B P - d i h y d r o d i o l e p o x i d e s was found t o o c c u r w i t h h i g h s t e r e o s e l e c t i v i t y . These i n v e s t i g a t o r s i s o l a t e d the polymer adducts t h a t were formed when [3H]BP was a p p l i e d t o t h e s k i n o f mice. There a r e two s t e r e o c h e m i c a l c o n f i g u r a t i o n s f o r t h e B P - 7 , 8 - d i h y d r o d i o l s , and t h e y may both be m e t a b o l i z e d t o t h e r e s p e c t i v e 9,10-epoxide. The epoxide may then r e a c t w i t h c e l l u l a r n u c l e o p h i l e s such as DNA, RNA, o r p r o t e i n s . For n u c l e i c a c i d s , t h e i n v i v o b i n d i n g o c c u r r e d p r e f e r e n t i a l l y t o guanine a t the 2-amino group ( i n b o t h DNA and RNA). Both s t e r e o i s o m e r formed mostg o f the c o v a l e n t l compound such as benzo[a]pyrene t h a t p e n e t r a t e s t h e s k i n must f i r s t pass i n t o and through t h e e p i d e r m i s and would be s u b j e c t t o an extensive metabolic reaction. Percutaneous

A b s o r p t i o n and E p i d e r m a l Tumors

Wester and co-workers (5) determined t h e e f f e c t o f frequency o f a p p l i c a t i o n on percutaneous a b s o r p t i o n o f h y d r o c o r t i s o n e (Table I I ) . When m a t e r i a l was a p p l i e d once o r t h r e e t i m e s per day t h e r e was a s t a t i s t i c a l d i f f e r e n c e (p 0.05) i n t h e percutaneous a b s o r p t i o n o f h y d r o c o r t i s o n e . One a p p l i c a t i o n per 24-hour exposure gave a h i g h e r percutaneous a b s o r p t i o n than i f t h e m a t e r i a l was a p p l i e d a t a lower c o n c e n t r a t i o n but more f r e q u e n t l y , namely, t h r e e t i m e s per day. T h i s was c o n f i r m e d w i t h a second c h e m i c a l , t e s t o s t e r o n e ( 6 ) .

T a b l e I I . A p p l i c a t i o n Frequency and Percutaneous Absorption of Hydrocortisone

Dose (ug/cm2)

Application (times/day)

T o t a l dose (ug/cm2)

13.3 13.3 40

1 3 1

13.3 40 40

Absorption (ug/cm2) 0.18 0.29 0.84

There i s a c o r r e l a t i o n between f r e q u e n c y o f a p p l i c a t i o n , percutaneous a b s o r p t i o n , and t o x i c i t y o f a p p l i e d c h e m i c a l . Wilson and H o l l a n d (7) determined t h e e f f e c t o f a p p l i c a t i o n f r q u e n c y i n e p i d e r m a l c a r c i n o g e n i c a s s a y s . A p p l i c a t i o n o f a s i n g l e l a r g e dose o f a h i g h l y complex m i x t u r e o f p e t r o l e u m o r s y n t h e t i c f u e l s t o a s k i n s i t e i n c r e a s e d the c a r c i n o g e n i c p o t e n t i a l o f t h e c h e m i c a l compared t o s m a l l e r o r more f r e q u e n t a p p l i c a t i o n s (Table I I I ) .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

10.

WESTER AND

MAIBACH

135

Inherent Toxicity

This carcinogenic t o x i c i t y c o r r e l a t e d w e l l with the r e s u l t s of Wester e t a l ( 6 ) / where a s i n g l e a p p l i e d dose i n c r e a s e d t h e percutaneous a b s o r p t i o n o f t h e m a t e r i a l compared t o s m a l l e r o r intermittent applications.

Table I I I .

S h a l e O i l - I n d u c e d I n c i d e n c e o f E p i d e r m a l Tumors

Dose (mg)

Frequency (per week)

T o t a l dose p e r week (mg)

No. o f a n i m a l s with carcinog e n i e tumors

OCSO No. 6

10 10 40

4 x 2 x 1

40 40 40

2 4 13

PCSO I I

10 30 40

4 4 x 1 x

40 40

17 19

Shale o i l

Cosmetic C h e m i c a l s and T o x i c i t y T a b l e IV shows t h e r e l a t i o n s h i p between percutaneous a b s o r p t i o n and erythema f o r s e v e r a l o i l s used i n c o s m e t i c s . The a u t h o r s attempted t o c o r r e l a t e a b s o r b a b i l i t y w i t h erythema. The most absorbed o i l / i s o p r o p y l m y r i s t a t e / produced t h e most erythema. The l o w e s t a b s o r b i n g oil, 2 - h e x y l - decanoxyoctane/ produced t h e l e a s t erythema. A b s o r b a b i l i t y and erythema f o r t h e o t h e r o i l s d i d not c o r r e l a t e ( 8 ) . The l e s s o n t o remember w i t h p e r c u t a n e o u s t o x i c i t y i s t h a t a t o x i c response r e q u i r e d b o t h an i n h e r e n t t o x i c i t y i n t h e c h e m i c a l and t h e percutaneous a b s o r p t i o n o f t h e c h e m i c a l . The degree o f t o x i c i t y w i l l depend on t h e c o n t r i b u t i o n o f b o t h c r i t e r i a .

T a b l e IV.

R e l a t i o n s h i p o f Percutaneous A b s o r p t i o n and Erythema f o r S e v e r a l O i l s Used i n C o s m e t i c s

Absorbability (greatest to least) Isopropyl myristate Glycerol tri(oleate) n -Octadecane Decanoxydecane 2-Hexyldecanoxyoctane

Erythema +

+

+; +

Ten S t e p s t o P e r c u t a n e o u s A b s o r p t i o n The p r e c e d i n g examples have shown t h a t two components/ namely i n h e r e n t c h e m i c a l a c t i v i t y and percutaneous a b s o r p t i o n / a r e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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n e c e s s a r y t o g e t a t o x i c o l o g i c a l r e a c t i o n . I t i s t h e r a t e and extent that a r e a c t i v e chemical i s d e l i v e r e d t o the target t i s s u e t h a t d e t e r m i n e s t h e degree o f t o x i c o l o g i c a l r e s p o n s e . However, percutaneous a b s o r p t i o n i s not a simple a c t o f apply chemical t o s k i n and t h e n " s e e i n g what happens". P e r c u t a n e o u s a b s o r p t i o n i s a complex p r o c e s s c o n s i s t i n g o f many f a c t o r s , any o f w h i c h c a n a f f e c t t h e f i n a l outcome. Wester and Maibach (9) have summarized t h e p r o c e s s i n t e n s t e p s ( o u r c u r r e n t v i e w , b u t presumably many s t e p s remain t o be d i s c o v e r e d ) .

Ten S t e p s t o P e r c u t a n e o u s A b s o r p t i o n 1. 2.

3. 4. 5. 6. 7. 8. 9. 10.

Vehicle release Absorption k i n e t i c s a. S k i n s i t e o f a p p l i c a t i o n b. I n d i v i d u a c. S k i n c o n d i t i o d. O c c l u s i o n e. Drug c o n c e n t r a t i o n and s u r f a c e a r e a s f. Multiple-dose application Excretion kinetics E f f e c t i v e c e l l u l a r and t i s s u e d i s t r i b u t i o n S u b s t a n t i v i t y (nonpenetrating surface adsorption) Wash and r u b r e s i s t a n c e Volatility Binding Anatomic pathways Cutaneous m e t a b o l i s m

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

Wester, R.C.; Maibach, H.I. In Environmental Pathology, N.K. Mottet, Ed.; Oxford University Press: New York, 1985;181-194. Kim, O.K.; Wester, R.C.; McMaster, J.A., Bucks, D.A.W.; Maibach, H.I. Contact Dermatitis 1987 17 (in press). Polikandriton, M.; Conine, D.L. J. Soc. Cosmetic Chem. 1985, 36, 159-168. Noonan, P.K.; Wester, R.C. In Dermatotoxicology, F.N. Marzulli and H.I. Maibach, Eds.; Hemisphere Publishing: New York, 1983; pp 71-90. Wester, R.C.; Noonan, P.K.; Maibach, H.I. Arch. Dermatol. Res.,1977 113, 620-622. Wester, R.C.; Noonan, P.K.; Maibach, H.I. Arch. Dermatol. Res.1980 267, 229-235. Wilson, J.S.; Holland, L.M. Toxicology 1982, 24, 45-54. Suzuki, M.; Asaba, K.; Komatsu, H.; Mockizuki, M. J. Soc. Cosmet. Chem. 1978 29, 265-271. Wester, R.C.; Maibach, H.I. Cutaneous Pharmacokinetics: 10 Steps to Percutaneous Absorption. Drug Metab. Disposition, 1983.

RECEIVED January 25, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 11

Dinocap Dermal Absorption in Female Rabbits and Rhesus Monkeys Implications for Humans 1

1

2

Stephen L. Longacre , Laura J. DiDonato , Ronald C. Wester , Howard I. Maibach , Susan S. Hurt , and Richard D. Costlow 2

1

2

1

Toxicology Department, Rohm and Haas Company, Spring House, PA 19477 Department of Dermatology University of California School of Medicine

2

The dermal absorption of dinocap was determined in female rabbits and rhesus monkeys as part of the risk assessment for Karathane Fungicide/Miticide. C-2,4-Dinitro-6-(1-methylheptyl)phenyl crotonate (2,4-DNHPC) was used as the model isomer for dinocap in these studies. In r a b b i t s , the dermal absorption of 25 mg/kg (approximately 2500 ug/cm ) 2,4-DNHPC was 4-9%, whether 2,4-DNHPC was applied neat (undiluted) or dissolved in acetone, or applied as the wettable powder or l i q u i d concentrate formulation. The total absorption of 13 d a i l y 6-hr dermal doses of 25 mg/kg/day (2500 ug/cm /day) of neat 2,4-DNHPC in rabbits was 6%, similar to the percent absorption observed following a single dermal dose. In rhesus monkeys, the dermal absorption of 2,4-DNHPC at 2500 ug/cm (in acetone) was similar to the dermal absorption in rabbits (5%). Dermal absorption of 2,4-DNHPC in monkeys at 40 ug/cm was 16%; this dose approxmated a u s e - d i l u t i o n . The absorption data for 2,4-DNHPC in rabbits and monkeys support the conclusion that, under expected use conditions, dermal exposure to dinocap does not pose an unreasonable developmental r i s k to man. R

14

2

2

2

2

R

Dinocap (Karathane Fungicide/Miticide) was registered by Rohm and Haas Company in the United States in 1951, and is p r i n c i p a l l y used as a fungicide for control of powdery mildew and as a m i t i c i d e . Dinocap contains as i t s active ingredients a mixture of 2,4- and 2,6-dinitrooctylphenyl crotonates in an approximate 2:1 r a t i o , where "octyl" refers to a mixture of 1-methylheptyl, 1-ethylhexyl, NOTE: A more detailed version of these data will be published in a toxicology journal. 0097-6156/89/0382-0137$06.00/0 © 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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and 1 - p r o p y l p e n t y l i s o m e r s ( F i g u r e 1 ) . S m a l l amounts o f t h e c o r r e s p o n d i n g f r e e p h e n o l s a r e a l s o p r e s e n t as a c t i v e i n g r e d i e n t s . D e v e l o p m e n t a l t o x i c i t y s t u d i e s i n New Z e a l a n d w h i t e r a b b i t s i n d i c a t e d that oral exposure to dinocap t e c h n i c a l during the p e r i o d o f o r g a n o g e n e s i s p r o d u c e d t e r a t a , w h i c h were m a n i f e s t e d as h y d r o ­ c e p h a l u s and/or m a l f o r m a t i o n s o f t h e n e u r a l t u b e and s k u l l ( 1 ) . The n o - o b s e r v e d e f f e c t l e v e l (NOEL) and t h e minimum e f f e c t l e v e l (MEL) f o r d i n o c a p o r a l d e v e l o p m e n t a l t o x i c i t y i n r a b b i t s were j u d g e d t o be 0.5 and 3 mg/kg/day, r e s p e c t i v e l y ( 1 ) . R e s i d u e s o f d i n o c a p on c r o p s a r e s u c h t h a t t h e r i s k o f d i n o c a p t o humans by t h e o r a l r o u t e i s n e g l i g i b l e . To a s c e r t a i n t h e p o s s i b l e r i s k o f dinocap to a g r i c u l t u r a l workers, s e v e r a l s t u d i e s were p e r f o r m e d . T h e s e s t u d i e s i n c l u d e d a d i n o c a p d e r m a l / o r a l a b s o r p t i o n s t u d y i n r a b b i t s , and a d i n o c a p dermal a b s o r p t i o n s t u d y i n r h e s u s monkeys. Our o b j e c t i v e s i n t h e s e a b s o r p t i o n s t u d i e s were a) t o compare t h e p e r c e n t dermal a b s o r p t i o n o f d i n o c a p i n t h e r a b b i t and r h e s u s monkey t o x i c o l o g y s t u d i e s , and b a b s o r p t i o n i n t h e r h e s u s monkey a t p o t e n t i a l worker e x p o s u r e c o n c e n t r a t i o n s . To f a c i l i t a t e t h e i n t e r p r e t a t i o n o f t h e d a t a , t h e a b s o r p t i o n s t u d i e s were p e r f o r m e d u s i n g C - 2 , 4 - d i n i t r o - 6 ( 1 - m e t h y l h e p t y l ) - p h e n y l c r o t o n a t e (2,4-DNHP'C) as t h e model compound f o r the alkyl s u b s t i t u t e d isomers of dinocap (Figure 2 ) . M a t e r i a l s and Methods T e s t Compounds. C-2,4-DNHPC (7.58 mCi/g; r a d i o p u r i t y = 96.5%; m o l e c u l a r w e i g h t = 256 g / m o l e ) , and n o n r a d i o l a b e l e d 2,4-DNHPC p u r i t y = 97%) used t o d i l u t e t h e s p e c i f i c a c t i v i t y o f t h e 4C-2,4-DNHPC, were b o t h s y n t h e s i z e d a t Rohm and Haas Company ( S p r i n g House, P A ) . The t e s t compounds were l i g h t amber c o l o r e d oils. A n i m a l s . F e m a l e New Z e a l a n d w h i t e r a b b i t s (34-42 weeks o l d ; 2.9-3.9 kg; H a z l e t o n D u t c h l a n d ; Denver, P A ) , and f e m a l e r h e s u s monkeys (5.4-10.7 kg; U n i v e r s i t y o f C a l i f o r n i a a t D a v i s P r i m a t e C e n t e r ; D a v i s , CA) were used i n t h e s e s t u d i e s (3 o r 4 a n i m a l s p e r group). 14 Rabbit Intravenous Study. C-2,4-DNHPC, d i s s o l v e d i n d i m e t h y l s u l f o x i d e (DMSO), was a d m i n i s t e r e d i n t h e j u g u l a r v e i n (0.1 m l / k g ) at 3 mg/kg (3 u C i / k g ) . U r i n e and f e c e s were c o l l e c t e d a t i n t e r v a l s up t o 4 days a f t e r d o s i n g and a n a l y z e d f o r t o t a l ' C - l a b e l . A l l r a b b i t s were h u m a n e l y k i l l e d by an i n t r a c a r d i a c i n j e c t i o n o f e u t h a n a s i a s o l u t i o n (T-61 ; A m e r i c a n H o e c h s t C o r p . , S o m e r v i l l e , NJ). R a b b i t O r a l S t u d i e s . C-2,4-DNHPC, s u s p e n d e d i n aqueous 1% gum t r a g a c a n t h , was a d m i n i s t e r e d by gavage (5 m l / k g ) a t 0.5, 3, o r 25 mg/kg (3-17 u C i / k g ) . U r i n e , f e c e s , and p l a s m a were c o l l e c t e d at i n t e r v a l s up t o 4 days a f t e r d o s i n g and a n a l y z e d f o r ^ C - l a b e l . R a b b i t 6 Hr Dermal E x p o s u r e S t u d i e s . The f u r on t h e d o r s a l s i d e o f e a c h r a b b i t was c l i p p e d t o e x p o s e an a r e a o f a p p r o x i m a t e l y 280 cm 1-3 days p r i o r t o d o s i n g . P l a s t i c c o l l a r s were p l a c e d on e a c h rabbit to prevent preening of the application site.p C-2,4-DNHPC (23-31 u C i / k g ) was a p p l i e d d i r e c t l y o n t o a 5 x 8 cm a r e a o f t h e 14

4

14

4

2

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

LONGACRE ET AL.

Dinocap Dermal Absorption

F i g u r e 1. S t r u c t u r a l f o r m u l a e o f t h e a c t i v e i n g r e d i e n t components o f d i n o c a p [ 2 , 4 - d i n i t r o o c t y l p h e n y l c r o t o n a t e s ( t o p ) and 2 , 6 - d i n i t r o o c t y l p h e n y l c r o t o n a t e s ( b o t t o m ) ] ; "n" e q u a l s 0, 1, and 2.

F i g u r e 2. S t r u c t u r a l f o r m u l a o f C-2,4-DNHPC; t h e a s t e r i s k s indicate location of the C-label.

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c l i p p e d d o r s a l s i t e as a) t h e n e a t ( u n d i l u t e d ) m a t e r i a l a t 25 ( 2 . 2 ) , 100 ( 8 . 3 ) , and 220 mg/kg ( 1 8 . 3 mg/cra ); b) t h e w e t t a b l e d u s t (WD) f o r m u l a t i o n a t 25 mg a i / k g (2.4 mg/cm ) a p p l i e d as a 2 0 . 4 % a i p a s t e (1:1.3 p a s t e : w a t e r ) a t 0.28 g p a s t e / k g ; c ) t h e l i q u i d c o n c e n t r a t e ( L C ) f o r m u l a t i o n a t 25 mg a i / k g ( 2 . 5 mg/cm a p p l i e d as a 3 9 . 8 % a i s o l u t i o n ( 0 . 0 6 m l / k g ) ; o r d) i n a c e t o n e a t 25 mg/kg (2.2 mg/cm ) a p p l i e d as a 3 9 . 8 % a i s o l u t i o n ( 0 . 0 6 m l / k g ) . S i x ( 6 ) h r a f t e r d o s e a p p l i c a t i o n , t h e a p p l i c a t i o n s i t e s were w i p e d w i t h gauze pads s o a k e d w i t h aqueous 9 5 % e t h a n o l , f o l l o w e d by w i p i n g w i t h pads s a t u r a t e c L w i t h w a t e r . A p p l i c a t i o n s i t e s o f r a b b i t s t r e a t e d w i t h t h e C-2,4-DNHPC-WD f o r m u l a t i o n were w i p e d w i t h d i s t i l l e d w a t e r o n l y . The w i p e s were e x t r a c t e d w i t h a c e t o n e and a n a l y z e d f o r t o t a l C - l a b e l . U r i n e , f e c e s , and p l a s m a were c o l l e c t e d a t i n t e r v a l s up t o 7 d a y s and a n a l y z e d f o r ^ C - l a b e l . A t t e r m i n a t i o n , t h e a p p l i c a t i o n s i t e s k i n s were c o l l e c t e d , d i s s o l v e d i n t i s s u e s o l u b i l i z e r ( U n i s o l ; I s o l a b I n c . ; A k r o n OH) and analyzed f o r t o t a l C-label z

2

4

R

14

R a b b i t 7 Day ( L e a v e - O n ) Dermal E x p o s u r e S t u d y . C-2,4-DNHPC was a p p l i e d d e r m a l l y as t h e n e a t m a t e r i a l a t 25 mg/kg (2.1 mg/cm ; 30 u C i / k g ) , and was l e f t on f o r 7 d a y s . U r i n e , f e c e s , and p l a s m a were c o l l e c t e d a t i n t e r v a l s o v e r t h e 7-day p e r i o d and a n a l y z e d f o r C - l a b e l . A t t e r m i n a t i o n , . t h e a p p l i c a t i o n s i t e s k i n s were c o l l e c t e d and a n a l y z e d f o r C - l a b e l . The a p p l i c a t i o n s i t e s were not w i p e d p r i o r t o ' C - a n a l y s i s o f t h e s k i n , s i n c e a l a r g e amount o f f u r had grown b a c k . R a b b i t M u l t i p l e Dermal Dose S t u d y . Neat C-2,4-DNHPC was a p p l i e d as 13 d a i l y 6 h r d o s e s a t 25 mg/kg/day. T h e 5 x 8 cm a p p l i c a t i o n a r e a was r o t a t e d among 7 s i t e s s u c h t h a t c o n s e c u t i v e d o s e s were n o t a p p l i e d t o t h e same o r . a d j a c e n t s i t e s . The a p p l i c a t i o n s i t e s were wiped 6 hr a f t e r each C-dose a p p l i c a t i o n , and t h e w i p e s were a n a l y z e d f o r C - l a b e l . U r i n e , f e c e s , and p l a s m a were c o l l e c t e d a t i n t e r v a l s d u r i n g t h e 13 d a y d o s i n g p h a s e and f o r up t o 10 days a f t e r t h e l a s t d o s e , and a n a l y z e d f o r ' C - l a b e l . R a b b i t s were k i l l e d 10 days a f t e r t h e l a s t d o s e , and t h e a p p l i c a t i o n s i t e s were c o l l e c t e d and a n a l y z e d f o r ^ C - l a b e l . 4

14

4

4

14

Monkey I n t r a v e n o u s S t u d y . C-2,4-DNHPC d i s s o l v e d i n DMS0, was a d m i n i s t e r e d i n t h e f e m o r a l v e i n (0.1 m l / k g ) a t 0.2 mg/kg (0.15 u C i / k g ) . P l a s m a was c o l l e c t e d a t i n t e r v a l s up t o 2 days a f t e r d o s i n g , w h i l e u r i n e and f e c e s were c o l l e c t e d a t i n t e r v a l s up t o 4 days a f t e r d o s i n g and a n a l y z e d f o r C - l a b e l . F e c a l s a m p l e s were p o o l e d p r i o r t o a n a l y s i s . T h e s e a n i m a l s were u s e d i n s u b s e q u e n t dermal s t u d i e s o n c e . i t was d e t e r m i n e d t h a t p l a s m a and u r i n e c o n ­ t a i n e d no r e s i d u a l C-label. Monkey P e r c u t a n e o u s S t u d y (40 u g / c m ) . C-2,4-DNHPC, d i s s o l v e d i n 400 u l a c e t o n e , was a p p l i e d t o 40 c m o f a b d o m i n a l s k i n a t 40 ug/cm ( 1 . 6 mg/monkey; a p p r o x i m a t e l y 0.2 mg/kg; 12 u C i / m o n k e y ) . The a n i m a l s were r e s t r a i n e d i n m e t a b o l i c c h a i r s d u r i n g t h e dermal e x p o s u r e p e r i o d , and were r e t u r n e d t o m e t a b o l i c c a g e s i m m e d i a t e l y f o l l o w i n g t h e dose wipe-off. T h e a p p l i c a t i o n s i t e s were wiped w i t h c o t t o n b a l l s l a d e n e d w i t h w a t e r , o r w i t h aqueous 9 5 % e t h a n o l f o l l o w e d by a w a t e r r i n s e 6 h r a f t e r a p p l i c a t i o n ; t h e w i p e s were 2

14

2

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

11. LONGACREETAL.

141

Dinocap Dermal Absorption

a n a l y z e d f o r 1 ^ C - 1 a b e l . P l a s m a was c o l l e c t e d a t i n t e r v a l s up t o 2 d a y s , w h i l e u r i n e and f e c e s were c o l l e c t e d a t i n t e r v a l s up t o 4 days and a n a l y z e d f o r C - l a b e l . Monkey P e r c u t a n e o u s S t u d y (2500 u g / c m ) . C-2,4-DNHPC d i s s o l v e d i n 14 u l a c e t o n e , was a p p l i e d t o 0.64 cm o f a b d o m i n a l s k i n a t 2500 ug/ciTr ( 1 . 6 mg/monkey; a p p r o x i m a t e l y 0.2 mg/kg; 12 u C i / m o n k e y ) . The a p p l i c a t i o n s i t e s were w i p e d w i t h aqueous 9 5 % e t h a n o l , f o l l o w e d by w a t e r , 6 h r a f t e r dose a p p l i c a t i o n ; t h e w i p e s were a n a l y z e d f o r C - l a b e l . P l a s m a was c o l l e c t e d a t i n t e r v a l s up t o 2 d a y s , w h i l e u r i n e and f e c e s were c o l l e c t e d a t i n t e r v a l s up t o 4 days and analyzed f o r C - l a b e l . P e r c e n t A b s o r p t i o n . The f r a c t i o n o f an o r a l o r dermal dose o f i4C-2,4-DNHPC a b s o r b e d r e p r e s e n t e d t h e u r i n a r y 1 4 c - e x c r e t i o n a f t e r o r a l o r dermal a d m i n i s t r a t i o n d i v i d e d b y t h e u r i n a r y ^ C - e x c r e t i o n after i v administration (2 3 ) 1 4

2

14

1

4

Results R a b b i t I n t r a v e n o u s S t u d y . Most (58-85%) o f an i v d o s e o f ' C-2,4-DNHPC was e x c r e t e d f r o m r a b b i t s w i t h i n 24-48 h r ( F i g u r e 3 ) . Urinary C - e x c r e t i o n was 5 - f o l d g r e a t e r t h a n f e c a l e x c r e t i o n ; 7 3 % o f t h e d o s e was r e c o v e r e d i n t h e u r i n e b y 96 h r . R a b b i t O r a l S t u d i e s . The p e r c e n t a b s o r p t i o n o f an o r a l dose o f 14C-2,4-DNHPC (0.5-25 mg/kg) was 60-69% { T a b l e I ) . P r a c t i c a l l y a l l o f an o r a l dose o f C-2,4-DNHPC was e l i m i n a t e d i n t h e e x c r e t a o f r a b b i t s w i t h i n 48 h r ( F i g u r e 3 ) . T h e p e r c e n t c u m u l a t i v e u r i n a r y and f e c a l C - e x c r e t i o n . w e r e each s i m i l a r among t h e t h r e e o r a l d o s e g r o u p s . F e c a l C-excretion ( 5 7 - 7 2 % o f d o s e ) was s l i g h t l y g r e a t e r t h a n u r i n a r y ^ C - e x c r e t i o n ( 3 5 - 5 1 % o f d o s e ) b y 96 h r . ' C-2,4-DNHPC-derived C - l a b e l was r a p i d l y a b s o r b e d a f t e r o r a l a d m i n i s t r a t i o n , r e a c h i n g peak p l a s m a C-concentrations within 1-3 h r ( T a b l e I I ) . Peak p l a s m a ^ - c o n c e n t r a t i o n was p r o p o r t i o n a l t o d o s e ( T a b l e I I ) . E l i m i n a t i o n o f C - l a b e l was b i p h a s i c f o r a l l g r o u p s ; a r a p i d e l i m i n a t i o n p h a s e ( t 1/2 = 2.2-3.8 h r ) , l a s t i n g a p p r o x i m a t e l y 24 h r a f t e r d o s i n g , p r e c e d e d a s l o w e r t e r m i n a l e l i m i n a t i o n p h a s e ( t 1/2 = 33.3-55.1 h r ) . +#

1

4

4

1 4

R a b b i t 6 Hr Dermal E x p o s u r e S t u d i e s . The p e r c e n t a b s o r p t i o n o f a 6 h r dermal e x p o s u r e o f C-2,4-DNHPC was 4-9%, r e g a r d l e s s o f t h e dose o r t h e f o r m u l a t i o n / v e h i c l e ( T a b l e I ) . A m a j o r i t y o f t h e a p p l i e d 1 C-2,4-DNHPC ( 6 0 - 9 5 % o f d o s e ; 89-95% o f r e c o v e r e d C - l a b e l ) was r e c o v e r e d i n t h e 6 h r w i p e - o f f r e g a r d l e s s o f t h e dose o r t h e f o r m u l a t i o n / v e h i c l e ( T a b l e I I I ) . Little C - l a b e l ( 0 . 0 2 - 1 % o f d o s e ) was r e c o v e r e d i n t h e a p p l i ­ c a t i o n s i t e s k i n s a t t h e end o f t h e 7-day i n - l i f e p b a s e ( T a b l e I I I ) . A t o t a l o f 5-12% o f a 6 h r dermal e x p o s u r e o f C-2,4-DNHPC was e l i m i n a t e d i n t h e e x c r e t a w i t h i n 7 d a y s r e g a r d l e s s o f t h e dose or t h e f o r m u l a t i o n / v e h i c l e (Table I I I ) . A l s o , t h e percent c u m u l a t i v e u r i n a r y and f e c a l ' C - e x c r e t i o n were c o m p a r a b l e among t h e dermal g r o u p s , r e g a r d l e s s o f t h e d o s e o r t h e f o r m u l a t i o n / v e h i c l e . Most o f t h e e x c r e t e d C - l a b e l was e l i m i n a t e d w i t h i n 2-4 days ( F i g u r e 3 ) , and was e s s e n t i a l l y e v e n l y d i s t r i b u t e d between t h e u r i n e and f e c e s . 14

4

14

4

1 4

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

142

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE Table I .

mg/kg

Dose ug/cm

2

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14

P e r c e n t A b s o r p t i o n o f C-2,4-DNHPC i n Female R a b b i t s and Rhesus Monkeys

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Percent Absorption

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— — — —

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4

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4

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—

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suspension.

F i g u r e 3. C u m u l a t i v e e x c r e t i o n o f C - l a b e l i n t h e u r i n e ( O h f e c e s ( • ) , and combined u r i n e and f e c e s ( • ) o f f e m a l e r a b b i t s a d m i n i s t e r e d a n ^ v , o r a l , 6 h r d e r m a l , o r 7-day ( l e a v e - o n ) dermal dose o f C-2,4-DNHPC.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

a

11. LONGACRE ET AL.

143

Dinocap Dermal Absorption

1 4

Table I I .

Peak P l a s m a C - C o n c e n t r a t i o n i n Female R a b b i t s and Rhesus Monkeys A d m i n i s t e r e d C-2,4-DNHPC 14

Dose mg/kg u g / c m

2

— — —

0. 5 3 25

Exposure Route Time

Oral Oral Oral

— — —

Formulation/ Vehicle n

Gum Gum Gum

Trag Trag Trag

Peak P l a s m a C-Concentration (ppm) (hr)

4 3 4

0.29 + 0.04 1.26 + 0.42 14.55 + 6.62

1 3 3

3 3 3

0.15 + 0.05 0.47 + 0.10 1.51 + 1.12

6 24

3 3 4

0.19 + 0.03 0.24 + 0.07 0.28 + 0.00

24 6 24

25 100 220

2150 8310 18i,300

Dermal Dermal Dermal

6 Hr 6 Hr 6 Hr

25 25 25

2390 2450 2220

Dermal Dermal Dermal

6 Hr 6 Hr 6 Hr

25

2060

Dermal

7 Day

Neat

4

0.24 + 0.19

6

18502313

Dermal

6 Hr (xl3)

Neat

4

0.89

+ 0.33

318

25 (X13)

Neat Neat Neat

b

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a

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1 4

Not d e t e c t e d . P l a s m a C - c o n c e n t r a t i o n s were i n t h e r a n g e of background t o t w i c e background.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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1 4

145

C - 2 , 4 - D N H P C - d e r i v e d C - l a b e l was m o d e r a t e l y t o s l o w l y a b s o r b e d f o l l o w i n g a 6 h r dermal e x p o s u r e ; peak p l a s m a 1 C - c o n c e n t r a t i o n s were a c h i e v e d w i t h i n 6-24 h r ( T a b l e I I ) . Peak plasma ' C - c o n c e n t r a t i o n f o l l o w i n g a p p l i c a t i o n o f t h e neat m a t e r i a l was p r o p o r t i o n a l t o d o s e ( T a b l e I I ) . S i m i l a r peak p l a s m a ' ^ - c o n c e n t r a t i o n s were a c h i e v e d when ^ C-2,4-DNHPC was a p p l i e d a s t h e WD o r LC f o r m u l a t i o n , o r d i s s o l v e d i n a c e t o n e compared t o an e q u i v a l e n t d e r m a l d o s e o f t h e neat m a t e r i a l ( T a b l e I I ) . E l i m i n a t i o n o f C - l a b e l was b i p h a s i c f o r a l l 6 h r d e r m a l l y e x p o s e d g r o u p s ; a r a p i d e l i m i n a t i o n p h a s e ( t 1/2 = 7.9-21.8 h r ) , l a s t i n g a p p r o x i m a t e l y 2-3 days a f t e r d o s i n g , p r e c e d e d a s l o w t e r m i n a l e l i m i n a t i o n p h a s e ( t 1/2 = 119-369 h r ) . The peak p l a s m a ' C - c o n c e n t r a t i o n (0.15-0.28 ppm) f o l l o w i n g a 6 h r dermal e x p o s u r e o f 25 mg/kg C-2,4-DNHPC was 52- t o 9 7 - f o l d l e s s t h a n t h e . p e a k p l a s m a C - c o n c e n t r a t i o n o f an e q u i v a l e n t o r a l d o s e o f C-2,4-DNHPC (14.55 ppm) R a b b i t 7 Day ( L e a v e - O n ) a 7-day l e a v e - o n dermal d o s e o f C-2,4-DNHPC was 1 3 % ( T a b l e I ) . Most o f t h e r e c o v e r e d ' C - l a b e l ( 7 1 % ; 3 9 % o f a p p l i e d d o s e ) was p r e s e n t i n o r on t h e a p p l i c a t i o n s i t e s k i n a t t h e end o f t h e 7-day i n - l i f e p h a s e ( T a b l e I I I ) . A t o t a l o f 17% o f t h e d o s e had been e l i m i n a t e d i n t h e e x c r e t a b y 7 days ( F i g u r e 3 ) . The e x c r e t e d ' C - l a b e l was e l i m i n a t e d i n a l i n e a r f a s h i o n a t a r a t e o f 1.4 and 1.0% o f t h e d o s e e x c r e t e d p e r d a y f r o m t h e u r i n e and f e c e s , respectively. C - 2 , 4 - D N H P C - d e r i v e d C - l a b e l was m o d e r a t e l y t o s l o w l y a b s o r b e d f o l l o w i n g a p p l i c a t i o n o f a l e a v e - o n dermal d o s e o f C-2,4-DNHPC, s i m i l a r t o 6 h r d e r m a l l y e x p o s e d r a b b i t s . A l s o , 7-day e x p o s e d a n i m a l s a c h i e v e d a s i m i l a r peak p l a s m a ^ - c o n c e n t r a ­ t i o n b y 6 h r compared t o 6 h r d e r m a l l y e x p o s e d a n i m a l s ( T a b l e I I ) . The 7-day e x p o s e d a n i m a l s e x h i b i t e d a s h o r t r a p i d e l i m i n a t i o n p h a s e ( t 1/2 = 3.3 h r ) , w h i c h p r e c e d e d a s t e a d y s t a t e p h a s e ; t h e l a t t e r p e r s i s t e d f o r t h e remainder o f t h e i n - l i f e phase. R a b b i t M u l t i p l e Dermal Dose S t u d y . S i x ( 6 ) % o f 13 d a i l y 6 h r dermal d o s e s o f 25 mg/kg/day C-2,4-DNHPC was a b s o r b e d ( T a b l e I ) . Most o f t h e m u l t i p l e dermal d o s e s was r e c o v e r e d i n t h e d a i l y 6 h r w i p e - o f f s ( 8 2 % o f d o s e ; 9 2 % o f r e c o v e r e d C - l a b e l ) ; 0.1% o f t h e t o t a l d o s e was r e c o v e r e d i n t h e a p p l i c a t i o n s i t e s k i n s 10 d a y s a f t e r t h e l a s t ( 1 3 t h ) dose ( T a b l e I I I ) . C - E x c r e t i o n was g r e a t e s t between 6 and 48 h r a f t e r t h e i n i t i a l d o s e ; e x c r e t i o n t h e n s l o w e d f o r t h e r e m a i n d e r o f t h e i n - l i f e p h a s e ( F i g u r e 4 ) . O n l y 8% o f t h e m u l t i p l e d o s e s had been e l i m i n a t e d i n t h e e x c r e t a b y t h e end o f t h e 10-day p o s t - d o s e p h a s e ( T a b l e I I I ) . Plasma ' C - c o n c e n t r a t i o n i n c r e a s e d approximately 6 - f o l d from 0.16 ppm 6 h r a f t e r t h e f i r s t d o s e t o a peak p l a t e a u ^ - c o n c e n ­ t r a t i o n o f 0.89 ppm 6 h r a f t e r t h e 1 3 t h ( l a s t ) d o s e ( T a b l e I I ) . The e l i m i n a t i o n o f ' C - l a b e l f r o m t h e p l a s m a f o l l o w i n g c o m p l e t i o n o f t h e d o s i n g p h a s e ( t 1/2 = 131 h r ) was c o m p a r a b l e t o t h e t e r m i n a l e l i m i n a t i o n r a t e f o l l o w i n g a s i n g l e 6 h r dermal e x p o s u r e o f C-2,4-DNHPC. Monkey I n t r a v e n o u s S t u d y . A t o t a l o f 49 and 3 5 % o f t h e d o s e was e x c r e t e d i n t h e u r i n e and f e c e s , r e s p e c t i v e l y , w i t h i n 4 d a y s . U r i n a r y ' C - e x c r e t i o n was e s s e n t i a l l y c o m p l e t e d w i t h i n 24 h r . C-2,4-DNHPC-derived C - l a b e l was r a p i d l y e l i m i n a t e d f r o m 4

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14 F i g u r e 4. C u m u l a t i v e e x c r e t i o n o f ' ^ C - l a b e l i n t h e u r i n e ( A ) , f e c e s ( • ) , and combined u r i n e a n d . f e c e s ( # ) f o l l o w i n g 13 d a i l y 6 h r dermal d o s e s o f 25 mg/kg/day C-2,4-DNHPC a p p l i e d a s t h e n e a t m a t e r i a l . D a t a p o i n t s r e p r e s e n t t h e mean + s t a n d a r d deviation. r

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t h e p l a s m a i n a b i p h a s i c manner; a r a p i d e l i m i n a t i o n phase ( t 1/2 = 1.4 h r ) l a s t i n g a p p r o x i m a t e l y 10 hr p r e c e d e d a s l o w e r e l i m i n a t i o n phase ( t 1/2 = 24 h r ) . Monkey 6 Hr Dermal E x p o s u r e S t u d i e s . The p e r c e n t dermal a b s o r p t i o n o f 40 and 2500 ug/cm^ i n monkeys was 16 and 5%, r e s p e c t i v e l y Table I ) . Most o f a dermal dose o f 1 C-2,4-DNHPC was r e c o v e r e d i n t h e 6 h r w i p e - o f f [69 and 95% o f t h e r e c o v e r e d C - l a b e l (29 and 75% o f t h e a p p l i e d d o s e ) f o l l o w i n g 40 and 2500 ug/cm , r e s p e c t i v e l y ] . A t o t a l o f 13 and 4% o f t h e a p p l i e d dose was e l i m i n a t e d i n t h e e x c r e t a w i t h i n 7 days f o l l o w i n g 40 and 2500 ug/cm , r e s p e c t i v e l y ( T a b l e I I I ) . The e x c r e t e d ' C - l a b e l was e l i m i n a t e d a t a f a i r l y l i n e a r r a t e o v e r most o f t h e 4-day i n - l i f e p h a s e , e s p e c i a l l y f r o m t h e 40 ug/crrr d o s e d a n i m a l s ( F i g u r e 5 ) . The e x c r e t e d ' C - l a b e l was e s s e n t i a l l y e v e n l y d i s t r i b u t e d between t h e u r i n e and f e c e s , s i m i l a r to the ' C - e x c r e t i o p a t t e r observed f o 6 h dermall exposed r a b b i t s . Plasma ' ^ - c o n c e n t r a t i o n rang backgroun t w i c e b a c k g r o u n d ( l i m i t o f s e n s i t i v i t y ^ 0.002 ppm) f o l l o w i n g dermal a p p l i c a t i o n o f C-2,4-DNHPC ( T a b l e I I ) . 4

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Discussion A c o n c u r r e n t l y performed study indicated that dinocap technical d i d n o t p r o d u c e d e v e l o p m e n t a l t o x i c i t y i n r a b b i t s f o l l o w i n g dermal a p p l i c a t i o n o f 25, 50, and 100 mg/kg/day ( 1 ) . D i n o c a p d i d c a u s e i r r i t a t i o n a t a l l dose l e v e l s , however. B e c a u s e o f t h e s e v e r i t y o f t h e i r r i t a t i o n , 100 mg/kg/day was t h e h i g h e s t dose s t u d i e d . Even a t t h a t , t h e a p p l i c a t i o n s i t e needed t o be r o t a t e d among 7 s i t e s s u c h t h a t c o n s e c u t i v e d o s e s were n o t a p p l i e d t o t h e same o r a d j a c e n t s i t e s . M a t e r n a l t o x i c i t y , m a n i f e s t e d by r e d u c e d f e e d c o n s u m p t i o n and r e d u c e d w e i g h t g a i n , was p r o d u c e d a t 100 mg/kg/day. The NOEL f o r d i n o c a p - i n d u c e d d e v e l o p m e n t a l t o x i c i t y by t h e dermal r o u t e was > 100 mg/kg/day. R e s u l t s o f t h e a b s o r p t i o n s t u d i e s s u p p o r t t h e c o n c l u s i o n t h a t d i n o c a p does n o t c a u s e d e v e l o p m e n t a l t o x i c i t y i n r a b b i t s by t h e dermal r o u t e i n t h a t a) t h e dermal a b s o r p t i o n o f d i n o c a p i s s i g n i f i c a n t l y l e s s t h a n t h e o r a l a b s o r p t i o n , b) peak p l a s m a ' C - l e v e l s a r e much l e s s a f t e r dermal a p p l i c a t i o n compared t o o r a l a d m i n i s t r a t i o n o f an e q u i v a l e n t d o s e , and c ) d i n o c a p does n o t a c c u m u l a t e upon m u l t i p l e dermal dose a d m i n i s t r a t i o n . The r a b b i t o r a l and dermal a b s o r p t i o n s t u d i e s d e s c r i b e d h e r e were d e s i g n e d t o s i m u l a t e t h e d e s i g n s o f t h e o r a l and dermal d e v e l o p m e n t a l t o x i c i t y s t u d i e s . The o r a l d o s e s i n t h e a b s o r p t i o n s t u d y i n c l u d e d t h e NOEL and MEL d o s e s o f t h e o r a l d e v e l o p m e n t a l t o x i c i t y s t u d i e s . The dermal d o s e s i n t h e a b s o r p t i o n s t u d y b r a c k e t e d t h o s e used i n t h e dermal d e v e l o p m e n t a l t o x i c i t y s t u d y ; t h e h i g h dermal d o s e o f 220 mg/kg i n t h e a b s o r p t i o n s t u d y r e p r e s e n t e d t h e maximum d o s e o f c o m m e r c i a l d i n o c a p LC f o r m u l a t i o n a t p h y s i c a l l y c o u l d be a p p l i e d t o a 40 cm d o s e a r e a . C-2,4-DNHPC was d e r m a l l y a p p l i e d as t h e n e a t m a t e r i a l t o s i m u l a t e t h e e x p o s u r e t o d i n o c a p t e c h n i c a l i n t h e dermal d e v e l o p m e n t a l t o x i c i t y s t u d y . F i n a l l y , 13 d a i l y 6 hr dermal d o s e s o f 4

American Chemical Society Library 1155 ISth St., N.W. In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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F i g u r e 5. C u m u l a t i v e e x c r e t i o n o f C - l a b e l i n t h e u r i n e ( 0 ) » f e c e s ( # ) , and combined u r i n e and f e c e s (D^-of f e m a l e r h e s u s monkeys f o l l o w i n g a 6 h r dermal e x p o s u r e o f C-2,4-DNHPC i n acetone.

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C-2,4-DNHPC were a p p l i e d t o 7 r o t a t i n g a p p l i c a t i o n s i t e s t o s i m i l a t e the dosing regimen o f the developmental t o x i c i t y study. Results o f the C-2,4-DNHPC m u l t i p l e dermal d o s e s t u d y i n d i c a t e d t h a t d i n o c a p does not a c c u m u l a t e i n t h e s k i n o r a l t e r t h e b a r r i e r f u n c t i o n o f the s k i n as t o permit greater absorption f o l l o w i n g m u l t i p l e d o s e a p p l i c a t i o n compared t o s i n g l e d o s e a p p l i c a t i o n . S i m i l a r r e s u l t s have been o b s e r v e d f o r m a l a t h i o n and s e v e r a l s t e r o i d s i n humans (4, 5 ) . A d d i t i o n a l g r o u p s were i n c l u d e d i n t h e d e s i g n o f t h e dermal a b s o r p t i o n study to i n v e s t i g a t e other parameters u s e f u l i n the r i s k e s t i m a t i o n o f dinocap. For example, dinocap i s s o l d commercially as both t h e WD and LC f o r m u l a t i o n s . The s i m i l a r i t y i n t h e dermal a b s o r p t i o n and p h a r m a c o k i n e t i c s o f C-2,4-DNHPC a p p l i e d a s t h e WD and LC f o r m u l a t i o n s compared t o t h e n e a t m a t e r i a l s t r o n g l y s u g g e s t s t h a t t h e s e f o r m u l a t i o n s do n o t p o s e any more o f a d e v e l o p m e n t a l r i s k i n r a b b i t s t h a n does t h e t e c h n i c a l m a t e r i a l The e f f e c t o f a c e t o n e on t h e dermal a b s o r p t i o s t u d i e d t o compare t h e s The monkey d i n o c a p dermal s t u d y u t i l i z e d a c e t o n e a s t h e v e h i c l e , s i n c e a s i g n i f i c a n t d a t a b a s e o f human and monkey dermal a b s o r p t i o n s t u d i e s e x i s t s which u t i l i z e d acetone as the v e h i c l e (6-10). It i s n o t e w o r t h y t h a t t h e dermal a b s o r p t i o n o f d i n o c a p i n r a b b i t s and monkeys was s i m i l a r , s i n c e a number o f compounds a r e known t o be more p e r m e a b l e i n r a b b i t s t h a n i n monkeys and humans ( 1 0 ) . R e s u l t s i n d i c a t e d t h a t o r a l and dermal doses o f C-2,4-DNHPC i n r a b b i t s were each h a n d l e d i n a s i m i l a r ( l i n e a r ) f a s h i o n p h a r m a c o k i n e t i c a l l y . F i r s t , t h e p e r c e n t a b s o r p t i o n was s i m i l a r f o r a l l doses a f t e r each r o u t e o f a d m i n i s t r a t i o n ( T a b l e I ) . S e c o n d , the f r a c t i o n o f the excreted ' C - l a b e l recovered i n the urine o r f e c e s was s i m i l a r f o r a l l d o s e l e v e l s f o r each r o u t e o f a d m i n i s t r a t i o n ( T a b l e I I I ) . T h i r d , t h e peak p l a s m a ' ^ - c o n c e n t r a ­ t i o n was p r o p o r t i o n a l t o d o s e a f t e r each r o u t e o f e x p o s u r e . F o u r t h , t h e e l i m i n a t i o n o f ^ C - l a b e l f r o m t h e p l a s m a and t h e p a t t e r n s o f e l i m i n a t i o n o f C - l a b e l i n t h e e x c r e t a were s i m i l a r f o r a l l doses a f t e r each r o u t e o f a d m i n i s t r a t i o n . T h u s , t h e observed t o x i c i t y a t the h i g h e r dose l e v e l s f o l l o w i n g o r a l ( d e v e l o p m e n t a l t o x i c i t y ) o r dermal ( m a t e r n a l t o x i c i t y ) a d m i n i s t r a t i o n d i d not a p p e a r t o a l t e r t h e a b s o r p t i o n , d i s t r i b u t i o n , o r e l i m i n a t i o n o f C-2,4-DNHPC compared t o l o w e r , n o - e f f e c t d o s e s . The l i n e a r p h a r m a c o k i n e t i c s i n d i c a t e t h a t e x t r a p o l a t i o n f r o m h i g h , e f f e c t - l e v e l d o s e s t o l o w e r n o - e f f e c t l e v e l doses may be p e r f o r m e d with greater confidence. The monkey was used a s a model t o e s t i m a t e t h e dermal a b s o r p t i o n o f d i n o c a p i n humans, s i n c e t h e monkey has been shown t o be an a p p r o p r i a t e model f o r s t u d y i n g dermal a b s o r p t i o n o f v a r i o u s x e n o b i o t i c s i n man (10, 1 1 ) . The dermal a b s o r p t i o n o f d i n o c a p i n monkeys was i n v e s t i g a t e d a t two c o n c e n t r a t i o n s w h i c h a p p r o x i m a t e p o t e n t i a l w o r k e r e x p o s u r e c o n c e n t r a t i o n s . The h i g h c o n c e n t r a t i o n (2500 ug/cm ), w h i c h was a l s o used i n t h e r a b b i t dermal d e v e l o p ­ mental and a b s o r p t i o n s t u d i e s , a p p r o x i m a t e s t h e e x p o s u r e w h i c h might r e s u l t from contact with u n d i l u t e d dinocap product ( S t r e e l m a n , D. R., Rohm and Haas Company I n t e r - C o m p a n y Communica­ t i o n , 1 9 8 6 ) . The low c o n c e n t r a t i o n (40 ug/cnrr) a p p r o x i m a t e s t h e exposure which might r e s u l t from c o n t a c t with dinocap f o r m u l a t i o n s 14

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at t h e i r u s e d i l u t i o n . The p e r c e n t a b s o r p t i o n was 3 - f o l d g r e a t e r at t h e l o w e x p o s u r e c o n c e n t r a t i o n compared t o t h e h i g h c o n c e n t r a ­ t i o n (16 v e r s u s 5 % ) . A g r e a t e r e f f i c a c y o f dermal a b s o r p t i o n a t lower e x p o s u r e c o n c e n t r a t i o n s h a s been o b s e r v e d f o r a number o f compounds ( 9 , 1 2 ) . S i n c e d i f f e r e n t w o r k e r e x p o s u r e s i t u a t i o n s ( i . e . , working with t h e f u l l concentrate o r t h e u s e - d i l u t i o n ) can r e s u l t i n s u b s t a n t i a l l y d i f f e r e n t c o n t a c t e x p o s u r e s , i t i s i m p e r a t i v e t h a t t h e a p p r o p r i a t e a b s o r p t i o n f a c t o r s be used when b a s i n g t h e r i s k e s t i m a t i o n o f d i n o c a p on a b s o r b e d d o s e . Conclusions The r i s k a s s e s s m e n t f o r d i n o c a p i n c l u d e d a) t h e d e t e r m i n a t i o n t h a t t h e p e r c e n t dermal a b s o r p t i o n o f d i n o c a p i n r a b b i t s ( a s e n s i t i v e s p e c i e s f o r d i n o c a p - i n d u c e d d e v e l o p m e n t a l t o x i c i t y ) was s i m i l a r t o t h e dermal a b s o r p t i o n i n monkeys ( a non-human p r i m a t e m o d e l ) and b) t h e measurement o f t h p o t e n t i a l worker exposur m o d e l . T h i s a p p r o a c h i s both p r a c t i c a l and a p p r o p r i a t e f o r u s e i n e v a l u a t i n g t h e r i s k o f d i n o c a p t o man by t h e dermal r o u t e . U s i n g t h i s a p p r o a c h , t h e dermal a b s o r p t i o n o f f u l l c o n c e n t r a t e d i n o c a p f o r m u l a t i o n s i n man i s e s t i m a t e d t o be 5% o f t h e a p p l i e d d o s e . T h e dermal a b s o r p t i o n o f d i n o c a p a t i t s u s e - d i l u t i o n i n man i s e s t i m a t e d t o be 1 6 % o f t h e a p p l i e d d o s e . T h e s e a b s o r p t i o n d a t a , i n c o n j u n c t i o n with measured f i e l d exposures, support t h e c o n c l u s i o n t h a t a g r i c u l t u r a l uses o f d i n o c a p do n o t p o s e an u n r e a s o n a b l e d e v e l o p m e n t a l r i s k t o man. Acknowledgments. The authors acknowledge t h e expert t e c h n i c a l help o f Ms. Donna M. A n d e r s o n , Mr. Howard L. T o m l i n s o n , and Mr. Thomas W. G r e g e r o f Rohm and Haas Company; Mr. D a n i e l A. W. Bucks and Mr. James M c M a s t e r o f U n i v . o f C a l i f o r n i a San F r a n c i s c o ; and Mr. R o b e r t Sarason o f t h e Primate Center o f t h e U n i v e r s i t y o f C a l i f o r n i a a t D a v i s . D r . D i a n a D. Bender and Mr. R o n a l d P. Owen o f Rohm and Haas Company s y n t h e s i z e d t h e C-2,4-DNHPC and n o n r a d i o l a b e l e d 2,4-DNHPC, r e s p e c t i v e l y .

Literature Cited 1. Costlow, R. D . ; Lutz, M. F.; Kane, W. W.; Hurt, S. S . ; O'Hara, G. P. Toxicologist 1986, 6, 85. 2. Shah, P. V.; Guthrie, F . E . J. Invest. Dermatol. 1983, 80, 291-293. 3. Wester, R. C.; Maibach, H. I . In Dermatotoxicity, 2nd Edition; M a r z u l l i , F . N.; Maibach, H. I., E d s . ; Hemisphere Publishing Corporation: New York, 1983; pp 131-146. 4. Wester, R. C.; Maibach, H. I.; Bucks, D. A. W.; Guy, R. H. Toxicol. Appl. Pharmacol. 1983, 68, 116-119. 5. Bucks, D. A. W.; Maibach, H. I.; Guy, R. H. J. Pharm. S c i . 1985, 74, 1337-1339. 6. Feldmann, R. J.; Maibach, H. I. J. Invest. Dermatol. 1970, 54, 399-404. 7. Bartek, M. J.; LaBudde, J. A.; Maibach, H. I. J. Invest. Dermatol. 1972, 58, 114-123.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

11. LONGACREETAL.

Dinocap Dermal Absorption

8.

Feldmann, R. J.; Maibach, H. I. T o x i c o l . Appl. Pharmacol. 1974, 28, 126-132. 9. Wester, R. C.; Maibach, H. I. J. T o x i c o l . Environ. Health 1985, 16, 25-37. 10. Wester, R. C.; Maibach, H. I. In Percutaneous Absorption; Bronaugh, R. L.; Maibach, H. I., E d s . ; Marcel Dekker, I n c . : New York, 1985; Chapter 20. 11. Wester, R. C.; Maibach, H. I. T o x i c o l . Appl. Pharmacol. 1975, 32, 394-398. 12. Wester, R. C.; Maibach, H. I. In Percutaneous Absorption; Bronaugh, R. L.; Maibach, H. I., E d s . ; Marcel Dekker, I n c . : New York, 1985; Chapter 26. RECEIVED December 4, 1987

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 12

Percutaneous Absorption in Rhesus Monkeys and Estimation of Human Chemical Exposure 1

1

1

Ronald C. Wester , James McMaster , Daniel A. W. Bucks , Eugene M . Bellet , and Howard I. Maibach 2

1

1

Department of Dermatology, University of California School of Medicine, San Francisco, CA 94143 Chemical Consultants International, Overland Park, KS 66204 2

Skin absorption estimat for predicting huma entering the body through skin. An example is Dinoseb, which, when applied to skin of rats (acetone vehicle; 72 hr exposure) is 90% absorbed. The literature shows absorption through rat skin to be much higher than in man. Dinoseb applied to the skin of rhesus monkeys (Premerge-3; 24 hr exposure) gave absorption of less than 10 percent. Balance in the monkey study accounted for greater than 80 percent of the applied dose, the majority of which was unabsorbed Dinoseb removed from the skin with the post 24 hr soap and water wash. Comparative absorption data for other chemicals show skin absorption in rhesus consistent with man. Central to the determination of human health hazard effects for skin exposure of chemicals is the estimate for percutaneous absorption. This value estimates the rate and extent that a chemical will penetrate the living human epidermis and becomes systemically available for distribution in the human body. Percutaneous absorption is a complex process involving many variables which can affect the estimate for systemic availability (1). However, the objective is crystal clear; an estimate of percutaneous absorption in vivo in man. Therefore, the study design should minimize variables and include aspects most relevant to man. This paper focuses on the variables of the animal model, and on accountability for applied dose. It is hoped that the conclusions derived from the information presented here will aid in producing relevant study designs and continued relevant data. Animal Model The best estimate for human percutaneous absorption would be an in vivo study in man. Factors such as risk, cost, and access to human volunteers necessitate use of an animal model. Cost and access c

0097-6156/89/0382-0152$06.00/0 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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WESTER ET AL.

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153

suggest a s m a l l l a b o r a t o r y a n i m a l . The h i s t o r i c a l use o f t h e r a t i n t o x i c o l o g i c a l s t u d i e s s u g g e s t s i t s u s e . What may g e t l o s t i n t h e c h o i c e o f a n i m a l i s t h e p r i o r i t y o f r e l e v a n c e t o man f o r percutaneous absorption. T a b l e I shows t h e percutaneous a b s o r p t i o n o f s e v e r a l c h e m i c a l s by r a t / r a b b i t / p i g and man ( 2 ) . Q u e s t i o n s f o r r e l e v a n c y s h o u l d be i f t h e s k i n a b s o r p t i o n i n t h e a n i m a l i s t h e same a s i n man and/ i f not t h e same, then i s t h e s k i n a b s o r p t i o n c o n s i s t e n t l y p r o p o r t i o n a l l y d i f f e r e n t from man? The answer t o b o t h q u e s t i o n s f o r r a t and r a b b i t i s no. F o r t h e p i g , t h e answer i s a maybe.

T a b l e I . Percutaneous A b s o r p t i o n o f S e v e r a l Compounds by R a t , R a b b i t , P i g and Man ( i n v i v o ) Penetrant

P e r c e n t Dose Absorbed ra

Haloprogin Acetylcysteine Cortisone Caffeine Butter yellow Testosterone

95 .8 3 .5 24 .7 53 .1 48 .2 47 .4

113. 0 2.0 30. 3 69. 2 100. 0 69. 6

19 .7 6 .0 4 .1 32 .4 41 .9 29 .4

11..0 2..4 3..4 47,.6 21,.6 13..2

Source: B a r t e k e t a l (2)

I t i s beyond t h i s paper's scope t o p r e s e n t a l l c o m p a r a t i v e d a t a i n v o l v i n g i n v i v o and i n v i t r o s k i n a b s o r p t i o n i n l a b o r a t o r y a n i m a l s and man. D e t a i l s a r e i n r e f e r e n c e s ( 3 - 7 ) . T h i s paper f o c u s e s on t h e use o f t h e r a t and t h e r h e s u s monkey a s r e l e v a n t a n i m a l models. T a b l e I I shows t h e c o m p a r a t i v e i n v i v o percutaneous a b s o r p t i o n of t e s t o s t e r o n e i n s e v e r a l s m a l l s p e c i e s . Absorption i n the l a b o r a t o r y s p e c i e s ( r a t , r a b b i t , g u i n e a p i g ) a r e on t h e upper end o f t h e a b s o r p t i o n s c a l e , w h i l e t h o s e i n t h e p i g , r h e s u s monkey and man a r e on t h e lower end o f t h e s c a l e .

T a b l e I I . Comparative Percutaneous A b s o r p t i o n o f T e s t o s t e r o n e on S e v e r a l S p e c i e s Species Rat Rabbit Guinea p i g Rhesus monkey Man

P e r c e n t o f Dose Absorbed 47.4 69.6 34.9 18.4 13.2

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

T a b l e I I I summarizes t h e e x i s t i n g c o m p a r a t i v e i n v i v o percutaneous a b s o r p t i o n i n t h e r h e s u s monkey and man. The l i s t i n c l u d e s a v a r i e t y o f c h e m i c a l s and a broad range o f s k i n a b s o r p t i o n e s t i m a t e s . I n a l l c a s e s , t h e percutaneous a b s o r p t i o n i n t h e r h e s u s monkey i s c o n s i s t e n t w i t h t h a t found i n man. Table I I I .

I n V i v o Percutaneous A b s o r p t i o n i n t h e Rhesus Monkey and Man

Chemical

P e r c e n t Dose Absorbed Rhesus Monkey

2,4-Dinitrochlorobenzene Nitrobenzene Cortisone Testosterone H y d r o c o r t i sone Benzoic a c i d Resorcinol P-Phenylendiamine 2-Nitro-PPD HC-Blue #1

Reference Man

52.5 + 4.3 4.2 + 0.5 5.3 + 3.3

53.5 + 6.2 1.5 + 0.3 3.4 + 1.6

59.2 + 7.6 0.18 + 0.03 0.18 + 0.06 0.55 + 0.10 0.13 + 0.03

42.6 + 16.4 0.08 + 0.03 0.19 + 0.04 0.1 4+ 0.04 0.15 + 0.12

(8) (8) (9)

(10) (11) (11) (11) (11)

B a s i c t o s c i e n t i f i c i n q u i r y i s t h e dose r e s p o n s e . F o r s k i n a b s o r p t i o n i n man, t h e p e r c e n t dose absorbed w i l l be dependent on t h e a p p l i e d dose ( T a b l e IV, r e f e r e n c e s 10,12). The dose response i s s i m i l a r f o r t h e r h e s u s monkey.

T a b l e I V . Percutaneous A b s o r p t i o n o f I n c r e a s e d T o p i c a l Doses o f S e v e r a l Compounds i n t h e Rhesus Monkey and Man ( i n v i v o ) Penetrant

Dose ug/cm2

Percent of Dose Absorbed Man Rhesus

4 40

2.9 2.1

1.9 0.6

Benzoic A c i d

4 40 2,000

59.2 33.6 17.4

42.6 25.7 14.4

Testosterone

4 40 250 400 1,600 4,000

18.4 6.7 2.9 2.2 2.9 1.4

13.2 8.8a

Hydrocortisone

a

2.8

30 ug/cm2.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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WESTER ETAL.

155

Percutaneous Absorption in Monkeys

Dose A c c o u n t a b i l i t y The p r e c e d i n g s e c t i o n on a n i m a l models s u g g e s t s t h e p o s s i b i l i t y o f d i f f e r e n c e s i n s k i n a b s o r p t i o n and subsequent human h e a l t h h a z a r d e f f e c t e s t i m a t e s dependent upon a n i m a l s p e c i e s . The p e r c e n t s k i n p e n e t r a t i o n d a t a from T a b l e V would e s t i m a t e , based on t h e r a t , t h a t a l l o f t h e Dinoseb which g e t s on s k i n i s absorbed. Human h e a l t h h a z a r d e s t i m a t e s would be p r e j u d i c e d i n t h i s d i r e c t i o n . A b s o r p t i o n i n the r h e s u s monkey would suggest t h a t an a b s o r p t i o n o f no more t h a n t e n p e r c e n t would b e t t e r e s t i m a t e human h e a l t h h a z a r d s . The o b v i o u s q u e s t i o n w i t h t h e r h e s u s monkey d a t a i s an a c c o u n t a b i l i t y o f t h e m a j o r i t y o f the dose t o ensure t h a t n o t h i n g p e c u l i a r happened d u r i n g t h e s t u d y . T a b l e VI g i v e s an a c c o u n t a b i l i t y o f t h e a p p l i e d Dinoseb dose. The m a j o r i t y o f th e dose appeared i n t he 24 hour p o s t a p p l i c a t i o n soap and water s k i n wash, and t h u s was not absorbed. Thus, t he l e s s than t e n p e r c e n t a b s o r p t i o n i s r e a l and i s p r o b a b l y more r e l e v a n t t o man t h a n th

T a b l e V.

A p p l i e d Dose ug/cm2

I n V i v o Percutaneous A b s o r p t i o n o f Dinoseb i n Rhesus Monkey and R a t

Percent S k i n P e n e t r a t i o n

a

P e r c e n t Dose Accountabi1ity

RAT 51.5 128.8 643.5

86.4 90.5 93.2

+ + +

1.1 1.1 0.6

87.9 91.5 90.4

+ +

+

86.0 81.2 80.3

+ + +

1.8 0.6 0.7

RHESUS MONKEY 43.6 200.0 3,620.0

5.4 7.2 4.9

+ +

T

2.9 6.4 3.4

4.0 18.1 5.2

Rat = acetone v e h i c l e ; 72 h r a p p l i c a t i o n Monkey = Premerge-3 v e h i c l e ; 24 hour a p p l i c a t i o n Complete Dinoseb d a t a t o be p u b l i s h e d elsewhere R e f e r e n c e 13 f o r r a t d a t a Study D e s i g n O b j e c t i v e A s t u d y d e s i g n s h o u l d r e f l e c t a c l e a r o b j e c t i v e . I n t h e case o f Dinoseb, the r h e s u s monkey s t u d y was d e s i g n e d t o b e s t e s t i m a t e human s k i n a b s o r p t i o n f o r a 24 hour exposure p e r i o d . Dinoseb was a p p l i e d as i t s c o m m e r c i a l l y a v a i l a b l e f o r m u l a t i o n (Premerge-3), and t h e doses

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Table VI.

P e r c u t a n e o u s A b s o r p t i o n and A c c o u n t a b i l i t y o f Dinoseb I n V i v o Study i n t h e Rhesus M o n k e y 2

Disposition Parameter

1+3.6

A p p l i e d Dose (ug/cm ) 200.0

P e r c e n t A p p l i e d Dose Urine Feces Contaminated Pan wash S k i n wash

solids

Total Accountability a

Complete

a

3,620.0

Accountability

3.3 + 1.8 0.8+ 0.5 0.03±0.02 0.04+0.03 81.1+4.0

4.4 + 2.39 3.0 ± 2.1 1.0 + 0.6 3.0 +_ 1.7 0.07± 0.08 0.02+ 0.02 0.4 + 0.3 0.8 + 1.1 75.0 + 22.9 73.8 + 6.8

86.0+4.0

81.2 + 18.1

80.3 + 5.2

Dinoseb d a t

r e p r e s e n t c o n c e n t r a t i o n s which human s k i n would be exposed t o . The r a t d a t a was t a k e n out o f c o n t e x t , s i n c e i t s o b j e c t i v e was t o compare s k i n a b s o r p t i o n o f young and o l d s k i n f o r a 72 hour exposure o f Dinoseb when a p p l i e d t o s k i n i n a c e t o n e . The comparison o f t h e r a t s t u d y e x t r a p o l a t e d t o a h e a l t h h a z a r d assessment i n humans would be f o r human a p p l i c a t o r s t o use Dinoseb, mixed i n a c e t o n e , c o n t i n u a l l y f o r 72 hours w i t h o u t t h e b e n e f i t o f a shower o r b a t h f o r the 3 day p e r i o d . T h i s c l e a r l y p o i n t s up t h e f a l l a c y o f u s i n g i n a p p r o p r i a t e d a t a from a n i m a l models i n t h e e x t r a p o l a t i o n o f h e a l t h h a z a r d e f f e c t s t o man. The d e s i g n o f a p r o p e r s t u d y complete w i t h w e l l though out o b j e c t i v e s w h i c h a r e t i e d i n a r a t i o n a l s c i e n t i f i c manner t o human h e a l t h exposure i s what i s r e q u i r e d .

A n i m a l Percutaneous A b s o r p t i o n Data t o E s t i m a t e Human Exposure The o b j e c t i v e o f percutaneous a b s o r p t i o n d a t a i s t o e s t i m a t e t h e amount o f c h e m i c a l which w i l l t r a n s f e r from t h e s u r f a c e o f s k i n t h r o u g h t h e e p i d e r m i s i n t o t h e s y s t e m i c c i r c u l a t i o n . That which g e t s i n t o the e p i d e r m i s o r s y s t e m i c c i r c u l a t i o n d e t e r m i n e s t h e e x i s t e n c e or degree o f l o c a l o r s y s t e m i c t o x i c i t y . Thus, s k i n a b s o r p t i o n i s p i v o t a l and i m p o r t a n t i n e s t i m a t i n g p o t e n t i a l h e a l t h h a z a r d s f o r t o p i c a l exposure, and r e l e v a n t i n f o r m a t i o n i s n e c e s s a r y . Data i n a n i m a l models not r e l e v a n t t o man f o r s k i n a b s o r p t i o n c e r t a i n l y cannot g i v e a t r u e e s t i m a t e , and i n f a c t may l e a d t o f u r t h e r confusion. The b e s t a n i m a l model f o r e s t i m a t i n g percutaneous a b s o r p t i o n r e l e v a n t t o man i s man him(her) s e l f . Y e t e s p e c i a l l y w i t h a g r i c u l t u r e chemicals there i s c o n t i n u a l searching f o r "relevant" animal data, when t h e c o r r e c t answers can e a s i l y be o b t a i n e d i n man. Perhaps when r e g u l a t o r y and m a n a g e r i a l p e o p l e r e a l i z e t h i s , t h e emphasis w i l l s h i f t t o t h e p r o p e r human s t u d i e s .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

12.

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157

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Wester, R.C.; Maibach, H.I. Drug Metab. Rev. 1983, 14, 169-205 Bartek, M.J.; La Budde, J.A.; Maibach, H.I. J. Invest. Dermatol.1972, 58, 114-23. Wester, R.C.; Noonan, P.K. Int. J. Pharmaceutics, 1980, 7, 99-110. Wester, R.C.; Maibach, H.I. In Models in Dermatology; H.I. Maibach and N.J. Lowe, Eds.; S. Karger: Basel, 1985, 159-169. Wester, R.C.; Maibach, H.I. In Percutaneous Absorption; R.L. Bronaugh and H.I. Maibach, Eds.; Marcel Dekker: New York, 1985; 251-66. Wester,R.C.,Maibach, H.I. In Progress in Drug Metabolism; J.W. Bridges and L.F. Chasseaud, Eds.; Taylor and Francis: Philadelphia, 1986; Chapter 3. Wester, R.C.; Maibach Marzulli and H.I 135-52. Bronaugh, R.L. In Percutaneous Absorption; R.L. Bronaugh and H.I. Maibach, Eds.; Marcel Dekker: New York; 1985; 267-279. McMaster, J.; Maibach, H.I., Wester, R.C.; Bucks, D.A.W. In Percutaneous Absorption; R. Bronaugh and H. Maibach, Eds.; Marcel Dekker: New York; 1985; 359-61. Wester, R.C.; Maibach, H.I.; Toxicol. Appl. Pharmacol. 1975, 32; 394-98. Wolfram, L.J. In Percutaneous Absorption; R. Bronaugh and H.I. Maibach, Eds; Marcel Dekker: New York, 1985; 409-22. Wester, R.C.; Maibach, H.I. J. Invest. Dermatol. 1976, 67; 518-20. Shah, P.V.; Fisher, H.L.; Sumler, M.R.; Monroe, R.J.; Chernoff, N.;Hall, L.L. J. Toxicol. Environ. Hlth.; 1987, 353-66.

RECEIVED January 25, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter

13

Percutaneous Absorption In Vitro Technique as an Alternative to In Vivo Assessments R. C. Scott Central Toxicology Laboratory, Imperial Chemical Industries PLC, Alderley Park, Nr. Macclesfield, Cheshire SK10 4TJ, United Kingdom Chemicals, during manufacture and use may come into contact with human skin and have the potential to be absorbed. Hence evaluation shoul absorption. Currently, Regulatory Agencies require in vivo dermal absorption studies for this quantitation. Such in vivo data, obtained using animal skin, are used to predict the potential hazard to man. As the permeability of animal skin and human skin has been shown to be different (for many chemicals) extrapolation is difficult. In the studies reported in this paper in vivo and in vitro absorption data using rat skin have been obtained with two molecules, cypermethrin and carbaryl, which have different physicochemical properties. For both these chemicals the in vitro absorption data which were obtained predicted the in vivo data. In vitro carbaryl absorption data were also obtained with human skin and compared with published in vivo data. Again, the in vitro data predicted the available in vivo results. As in vitro data are more readily obtained and as human skin can be used (thus eliminating any extrapolation step from animal to man) the in vitro system offers a geniune alternative and improvement to in vivo animal experiments. Safety evaluation of pesticides and their formulations requires an integrated series of toxicity studies. By necessity, animals are used as models for man and the data are then extrapolated to predict the potential hazard to man. Toxicity after the percutaneous absorption of toxic active ingredients, such as pesticides, is assessed currently using in vivo absorption data determined using an animal skin (usually rat or rabbit). This method is used since chemicals with only a limited toxicology data base cannot, for ethical reasons, be tested in man directly. 0097-6156/89/0382-0158$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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However, once s u f f i c i e n t i n f o r m a t i o n has been g a t h e r e d t o enable a f u l l e r u n d e r s t a n d i n g of the t o x i c o l o g i c a l p r o f i l e of an a c t i v e i n g r e d i e n t , human v o l u n t e e r s t u d i e s might then be p o s s i b l e . I n e v i t a b l y t h e a b i l i t y t o conduct such s t u d i e s i s determined by c o n s i d e r a t i o n s of e t h i c a l committees who approve p r o t o c o l s . Often p e s t i c i d e s a r e prepared i n many f o r m u l a t i o n s and t h e problem o f , f o r example, a s s e s s i n g t h e s a f e t y of s e v e r a l n o v e l f o r m u l a t i o n s ( i e the f o r m u l a t i o n which r e s u l t e d i n the l o w e s t a b s o r p t i o n r a t e ) w i t h the same a c t i v e i n g r e d i e n t by t h e human v o l u n t e e r e x p e r i m e n t a l r o u t e would be a d a u n t i n g t a s k . I n v i v o , a n i m a l s t u d i e s a r e o n l y m a r g i n a l l y l e s s complex, a l t h o u g h the a c q u i s i t i o n of d a t a i s f a c i l i t a t e d by more ready a c c e s s t o t e s t s u b j e c t s . I n o r d e r t o f a c i l i t a t e the development of d a t a on t h e a b s o r p t i o n of p e s t i c i d e s from v a r i o u s f o r m u l a t i o n s , i n v i t r o t e c h n i q u e s have been developed t o p r e d i c t i n v i v o percutaneous absorption. The major advantag use of human t i s s u e , t h r e d u c t i o n i n t h e use of a n i m a l s . I t i s t h e i n t e n t i o n of t h i s paper t o show t h a t t h e percutaneous a b s o r p t i o n of p e s t i c i d e s can be measured i n v i t r o w i t h t h e same degree o f c e r t a i n t y and a c c u r a c y demanded from i n v i v o s t u d i e s . Some of t h e p o t e n t i a l e x p e r i m e n t a l p i t f a l l s f o r t h e unwary e x p e r i m e n t a l i s t a r e a l s o d i s c u s s e d and comparisons w i t h i n v i v o d a t a p r e s e n t e d . The i n v i v o and i n v i t r o a b s o r p t i o n , through r a t s k i n , of a p o o r l y absorbed l i p o p h i l i c m o l e c u l e , t h e i n s e c t i c i d e c y p e r m e t h r i n , has been s t u d i e d ( 1 ) . I n t h i s study 3\il of a p o t e n t i a l c o n c e n t r a t e f i e l d f o r m u l a t i o n (nominal c y p e r m e t h r i n c o n c e n t r a t i o n , 3 5 9 g / l ) , o r 2 0 u l of an o i l - b a s e d spray d i l u t i o n (nominal c y p e r m e t h r i n c o n c e n t r a t i o n , 2 9 g / l ) , was a p p l i e d t o t h e shorn backs of 4-5 week o l d r a t s (Alpk/AP s t r a i n , W i s t a r d e r i v e d ) . The a p p l i c a t i o n s i t e was l e f t open t o the atmosphere but p r o t e c t e d by a p l a s t i c guard. To f a c i l i t a t e the d e t e r m i n a t i o n of a b s o r p t i o n , [-^C] c y p e r m e t h r i n was added t o t h e f o r m u l a t i o n . A b s o r p t i o n was a s s e s s e d a f t e r 8 hours. T h i s time p e r i o d was s e l e c t e d t o r e p r e s e n t a t y p i c a l working day p e r i o d . With t h e spray d i l u t i o n t h e t i m e c o u r s e of a b s o r p t i o n over a 24 hour c o n t a c t p e r i o d was determined. I n b o t h s t u d i e s , d e t e r m i n a t i o n of t o t a l a b s o r p t i o n up t o a p a r t i c u l a r time p o i n t r e q u i r e d a n i m a l s t o be k i l l e d humanely p r i o r to t h e a n a l y s i s of the s k i n s i t e , u r i n e , f a e c e s and v a r i o u s t i s s u e s f o r [ C ] c o n t e n t (which was regarded as e q u i v a l e n t t o cypermethrin). D u r i n g t h e 8 hour c o n t a c t p e r i o d w i t h the c o n c e n t r a t e f o r m u l a t i o n a mean of 1.0% (SEM ± 0.4; n = 3) o f t h e a p p l i e d r a d i o l a b e l was d e t e c t e d t o have been absorbed. I n our experiments we d e f i n e d t h e absorbed c h e m i c a l as t h a t which had passed through the s k i n , i e e n t e r e d the c i r c u l a t o r y system and, t h e r e f o r e , c a p a b l e of e x e r t i n g s y s t e m i c t o x i c i t y . A c h e m i c a l which i s l o c a t e d i n t h e s t r a t u m corneum ( t h e outermost l a y e r of t h e e p i d e r m i s ) , a l t h o u g h p o t e n t i a l l y a b s o r b a b l e , cannot e x e r t s y s t e m i c t o x i c i t y , o t h e r than l o c a l , and so i s not regarded as absorbed. 1 4

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A s e r i e s of i n v i t r o experiments were done t o o b t a i n d a t a f o r comparison w i t h t h a t o b t a i n e d i n v i v o . The a p p l i c a t i o n c o n d i t i o n s of the f o r m u l a t i o n on the s k i n i n v i t r o d u p l i c a t e d those i n v i v o . The a p p a r a t u s used f o r the i n v i t r o experiments was a g l a s s d i f f u s i o n c e l l ( F i g u r e 1) i n which the s k i n membrane was sandwiched between a donor and r e c e p t o r chamber, w i t h the e p i d e r m a l s u r f a c e a d j o i n i n g the donor chamber. The donor chamber was open at the top i n o r d e r to expose the e p i d e r m a l s u r f a c e t o ambient atmospheric c o n d i t i o n s and the r e c e p t o r chamber was p l a c e d i n a w a t e r - b a t h m a i n t a i n e d a t a c o n s t a n t temperature (30° ± 1°C). Two v a r i a b l e s were i n t r o d u c e d i n t o the experiment. F i r s t l y , whole s k i n ( e p i d e r m i s and d e r m i s ) o r an e p i d e r m a l membrane (dermis removed) was used (2^). Secondly, s e v e r a l r e c e p t o r f l u i d s ( p h y s i o l o g i c a l s a l i n e , 6% V0LP0 i n s a l i n e , 6% PEG i n s a l i n e , 20% f o e t a l c a l f serum and 50% aqueous e t h a n o l ) were used w i t h b o t h whole and epidermal s k i n membranes. The i n v i t r o d a t a are p r e s e n t e d i n T a b l e 1 With whole s k i n i d e t e c t e d d u r i n g the 8 hou f l u i d s . These r e s u l t s do not agree w i t h the i n v i v o d a t a . However, when e p i d e r m a l membranes were used, a b s o r p t i o n ( s i m i l a r to t h a t measured i n v i v o ) was d e t e c t e d w i t h 3 of the 5 r e c e p t o r f l u i d s . C y p e r m e t h r i n i s e s s e n t i a l l y i n s o l u b l e i n the 2 r e c e p t o r f l u i d s ( p h y s i o l o g i c a l s a l i n e and 6% PEG i n s a l i n e ) i n t o which no a b s o r p t i o n was d e t e c t e d . These d a t a i n d i c a t e t h a t the i n v i v o percutaneous a b s o r p t i o n of a l i p o p h i l i c m o l e c u l e can be p r e d i c t e d i n v i t r o by u s i n g e p i d e r m a l membranes and a s u i t a b l e r e c e p t o r f l u i d i n which the p e n e t r a n t i s s o l u b l e . The i n a b i l i t y of l i p o p h i l i c m o l e c u l e s t o p e n e t r a t e i n v i t r o whole s k i n (though e s s e n t i a l l y the d e r m i s ) has been demonstrated p r e v i o u s l y ( 2 , 3 ) . I n v i v o c h e m i c a l s must d i f f u s e through the s t r a t u m corneum and v i a b l e e p i d e r m i s , t o reach the s y s t e m i c c i r c u l a t i o n , but not the f u l l w i d t h of the dermis. Thus the use of e p i d e r m a l membranes i n v i t r o mimics the i n v i v o pathway to the c i r c u l a t o r y system. The need f o r a r e c e p t o r f l u i d i n which the p e n e t r a n t i s s o l u b l e , (but which does not a l t e r the o v e r a l l i n t e g r i t y of the d i f f u s i o n b a r r i e r ) i s necessary and has been r e p o r t e d p r e v i o u s l y (4) but t h i s i s s t i l l not a p p r e c i a t e d f u l l y . The t e c h n i c a l m i s t a k e of u s i n g whole s k i n and an u n s u i t a b l e r e c e p t o r f l u i d might, i n the p a s t , have p r e j u d i c e d workers i n the i n v e s t i g a t o r y f i e l d of percutaneous a b s o r p t i o n from u s i n g the i n v i t r o technique. C e r t a i n l y , the i n a b i l i t y to reproduce i n v i v o a b s o r p t i o n r e s u l t s i n v i t r o would have been a f r u s t r a t i n g experience. The p r o f i l e s of i n v i v o and i n v i t r o c y p e r m e t h r i n a b s o r p t i o n a g a i n s t time, through r a t s k i n , from the o i l - b a s e d spray d i l u t i o n are shown i n F i g u r e 2. D u r i n g the i n i t i a l 12 hour c o n t a c t p e r i o d t h e r e was no d i f f e r e n c e between the i n v i v o and i n v i t r o d a t a , but a f t e r t h i s time the data o b t a i n e d w i t h 6% V0LP0 20 as the r e c e p t o r f l u i d i n v i t r o underestimated the i n v i v o a b s o r p t i o n . The 50% aqueous e t h a n o l s o l u t i o n a g a i n demonstrated t h a t over prolonged c o n t a c t p e r i o d s , the i n v i t r o t e c h n i q u e i s capable of p r e d i c t i n g the i n v i v o a b s o r p t i o n .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Support Screen

Sampling Receptor C h a m b e r

F i g u r e _1

Arm

Diagram of t h e g l a s s d i f f u s i o n c e l l used i n i n v i t r o percutaneous a b s o r p t i o n experiments

T a b l e I . I n v i t r o A b s o r p t i o n of C y p e r m e t h r i n through Rat S k i n D u r i n g 8hr Contact P e r i o d from a C o n c e n t r a t e F i e l d Formulation % a p p l i e d dose absorbed

1 ND (5)

Whole s k i n Receptor 2 3 4 ND (5)

ND ND (5) (5)

1 5

1

ND (5) |

Epidermis Receptor 2 3

1.7 2.7 ±0.2 ±0.4 (4) (4)

ND

4 2.1 ±0.4 (3)

R e c e p t o r f l u i d s used: (1) 50% aqueous e t h a n o l (2) 6% V0LP0 20 i n s a l i n e (3) p h y s i o l o g i c a l s a l i n e (4) 20% f o e t a l c a l f serum (5) 6% PEG i n s a l i n e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

5 ND

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Such an i n v i v o time course e x p e r i m e n t , u s i n g t h i s type of p r o t o c o l , r e q u i r e s the use and s a c r i f i c e of many a n i m a l s (eg. 2-5 per time p o i n t ) , w h i l s t the i n v i t r o t e c h n i q u e r e q u i r e s much fewer (3-5 r e p l i c a t e s f o r each r e c e p t o r f l u i d w i t h 3-5 s k i n samples from each r a t ) . The a b i l i t y t o study the e f f e c t of a l t e r i n g the c o n c e n t r a t i o n , time of s k i n c o n t a c t or changes i n f o r m u l a t i o n s c o u l d be measured i n v i t r o much more e f f i c i e n t l y and the e f f e c t on i n v i v o a b s o r p t i o n p r e d i c t e d . In such a manner development of l e s s t o x i c f o r m u l a t i o n s , i e those which cause the l e a s t percutaneous a b s o r p t i o n , would be f a c i l i t a t e d . A s i m i l a r degree of a c c u r a t e p r e d i c t i o n of i n v i v o a b s o r p t i o n , u s i n g the i n v i t r o method, has a l s o been a c h i e v e d w i t h a more water s o l u b l e and w e l l absorbed p e s t i c i d e , c a r b a r y l . I n i t i a l l y , animals were g i v e n a s i n g l e i n t r a v e n o u s b o l u s i n j e c t i o n of c a r b a r y l and the u r i n e and f a e c e s c o l l e c t e d . The p r o p o r t i o n of the dose e l i m i n a t e d i n the u r i n e was then c a l c u l a t e d The k i n e t i c s of e l i m i n a t i o n f o l l o w i n g percutaneou the same as a f t e r i n t r a v e n o u a p p l i e d to r a t s k i n i n a v o l a t i l e v e h i c l e ( a c e t o n e ) and a b s o r p t i o n was a s s e s s e d by c o l l e c t i n g u r i n e and a s s a y i n g f o r [^ C] which was regarded as e q u i v a l e n t to c a r b a r y l . The amount of [^- C] i n the u r i n e a f t e r dermal c o n t a c t was then a d j u s t e d by the c a l c u l a t e d ' c o r r e c t i o n f a c t o r * from the i n t r a v e n o u s study ( i . e . p r o p o r t i o n i n the u r i n e ) t o determine percutaneous a b s o r p t i o n . T h i s t e c h n i q u e has been employed s u c c e s s f u l l y i n monkey and human s t u d i e s ( 5 ) . I n v i t r o a b s o r p t i o n was measured i n t o two r e c e p t o r f l u i d s (see F i g u r e 3 ) . The use of 50% aqueous e t h a n o l p r o v i d e d the b e t t e r agreement than the 6% V0LP0 i n s a l i n e r e c e p t o r f l u i d and p r e d i c t e d a c c u r a t e l y the i n v i v o d a t a . The percutaneous a b s o r p t i o n of c a r b a r y l has a l s o been s t u d i e d p r e v i o u s l y through human forearm s k i n (6^). We have d u p l i c a t e d the a b s o r p t i o n c o n d i t i o n s and repeated the study i n v i t r o w i t h human abdominal s k i n . A comparison of our i n v i t r o d a t a w i t h the p u b l i s h e d i n v i v o d a t a i s p r e s e n t e d i n F i g u r e 4. A g a i n , t h e r e i s agreement between the i n v i v o and i n v i t r o d a t a , e s p e c i a l l y when the s m a l l amount of c a r b a r y l a p p l i e d and absorbed i s c o n s i d e r e d t o g e t h e r w i t h the d i f f e r e n t a p p l i c a t i o n s i t e . I t i s p o s s i b l e that a repeat i n v i v o study u s i n g the abdomen as the a p p l i c a t i o n s i t e would g i v e an even b e t t e r c o r r e l a t i o n . These e x p e r i m e n t a l d a t a support the view t h a t the i n v i t r o t e c h n i q u e c o u l d be a genuine a l t e r n a t i v e to the i n v i v o d e t e r m i n a t i o n of percutaneous a b s o r p t i o n . In v i t r o d a t a which p r e d i c t e d i n v i v o data have been o b t a i n e d w i t h d i f f e r e n t types of c h e m i c a l s ( p h y s i c o c h e m i c a l p r o p e r t i e s ) and w i t h d i f f e r e n t s p e c i e s ( r a t and man). E x t r a p o l a t i o n of dermal t o x i c i t y d a t a from animal s t u d i e s to man i s d i f f i c u l t . The a b i l i t y of the i n v i t r o t e c h n i q u e to q u a n t i f y d i f f e r e n c e s i n a b s o r p t i o n between a n i m a l models and man i s i l l u s t r a t e d i n F i g u r e 5. T h i s shows the a b s o r p t i o n p r o f i l e s of [ l ^ C ] c a r b a r y l from a 40ug cm"^ dose, a p p l i e d i n a c e t o n e , t o r a t and human s k i n . The p r o f i l e and r a t e s of a b s o r p t i o n are very different. I f the r a t d a t a were used t o p r e d i c t human a b s o r p t i o n 4

4

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Figure

2_

Cumulative i n v i v o and i n v i t r o a b s o r p t i o n i n s e c t i c i d e c y p e r m e t h r i n from an o i l - b a s e d d i l u t i o n through r a t s k i n

of the spray

24 n 50% A q u e o u s Ethanol

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F i g u r e _3

A b s o r p t i o n o f c a r b a r y l f o l l o w i n g d e p o s i t i o n on s k i n i n a v o l a t i l e ( a c e t o n e ) v e h i c l e through r a t s k i n

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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165

Percutaneous Absorption Study Techniques

t h e r e would be a l a r g e o v e r e s t i m a t i o n . These d a t a show c l e a r l y t h a t when a b s o r p t i o n s t u d i e s a r e done to p r e d i c t human a b s o r p t i o n then the best s k i n t o use i s human; t h e r e i s , as y e t , no a n i m a l s k i n which has proved t o be a good model f o r human s k i n ( 7 , 8 ) . The d i f f i c u l t i e s of human i n v i v o s t u d i e s have been d i s c u s s e d . The most r a t i o n a l way f o r w a r d , t h e r e f o r e , t o study and understand percutaneous a b s o r p t i o n as i t r e l a t e s to man would appear t o be the use of an i n v i t r o system u s i n g human s k i n . T h i s approach a l l o w s the e f f e c t of f o r m u l a t i o n changes, dose, time of c o n t a c t and o t h e r v a r i a b l e s on percutaneous a b s o r p t i o n , as they r e l a t e t o man, t o be a s s e s s e d r e a d i l y . Most d e t e r m i n a t i o n s of a b s o r p t i o n , p a r t i c u l a r l y f o r the assessment of s y s t e m i c h a z a r d by R e g u l a t o r y A u t h o r i t i e s , l e a d t o p r e s e n t a t i o n of the d a t a i n terms of '% a p p l i e d dose a b s o r b e d a t a s p e c i f i e d t i m e . The p r e c i s e times of i n t e r e s t a r e i l l - d e f i n e d but o f t e n r e l a t e t o the w o r k i n da p e r i o d (8-10 h o u r s ) d t 24 hour p e r i o d . At p r e s e n t ' c l a s s i f i c a t i o n scheme' adopted amongst i n t e r e s t e d s c i e n t i s t s i n t h i s a r e a of i n v e s t i g a t i o n . The d a t a p r e s e n t e d i n F i g u r e s 2 and 3 a r e f u r t h e r a n a l y s e d i n T a b l e I I . With b o t h compounds, the e x t e n t of i n v i v o a b s o r p t i o n , t o g e t h e r w i t h the a p p r o p r i a t e '% a p p l i e d dose absorbed' would have been p r e d i c t e d from the i n v i t r o d a t a ( 6 % V0LP0 d a t a was l e s s a c c u r a t e than 50% aqueous e t h a n o l ) a t any of the r e l a t e d time p o i n t s . I f d a t a a r e r e q u i r e d i n t h i s form then the i n v i t r o system c l a s s i f i e d a c c u r a t e l y the i n v i v o a b s o r p t i o n . P r e c i s e l y how t h i s type of d a t a can be used f o r assessment of hazard i s , however, not e n t i r e l y c l e a r . T o x i c i t y i s not r e l a t e d s i m p l y t o 'the percentage of a p p l i e d dose absorbed' but a b s o r p t i o n r a t e and t o t a l amount absorbed or a c o m b i n a t i o n of t h e s e . Data p r e s e n t e d i n T a b l e I I I a r e from i n v i t r o experiments t o determine the a b s o r p t i o n of l^C] c a r b a r y l through r a t s k i n a f t e r a p p l i c a t i o n ( i n an acetone v e h i c l e ) a t doses of 4, 40, 400 and 4000 u-g/cm^. The a b s o r p t i o n r a t e s and '% a p p l i e d dose absorbed' have been determined and c a l c u l a t e d over two time p e r i o d s . I f the d a t a a r e c o n s i d e r e d i n terms of '% dose absorbed' t h e n , the h i g h e r v a l u e s a r e o b t a i n e d w i t h the lower doses. However, the h i g h e s t a b s o r p t i o n r a t e s , which would determine any t o x i c i t y , a r e from the h i g h e r doses. What i s a l s o o b v i o u s w i t h t h i s p a r t i c u l a r compound from t h i s v e h i c l e , i s the n o n - l i n e a r i t y of the a b s o r p t i o n r a t e s w i t h dose. The i n v i t r o system f a c i l i t a t e s the a c q u i s i t i o n of such d a t a . The '% a p p l i e d dose absorbed' can o n l y be r e l e v a n t t o any p a r t i c u l a r dose a p p l i e d and s p e c i f i c time p e r i o d . A b s o r p t i o n r a t e s a r e more m e a n i n g f u l f o r t o x i c o l o g i c a l / h a z a r d assessments and e n a b l e a b s o r p t i o n from d i f f e r e n t f o r m u l a t i o n s and dose l e v e l s (as can happen i n the f i e l d from s p r a y equipment) t o be compared. These r a t e s can be o b t a i n e d u s i n g the i n v i t r o t e c h n i q u e . I n our l a b o r a t o r y we have d e v e l o p e d , an e x p e r i m e n t a l i n v i t r o percutaneous a b s o r p t i o n scheme which we use t o p r e d i c t the p o t e n t i a l h a z a r d from p e s t i c i d e f o r m u l a t i o n s as a r e s u l t of dermal c o n t a c t ( T a b l e I V ) . A b s o r p t i o n measured from b u l k ' i n f i n i t e ' doses to the s k i n p r o v i d e s d a t a r e l e v a n t t o the worst exposure s i t u a t i o n . 1

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

166

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE 1

Table I I . Comparison o f t h e '% A p p l i e d Dose A b s o r b e d i n I n V i v o and I n V i t r o Experiments a t V a r i o u s Times A f t e r A p p l i c a t i o n t o the Skin

Compound

Expt

% A p p l i e d Dose Absorbed 20hr 5hr lOhr

Cypermethrin

In v i v o 50% aq. e t h 6% VOLPO

3 4 2.5

Carbaryl

In v i v o 50% aq. e t h 6% VOLPO

10 10 5

6 9 5

19 20 11

20 20 7.5

33 25 12

Table I I I . I n V i t r Doses Expressed as t h e Rate and % i A p p l i e d Dose A b s o r b e d Over S p e c i f i c Time P e r i o d s 1

Dose (u-g/cm^)

2 - 6

1

Time P e r i o d ( h r ) 20 - 40 %

Js

%

-

-

4

1.77

100

40

1.16

15

0.28

35

400

1.75

25

0.84

10

4000

5.56

1.17

2

0.8

J , approximate a b s o r p t i o n r a t e (u-g/cm^/hr) %, t o t a l ' % a p p l i e d dose a b s o r b e d a t end o f time p o i n t s

1

Table IV. In Vitro Dermal Application Regime for Predicting Potential Percutaneous Absorption From Different Formulations Formulation Concentrate Spray d i l u t i o n Spray d i l u t i o n ( x l O ) Spray d i l u t i o n ( i l O ) Spray d i l u t i o n Spray d i l u t i o n (unoccluded) Cone: Spray d i l u t i o n (1:3)

z

A p p p l i c a t i o n Volume ( u l / c m ) 100 100 100 100 10 10 100

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

13. SCOTT

Percutaneous Absorption Study Techniques

167

To study t h e e f f e c t s of o t h e r than t h e s p e c i f i e d s p r a y d i l u t i o n c o n t a c t i n g s k i n , as can happen i f l a b e l i n s t r u c t i o n s a r e not f o l l o w e d , a b s o r p t i o n i s measured from a range of s p r a y d i l u t i o n s which i n c l u d e s the recommended d i l u t i o n . A d d i t i o n a l l y , t o a s s e s s a b s o r p t i o n under exposure s i t u a t i o n s l i k e l y i n t h e f i e l d , t h e s p r a y d i l u t i o n i s a p p l i e d t o t h e s k i n a t a more r e a l i s t i c exposure r a t e (lOu-l/cm^) w i t h both t h e f o r m u l a t i o n s covered ( o c c l u d e d ) t o prevent v e h i c l e components e v a p o r a t i n g and a l s o open t o t h e atmosphere (unoccluded) t o mimic a c t u a l f i e l d exposure. A f u r t h e r a p p l i c a t i o n regime which we have r e c e n t l y i n t r o d u c e d i s t o measure a b s o r p t i o n from a m i x t u r e o f t h e c o n c e n t r a t e f o r m u l a t i o n and s p r a y d i l u t i o n (1:3) t o study p o t e n t i a l a b s o r p t i o n f o r t h e ' m i x e r - l o a d e r ' o p e r a t o r . O b v i o u s l y , t h i s approach i s geared t o t h e spray o p e r a t o r s i t u a t i o n which we b e l i e v e t o be the most r e l e v a n t f o r c o n s i d e r a t i o n of r i s k w i t h p e s t i c i d e f o r m u l a t i o n s . I n a l l our a p p l i c a t i o n s we a p p l y s p e c i f i e than amounts of a c t i v e d a t a can be r e - i n t e r p r e t e d i n terms of '% a p p l i e d dose i f desired. I t i s f i r m l y our b e l i e f t h a t the h a z a r d o f dermal exposure and subsequent a b s o r p t i o n t o p e s t i c i d e s needs t o be a s s e s s e d . C u r r e n t l y , we b e l i e v e t h a t t h e i d e a l way t o do t h i s i s w i t h f u l l human p h a r m a c o k i n e t i c v o l u n t e e r t r i a l s , however, t h e a c c u m u l a t i o n of s u f f i c i e n t d a t a by t h i s method p r e s e n t s many problems which makes t h i s approach i m p r a c t i c a l . I n order t o f u r t h e r a i d our u n d e r s t a n d i n g of t h e f a c t o r s g o v e r n i n g t h e a b s o r p t i o n o f p e s t i c i d e s , from d i f f e r e n t f o r m u l a t i o n s and t h e subsequent h a z a r d to spray o p e r a t o r s , we b e l i e v e t h a t t h e i n v i t r o t e c h n i q u e o f f e r s a genuine a l t e r n a t i v e t o i n v i v o s t u d i e s .

Literature cited 1.

2.

3.

4. 5.

Scott, R. C. and Ramsey, J. D. (1987). Comparison of the in vivo and in vitro absorption of a lipophilic molecule (cypermethrin, a pyrethroid insecticide). J. Invest. Dermatol. 89: 142-146 Scott, R. C., Walker, M. and Dugard, P. H. (1986). In vitro percutaneous absorption experiments: A technique for production of intact epidermal membranes from rat skin. J. Soc. Cosmet. Chem. 37: 35-41. Scheuplein, R. J. (1965). Mechanism of percutaneous absorption, iv. Penetration of non-electrolytes (alcohols) from aqueous solutions and from pure liquids. J. Invest. Dermatol. 60: 286-296. Bronaugh, R. L. and Stewart, R. F. (1984). Methods for in vitro percutaneous absorption studies. 111. Hydrophobic compounds. J. Pharm. Sci. 73: 1255-1258 Bronaugh, R. L. and Maibach, H. I. (1985). Percutaneous absorption of nitroaromatic compounds: in vivo and in vitro studies in human and monkey. J. Invest. Dermatol. 84: 180183.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

168 6. 7.

8.

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Feldman, R. J. and Maibach, H. I. (1970). Absorption of some organic compounds through skin in man. J. Invest. Dermatol. 54: 399-404. Scott, R. C., Walker, M. and Dugard, P. K. (1986). A comparison of the in vitro permeability properties of human and from laboratory animal skins. Int. J. Cos. Sci. 8 189-194 (1986). Bartek, M. J., La Budde, J. A. and Maibach, H. I. (1972). Skin permeability in vivo: comparison in rat, rabbit, pig and man. J. Invest. Dermatol. 58 114-123.

RECEIVED March 8, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 14

Dermal Absorption and Pharmacokinetics of Pesticides in Rats 1

2

3

P. V. Shah , H. L. Fisher , M . R. Sumler , and L. L. Hall

2

1Toxic Effects Branch, Health and Environmental Review Division (TS-796), Office of Toxic Substances, U.S. Environmental Protection Agency, Washington, DC 20460 Cellular Biology Branch, Health Effects Research Laboratory, U.S. Environmental Protectio Northrop Environmenta Triangle Park, NC 27709 2

3

Dermal a b s o r p t i o n and d i s p o s i t i o n o f carbaryl, c a r b o f u r a n , c h l o r d e c o n e , h e x a c h l o r o b i p h e n y l and d i n o s e b were s t u d i e d in female F i s c h e r 344 rats. Acetone s o l u t i o n o f t h e [14C] l a b e l e d pesticide was a p p l i e d to a p r e v i o u s l y c l i p p e d m i d - d o r s a l r e g i o n o f t h e back. At v a r i o u s t i m e i n t e r v a l s , a n i m a l s were killed and s e l e c t e d organs were a n a l y z e d f o r radioactive [14C] c o n t e n t . A physiological compartmental model was f i t t e d t o t h e s k i n p e n e t r a t i o n , tissue distribution, and e x c r e t i o n d a t a . The e q u i l i b r i u m t i s s u e t o b l o o d ratios and excretion r a t e c o n s t a n t s and s k i n a b s o r p t i o n rate were d e t e r m i n e d f r o m t h e model. In many c a s e s , differences in kinetics and retention were found between young and a d u l t rats. The dermal p e n e t r a t i o n function was s i n g l e o r biexponential, requiring a one o r two compartmental model f o r s i m u l a t i n g t h e s k i n penetration process. More t h a n one b i l l i o n pounds o f p e s t i c i d e s a r e b e i n g u s e d each year i n the U n i t e d S t a t e s alone t o i n c r e a s e the p r o d u c t i v i t y of a g r i c u l t u r a l l a n d ( 1 ) . G r e a t e r r e l i a n c e on use o f p e s t i c i d e s appears i n e v i t a b l e as t h e demand f o r f o o d p r o d u c t i o n i n c r e a s e s w i t h i n c r e a s i n g p o p u l a t i o n . O c c u p a t i o n a l exposure t o p e s t i c i d e s o c c u r s v i a o r a l , d e r m a l , and i n h a l a t i o n r o u t e s . A l t h o u g h t h e r a t e o f a b s o r p t i o n may be h i g h e r by pulmonary and o r a l r o u t e s , t h e t o t a l amount absorbed a f t e r s k i n c o n t a c t may be much g r e a t e r d u r i n g o c c u p a t i o n a l exposure t o p e s t i c i d e s (2,3). Knowledge of dermal a b s o r p t i o n ( r a t e s and e x t e n t ) o f p e s t i c i d e s i s v e r y i m p o r t a n t i n r i s k assessment and e s t a b l i s h m e n t 0097-6156/89/0382-0169$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

170

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

of r e e n t r y standards f o r f i e l d workers. With r e g a r d t o o c c u p a t i o n a l e x p o s u r e t o t o x i c a n t s i n g e n e r a l , r i s k assessment has u s u a l l y been b a s e d on s t u d i e s i n a d u l t s . S k i n a b s o r p t i o n depends on a number o f f a c t o r s , i n c l u d i n g t h e c h e m i c a l and p h y s i c a l n a t u r e o f t h e p e n e t r a n t and t y p e o f s k i n . The l i m i t e d l i t e r a t u r e on t h e e f f e c t s o f age on s k i n p e n e t r a t i o n has been r e c e n t l y summarized (4)» I t a p p e a r s t h a t t h e e f f e c t o f age on p e r c u t a n e o u s a b s o r p t i o n v a r i e s w i t h s p e c i e s , compound, and method o f assessment. I n c r e a s e d s k i n p e r m e a b i l i t y o f n e o s y n e p h r i n e (as measured by t h e b l a n c h i n g r e s p o n s e ) i n p r e m a t u r e i n f a n t s compared t o f u l l t e r m i n f a n t s has been documented (50. No d i f f e r e n c e s i n p e r c u t a n e o u s a b s o r p t i o n o f t e s t o s t e r o n e between t h e newborn and a d u l t r h e s u s monkey was n o t e d {6J, w h i l e more r a p i d l o s s o f t o p i c a l l y a p p l i e d t r i a d i m e f o n from t h e s k i n o f young Sprague-Dawley r a t s has been r e p o r t e d (7). A recent c o m p a r a t i v e s t u d y on p e r c u t a n e o u absorptio f pesticide i young and a d u l t F i s c h e percutaneous a b s o r p t i o We p r e s e n t h e r e t h e c o m p a r a t i v e p e r c u t a n e o u s a b s o r p t i o n and d i s p o s i t i o n of c a r b a r y l , carbofuran, chlordecone, hexachlorob i p h e n y l , and d i n o s e b i n young and a d u l t F i s c h e r 344 r a t s u t i l i z i n g i d e n t i c a l methodology. A l s o , comparison o f t h e r e s u l t s o f i n v i v o and two i n v i t r o methods f o r p e r c u t a n e o u s a b s o r p t i o n measurements a r e p r e s e n t e d . The u t i l i t y o f t h e p h y s i o l o g i c a l compartmental model i n s i m u l a t i n g dermal a b s o r p t i o n and disposition i s discussed. EXPERIMENTAL APPROACH; CHEMICALS The c h e m i c a l s s t u d i e d , t h e i r r e s p e c t i v e s p e c i f i c a c t i v i t i e s , r a d i o l a b e l i n g p o s i t i o n , and s o u r c e s a r e l i s t e d i n T a b l e I . The r a d i o c h e m i c a l p u r i t y was g r e a t e r t h a n 98% f o r a l l pesticides studied. U n l a b e l e d c h e m i c a l s were s u p p l i e d by t h e P e s t i c i d e and I n d u s t r i a l C h e m i c a l R e p o s i t o r y (U.S. E n v i r o n m e n t a l P r o t e c t i o n Agency, R e s e a r c h T r i a n g l e Park, NC). Minimum E s s e n t i a l Medium (MEM) E a g l e Formula w i t h E a r l ' s s a l t s a n d L - g l u t a m i n e and w i t h o u t p h e n o l r e d and sodium c a r b o n a t e was p u r c h a s e d from G i b c o L a b o r a t o r i e s , C h a g r i n F a l l s , OH. HEPES ( N- 2h y d r o x y e t h y l p i p e r a z i n e - N - 2 - e t h a n e s u l f o n i c a c i d ) , sodium p y r u v a t e , L- s e r i n e , L - a s p a r t i c a c i d , and g e n t a m i c i n were p u r c h a s e d from G i b c o , Grand I s l a n d , NY. Anhydrous sodium c a r b o n a t e , sodium h y d r o x i d e , e t h y l e t h e r ( a n h y d r o u s ) , and a c e t o n e ( r e a g e n t grade) were p u r c h a s e d from F i s h e r S c i e n t i f i c Co., F a i r l a w n , NJ. IN VIVO APPLICATION Female F i s c h e r 344 r a t s o f 33 (young) and 82 ( a d u l t ) days o f age were u t i l i z e d f o r d e r m a l a b s o r p t i o n e x p e r i m e n t s . Three r a t s were u s e d f o r each t i m e p o i n t . Randomized o f f s p r i n g f r o m t i m e d p r e g n a n t r a t s were u s e d i n o r d e r t o have b e t t e r c o n t r o l o v e r t h e age and w e i g h t o f t h e a n i m a l s u s e d i n t h e s e e x p e r i m e n t s . The i n v i v o e x p e r i m e n t a l t e c h n i q u e s employed i n t h i s s t u d y have been d e s c r i b e d i n d e t a i l by Shah e t a l . ( 8 ) .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988. position

C

C

,

,

Klng(U)

Ring(U)

,

1

4

C C £ / ;

]

Benzofuranyl r i n g

2 , 4 , 5 , 2 , 4 , 5 - [

1 4

1 4

^^C(U)

1 4

Naphthyl[1- C]

Radiolabeling

2,4,5,2*,4',5'-Hexachlorobiphenyl.

9.2

4.29

Dinoseb

1

3.2

Chlordecone

PCB

39.4

5.8

Carbofuran

Carbaryl

Specific activity (mCi/mM)

New E n g l a n d N u c l e a r , B o s t o n , MA

P a t h f i n d e r Lab., I n c . , S t . L o u i s , MO

Midwest R e s e a r c h I n s t i t u t e , Kansas C i t y , MO

FMC Corp., P r i n c e t o n , NJ

Amersham, A r l i n g t o n H e i g h t s , I L

Source

P e s t i c i d e s T e s t e d i n P r e s e n t S t u d y , w i t h S p e c i f i c A c t i v i t y , R a d i o l a b e l i n g P o s i t i o n , and Source

Common name

TABLE I .

172

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

B r i e f l y , t h e a n i m a l ' s h a i r was c l i p p e d 24 h p r i o r t o dermal a p p l i c a t i o n w i t h O s t e r a n i m a l h a i r c l i p p e r s ( O s t e r , Milwaukee, WI). D o s i n g s o l u t i o n s (0.1 mL young and 0.2 mL a d u l t ) c o n t a i n i n g r a d i o a c t i v i t y and a p p r o p r i a t e dose (285 nmol/cm ) i n a c e t o n e were a p p l i e d t o t h e p r e v i o u s l y c l i p p e d mid-back r e g i o n o f t h e r a t . The t r e a t e d a r e a , w h i c h r e p r e s e n t s a p p r o x i m a t e l y 2.3% o f t h e t o t a l body s u r f a c e a r e a (2.8 cm young , 5.6 cm a d u l t ) , was p r o t e c t e d f r o m grooming by p l a c i n g a n o n o c c l u s i v e d e v i c e o v e r i t . At t h e end o f the d e s i g n a t e d t i m e i n t e r v a l , the a n i m a l was a n e s t h e t i z e d w i t h e t h e r and a b l o o d specimen was o b t a i n e d from t h e h e a r t . V a r i o u s t i s s u e s were removed t o determine t h e d i s t r i b u t i o n o f dermally absorbed r a d i o - l a b e l e d p e s t i c i d e s . A l l t h e t i s s u e s , e x c r e t o r y p r o d u c t s , and c a r c a s s e s were a n a l y z e d f o r r a d i o a c t i v i t y by l i q u i d s c i n t i l l a t i o n c o u n t i n g as d e s c r i b e d by Shah e t a l . ( 8 ) . 2

2

2

IN VITRO PROCEDURE I n v i t r o a b s o r p t i o n o f p e s t i c i d e s t h r o u g h young and a d u l t s k i n was d e t e r m i n e d by u s i n g b o t h F r a n z c e l l s and c o n t i n u o u s flow-through cells. The d e t a i l e d e x p e r i m e n t a l and s k i n p r e p a r a t i o n t e c h n i q u e s have been d e s c r i b e d i n d e t a i l by Shah e t a l . (_9). B r i e f l y , back h a i r was c l i p p e d 24 h p r i o r t o e x p e r i m e n t . A n i m a l s were k i l l e d w i t h c a r b o n d i o x i d e , and back s k i n was c a r e f u l l y e x c i s e d . Skin was f l o a t e d on t i s s u e c u l t u r e medium (minimum e s s e n t i a l media, Eagle formula w i t h E a r l ' s S a l t s ) . S p l i t t h i c k n e s s (350 um t h i c k n e s s ) s k i n s o b t a i n e d w i t h a dermatome ( P a d g e t t Dermatome, Kansas C i t y , MO) were mounted i n t h e i n v i t r o c e l l s w i t h e p i d e r m i s exposed t o t h e e n v i r o n m e n t . Only one s k i n d i s c was used from each animal. The amount o f p e s t i c i d e ( i n a c e t o n e ) per cm a r e a was k e p t t h e same f o r b o t h i n v i v o and i n v i t r o e x p e r i m e n t s . Receptor f l u i d was m a n u a l l y removed p e r i o d i c a l l y from t h e F r a n z c e l l s t o d e t e r m i n e t h e r a t e and e x t e n t o f p e n e t r a t i o n , w h i l e c o n t i n o u s f l o w t h r o u g h c e l l s w i t h a media f l o w r a t e of 3 mL/h were a u t o m a t i c a l l y sampled every 3 h. Sample p r e p a r a t i o n f o r r a d i o a c t i v i t y d e t e r m i n a t i o n and o x i d a t i o n o f t r e a t e d s k i n were done a c c o r d i n g t o p r o c e d u r e d e s c r i b e d by Shah e t a l . (8_). B r i e f l y , t h e t r e a t e d s k i n was combusted i n t h e sample o x i d i z e r t o measure the r e m a i n i n g r a d i o a c t i v i t y i n o r on t h e s k i n . A l l o t h e r samples were mixed w i t h 10 mL I n s t a - G e l ( P a c k a r d I n s t r u m e n t Co., Downers Grove, I L ) for s c i n t i l l a t i o n counting. 2

Data A n a l y s i s S t a t i s t i c a l c o m p a r i s o n s t o d e t e r m i n e t h e e f f e c t o f age on s k i n p e n e t r a t i o n of p e s t i c i d e s were made u s i n g t h e S t a t i s t i c a l A n a l y s i s System package (10) w i t h age and age X t i m e as c l a s s v a r i a b l e s i n the g e n e r a l l i n e a r model (GLM). R e c i p r o c a l v a r i a n c e was used f o r weighting. The s i g n i f i c a n c e o f t h e l a b o r a t o r y method u s e d ( f l o w , s t a t i c , i n v i v o ) was d e t e r m i n e d w i t h t h e SAS u s i n g age, method, and age X method, and age X method X t i m e as c l a s s v a r i a b l e s i n the GLM and r e c i p r o c a l v a r i a n c e f o r w e i g h t i n g .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

14.

SHAH ET AL.

Absorption and Pharmacokinetics

173

A m a t h e m a t i c a l model was c o n s t r u c t e d a l o n g t h e l i n e s o f t h e p h y s i o l o g i c a l model d e v e l o p e d by Bungay e t a l . (11,12). L e a s t squares f i t t i n g o f t h e model t o t h e d a t a was done u s i n g SAAM 27, a computer package d e v e l o p e d a t t h e N a t i o n a l I n s t i t u t e s o f H e a l t h (NIH) by Berman and Weiss ( 1 3 ) . RESULTS S k i n a b s o r p t i o n o f c a r b o f u r a n as d e t e r m i n e d by t h e t h r e e methods i s shown i n T a b l e I I . The i n v i v o s k i n a b s o r p t i o n o f c a r b o f u r a n was 4.7% a t 6 h and 33.5% a t 72 h i n young a n i m a l s . I n a d u l t s , i t i n c r e a s e d from 2.0% a t 6 h t o 11.8% a t t h e 72 h t i m e p o i n t . The s t a t i c system gave 3.1% s k i n a b s o r p t i o n a t 6 h and 12.0% a t 72 h i n young a n i m a l s , w h i l e i n a d u l t s , a b s o r p t i o n ranged from 1.7% a t 6 h t o 8.8% a t t h e 72 h t i m e p o i n t . The s k i n a b s o r p t i o n o f c a r b o f u r a n as d e t e r m i n e d by t h e f l o w system i n young a n i m a l s ranged from 27.3% a t t h a d u l t s , i t ranged from 5.0 p o i n t . The y o u n g / a d u l t r a t i o o f s k i n p e n e t r a t i o n a t 72 h was 2.9 ( i n v i v o ) , 3.7 ( f l o w ) , and 1.4 ( s t a t i c ) . Thus, a l l t h r e e methods gave h i g h e r s k i n p e n e t r a t i o n v a l u e s i n young r a t s compared t o a d u l t s . However, t h e s t a t i c c e l l gave c o n s i s t e n t l y l o w e r s k i n p e n e t r a t i o n v a l u e s i n young r a t s compared t o i n v i v o o b s e r v a t i o n s . S k i n a b s o r p t i o n o f c a r b a r y l as d e t e r m i n e d by t h e t h r e e methods i s shown i n T a b l e I I I . The s k i n a b s o r p t i o n o f c a r b a r y l ( i n v i v o ) ranged from 2.4% a t 6 h t o 38.5% a t 72 h i n young a n i m a l s , and i n a d u l t s i t ranged from 3.6% a t 6 h t o 38.4% a t 72 h. The s k i n a b s o r p t i o n o f c a r b a r y l ( s t a t i c ) ranged from 5.3% a t 6 h t o 20.9% a t 72 h i n t h e young, and i n a d u l t s , i t ranged from 4.7% a t 6 h t o 20.3% a t 72 h. The s k i n a b s o r p t i o n o f c a r b a r y l ( f l o w ) ranged from 18.8% a t 6 h t o 29.2% a t 72 h i n young a n i m a l s , and i n a d u l t s , i t ranged from 8.1% a t 6 h t o 18.2% a t 72 h. Both i n v i v o and s t a t i c systems showed no s i g n i f i c a n t y o u n g / a d u l t d i f f e r e n c e s i n s k i n p e n e t r a t i o n o f c a r b a r y l a t t h e 72 h t i m e p o i n t , w h i l e t h e f l o w system gave s i g n i f i c a n t d i f f e r e n c e s i n s k i n p e n e t r a t i o n between young and a d u l t a n i m a l s , w i t h young a b s o r b i n g more c a r b a r y l compared t o a d u l t s . S k i n a b s o r p t i o n o f d i n o s e b as d e t e r m i n e d by t h e t h r e e methods i s shown i n T a b l e IV. Dinoseb showed r a p i d p e n e t r a t i o n i n b o t h young and a d u l t r a t s compared t o o t h e r p e s t i c i d e s . The r e s u l t s from t h e i n v i v o method showed a s t a t i s t i c a l l y s i g n i f i c a n t l o w e r s k i n a b s o r p t i o n o f d i n o s e b i n young a n i m a l s . In t h e young, s k i n a b s o r p t i o n ranged from 11.1% a t 1 h t o 55.7% a t 72 h, w h i l e i n a d u l t s , i t ranged from 22.3% a t 1 h t o 84.3% a t 72 h. The s t a t i c method i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s i n s k i n p e n e t r a t i o n o f d i n o s e b i n young and a d u l t , e x c e p t a t 1 h s a m p l i n g p e r i o d . In the young, t h e s k i n a b s o r p t i o n v a l u e ( s t a t i c ) , f o r d i n o s e b ranged from 3.0% a t 1 h t o 74.9% a t 72 h, and i n t h e a d u l t , i t ranged from 1.5% a t 1 h t o 70.8% a t 72 h. The f l o w system i n d i c a t e d s i g n i f i c a n t young/adult d i f f e r e n c e s i n s k i n absorption of dinoseb a t a l l t i m e p o i n t s , and young a b s o r b e d more t h a n t w i c e t h e amount o f d i n o s e b as a d u l t s . The s k i n a b s o r p t i o n v a l u e ( f l o w ) i n young ranged from 6.4% a t 1 h t o 47.3% a t 72 h, and 2.4% a t 1 h t o 20.6% a t 72 h i n t h e a d u l t .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

11.2±0.9

3.7**

4. 1**

NS

f o r the hypothesis

0.05.

S i g n i f i c a n t a t p < 0.05

from J . T o x i c o l . E n v i r o n .

NS Not s i g n i f i c a n t , p >

**

* Reprinted

Age X t i m e

Health.

12.0±1.1

9.8±1.0

7.0±0.8

8.8±0.8

7.1±0.7

4.6±0.5

1.7±0.2

o f no y o u n g - a d u l t d i f f e r e n c e .

22:207-223, 1987 w i t h p e r m i s s i o n

of publisher.

o

w w

o

H O

o •* *•

P 3E

o 5

s

**

2.9**

3.8**

4.2**

2.4

Young adult

**

11.8±0.9

6.8±0.1

5.7±1.2

2.0±0.2

Adult

In v i v o

o H o 2

33.5±2.7

26.1±2.6

23.9±1.8

4.7±0.7

Young

Carbofuran*

3.4**

1.4**

1.4**

1.5**

1.8

Young/ adult

**

41.1±5.9

72

9.7±0.9

4.6**

3.1±0.5

Adult

Age

39.4±6.0

48

7.8±0.9

5.5**

Young

1.4**

36.0±5.7

24

5.0±0.6

Young/ adult

System

Penetration

Static

% Skin

Comparison o f S k i n P e n e t r a t i o n o f

4.3**

27.3±4.5

6

Adult

Methods

All

Young

Time (h)

Flow System

TABLE I I .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

S i g n i f i c a n t a t p < 0.05 f o r t h e h y p o t h e s i s Not s i g n i f i c a n t , p > 0.05.

o f no y o u n g - a d u l t

difference.

*

*

Age X Time

* NS

NS

1.04

1.03

*

20.3±1.9

1.02

1.02

1.15

Age

20.9±1.5

16.1±1.6

11.1±1.1

4.7±0.6

Young Adult

1.80*

1.60*

System

Adult

Static

Penetration

All

18.2±2.6

29 2±2.9

72

16.5±1.2

1.70*

16.0±2.3

27 2±2.8

48

11.4±0.8

1.86*

13.3±1.9

24 6±2.6

24

5.3±0.5

2.33*

8.1±1.2

Young

Adult

18 .8±2.1

Young

6

Time (h)

Young Adult

Flow System

% Skin

38.5±2.4

23.6±1.0

14.9±2.5

2.4±0.3

Young

TABLE I I I . Methods Comparison o f S k i n P e n e t r a t i o n o f C a r b a r y l

Adult

Vivo

38.4±1.3

32.4±0.7

15.2±1.2

3.6±0.5

In

*

*

0.89*

1.00

0.73*

0.98

0.66*

Young Adult

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

29.0±3.5

41.0±3.7

45.0±3.8

47.3±3.9

6

24

48

72

20.6±3.5

18.6±3.0

15.7±2.5

11.19±2.2

2.4±0.6

Flow System Adult

0.70*

0.66*

difference.

*

84.3±2.7

0.65*

*

55.7±2.4

85.1±1.9

55.3±9.7

*

1.10

1.06

1.07

0.74*

0.90

43.1±9.4 85.5±1.8

0.50 22.3±8.8

In V i v o Ratio Adult

63.0±2.9

38.8±5.7

11.1±2.1

Young

*

70.8±3.8

66.2±3.8

1.13

1.22

17.0±1.1 52.3±3.1

2.00*

1.5±0.1

S t a t i c System Adult Ratio

*

74.9±8.0

70.9±8.4

59.1±7.4

20.7±2.0

3.0±0.2

Young

*

2.47*

2.29*

2.42*

2.62*

2.61*

2.64*

Ratio

Penetration

* S i g n i f i c a n t a t p < 0.05 f o r t h e h y p o t h e s i s o f no y o u n g - a d u l t

Age X Time

Age

All

6.4±1.0

1

\

Youna

Time (h)

% Skin

TABLE IV. Methods Comparison o f S k i n P e n e t r a t i o n o f Dinoseb

0

6

a

O

W

5

H

Gfl

W

o 2 2 o *i o

H

o

1

5

14.

Absorption and Pharmacokinetics

SHAH ET AL.

177

Methods c o m p a r i s o n o f h e x a c h l o r o b i p h e n y l s k i n p e n e t r a t i o n i n young and a d u l t r a t s i s shown i n T a b l e V. I n v i t r o s k i n a b s o r p t i o n v a l u e s o f h e x a c h l o r o b i p h e n y l i n young and a d u l t r a t s were s i g n i f i c a n t l y lower t h a n i n v i v o r e s u l t s . Skin absorption v a l u e s i n young a t 6 h were 0.012%, 0.071%, and 2.5% as d e t e r m i n e d by f l o w , s t a t i c , and i n v i v o methods, r e s p e c t i v e l y . In a d u l t s , s k i n a b s o r p t i o n v a l u e s a t 6 h were 0.015, 0.019, and 0.63% as d e t e r m i n e d by f l o w , s t a t i c , and i n v i v o methods, r e s p e c t i v e l y . S k i n a b s o r p t i o n v a l u e s i n young a t 72 h were 1.25, 2.50, and 33.2% as d e t e r m i n e d by f l o w , s t a t i c and i n v i v o methods, r e s p e c t i v e l y . In a d u l t s , 72 h s k i n a b s o r p t i o n v a l u e s were 1.36, 1.15, and 19.6% as d e t e r m i n e d by f l o w , s t a t i c and i n v i v o methods, r e s p e c t i v e l y . Both i n v i t r o methods showed n o n s i g n i f i c a n t d i f f e r e n c e s i n s k i n p e n e t r a t i o n o f h e x a c h l o r o b i p h e n y l between young and a d u l t r a t s , w h i l e i n v i v o r e s u l t s showed s i g n i f i c a n t d i f f e r e n c e s i n s k i n p e n e t r a t i o n between young and a d u l t r a t s a t a l l t i m e p o i n t s Methods c o m p a r i s o n (Kepone) i s shown i n T a b l c h l o r d e c o n e i n young and a d u l t r a t s were s i g n i f i c a n t l y lower t h a n i n v i v o v a l u e s . S k i n a b s o r p t i o n v a l u e s i n young a t 6 h were 0.45, 0.022, and 0.51% as d e t e r m i n e d by f l o w , s t a t i c , and i n v i v o methods, r e s p e c t i v e l y . I n a d u l t s , s k i n a b s o r p t i o n v a l u e s a t 6 h were 0.052, 0.026, and 0.398% as d e t e r m i n e d by f l o w , s t a t i c , and i n v i v o methods, r e s p e c t i v e l y . S k i n a b s o r p t i o n v a l u e s i n young a t 72 h were 1.4, 2.2, and 9.1% as d e t e r m i n e d by f l o w , s t a t i c and i n v i v o methods, r e s p e c t i v e l y . I n a d u l t s , 72 h s k i n a b s o r p t i o n v a l u e s were 1.2, 2.2, and 7.9% as d e t e r m i n e d by f l o w , s t a t i c , and i n v i v o methods, r e s p e c t i v e l y . A l l t h r e e methods showed h i g h e r s k i n p e n e t r a t i o n o f c h l o r d e c o n e i n young compared t o a d u l t animals. PHYSIOLOGICAL MODELING A p h y s i o l o g i c a l compartmental model f o r c a r b o f u r a n dermal a b s o r p t i o n and d i s p o s i t i o n i s shown i n F i g u r e 1. The compartmental volumes ( V ) were t h e average w e i g h t o f k i d n e y , l i v e r , and body f o u n d i n t h e p r e s e n t s t u d y . The o t h e r compartmental volumes were d e r i v e d from t h e body p r o p o r t i o n s g i v e n by L u t z e t a l . ( 14)• C a r d i a c o u t p u t and b l o o d f l o w r a t e s were s c a l e d d i r e c t l y w i t h compartmental s i z e from t h e v a l u e s g i v e n by Bungay e t a l . ( 1 1 ) . A b l o o d f l o w r a t e f o r a 1.9 g k i d n e y was c a l c u l a t e d t o be 8.33 mL/min from a plasma f l o w r a t e o f 5 mL/min and h e m a t o c r i t o f 40% ( 1 5 ) . The system o f d i f f e r e n t i a l e q u a t i o n s d e s c r i b i n g t h e t r a n s f e r of m a t e r i a l between organ compartments i s g i v e n below. F o r b l o o d T

d i=K,L M,S

V. R.

For muscle and n o n t r e a t e d dt

y l

V

Y

B

Y

B " V.R. i i i

skin where

i = M,S.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988. *

Age X t i m e

1.15±0.21

0.592±0.138

0.188±0.054

0.019±0.005

Adult

2.20

2.18

2.11

2.43

3.63

* *

33.2±2.2

28.1±3.5

13.62±0.53

2.50±0.20

Young/ a d u l t Young

* S i g n i f i c a n t a t p < 0.05 f o r t h e h y p o t h e s i s o f no y o u n g - a d u l t d i f f e r e n c e . NS Not s i g n i f i c a n t , p > 0.05.

NS

1.36±0.16 2.50±0.32

1.25±0.24

1.05

0.58±0.08 0.92

0.4610.12

1.00

Age

1.25±0.23

72

0.071±0.022

0.83

0.015±0.002

0.96

0.60±0.13

48

Young/ a d u l t Young

0.17±0.03

Penetration

S t a t i c System

% Skin

Adult

All

0.1710.04

0.012±0.003

Young

24

6

Time (h)

1

19.611.5

18.4±0.6

8.3510.53

0.63±0.06

Adult

In v i v o

*

*

1.65*

1.69*

1.53*

1.63*

3.94*

Young adult

Methods Comparison o f S k i n P e n e t r a t i o n o f 2 , 2 , 4 , 4 * , 5 , 5 ' - H e x a c h l o r o b i p h e n y l

Flow System

T a b l e V.

o

B W W

H Q

s

o

2

1

r 3 0

o

s

3

s

00

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

0.921±0.052

1.201±0.057

1.398±0.057

24

48

72

1.234±0. 310

0.699±0. 243

0.205±0. 090

0.052±0. 020

1.84*

1.13*

1.80*

4.49*

8.69*

Young Adult

2.213±0.435

1.100±0.238

2.47±0.072

0.022±0.007

1.09

1.03

1.21

1.24

0.86

Young Adult

difference.

2.153±0.320

0.912±0.161

0.199±0.039

0.026±0.007

S t a t i c System Young Adult

% Skin Penetration

9.066±0.664

6.825±0.219

3.570*0.453

0.513*0.063

7.922±1.061

5.626±0.686

2.508±0.606

3.98±0.108

In V i v o Method Young Adult

Methods Comparison o f S k i n P e n e t r a t i o n o f C h l o r d e c o n e

* S i g n i f i c a n t a t p < 0.05 f o r t h e h y p o t h e s i s o f no y o u n g - a d u l t

All

0.450±0.043

Flow System Young Adult

6

Time (h)

Table V I .

1.21

1.14

1.21

1.42

1.29

Young Adult

180

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Treated Skin

Blood

v

Liver

Feces

Kidney Urine

Skin

Muscle and Fat Figure 1. Physiological model for carbofuran dermal absorption and disposition in the rat. (Reproduced with permission from ref. 9. Copyright 1987 Hemisphere Publishing Corporation.)

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

14. For

181

Absorption and Pharmacokinetics

S H A H E T A L .

kidney _d_ dt K Y

and f o r

VK

=

liver Y

Y

dt L

k

Y

V^ B " V R L B L L For t r e a t e d s k i n y

dt T

y

T

=

Y (0)

" V i '

T

Nomenclature

v

Dermal dose (n moles R a d i o a c t i v i t y i n compartmen Blood flow r a t e to t i s s u (mL/h) Volume f o r t i s s u e compartment i : (mL) Equilibrum tissue/blood r a t i o for compartment i Dermal a b s o r p t i o n r a t e c o n s t a n t ( h ~ ) Urinary e x c r e t i o n r a t e constant (h~ ) F e c a l e x c r e t i o n r a t e c o n s t a n t (h"^)

i R-

k k k

Subscripts

M S

1

A

T F U

1

y

F

Muscle Nontreated skin Treated s k i n Feces Urine

C a r b o f u r a n l e v e l s from b l o o d , k i d n e y , l i v e r , s k i n , c a r c a s s , u r i n e , and f e c e s were f i t t e d t o the model u s i n g SAAM27 t o o p t i m i z e t h e parameters f o r e q u i l i b r u m t i s s u e / b l o o d r a t i o (R,p), t h e u r i n e ( K y ) , and f e c e s ( K ) , e x c r e t i o n - r a t e c o n s t a n t , and t h e d e r m a l a b s o r p t i o n - r a t e c o n s t a n t ( K ) . The parameter v a l u e s a r e shown i n Table V I I . The dermal a b s o r p t i o n - r a t e c o n s t a n t ( K ) f o r young, 0.0 0548 F

A

A

1

1

h " , i s about 3 t i m e s t h a t f o r a d u l t s , 0.00173 h " . The model r e s u l t s f o r dermal a b s o r p t i o n were i n agreement w i t h t h e o b s e r v e d v a l u e s a f t e r 24 h f o r b o t h young and a d u l t r a t s . B e f o r e 24 h, t h e model r e s u l t s were a p p r o x i m a t e l y h a l f o f t h e o b s e r v e d v a l u e s , perhaps i n d i c a t i n g an a d d i t i o n a l r a p i d component o f a b s o r p t i o n w h i c h was not i n c o r p o r a t e d i n t h e model. The u r i n a r y e x c r e t i o n r a t e c o n s t a n t s ( K ) were f o u n d t o be 36.1 h ~ f o r young and 26.1 h " f o r t h e a d u l t . The f e c a l e x c r e t i o n r a t e c o n s t a n t s ( K ) were found t o be 0.67 h ~ f o r young and 0.24 h ~ f o r the a d u l t . The i n c r e a s e d dermal a b s o r p t i o n - r a t e c o n s t a n t i n young compared t o a d u l t a n i m a l s appears t o be b a l a n c e d by the i n c r e a s e d e x c r e t i o n r a t e i n t h e young, such t h a t t h e body burdens i n young and a d u l t s a r e about t h e same a t 120 h. The e q u i l i b r i u m t i s s u e - t o - b l o o d r a t i o s ( T a b l e V I I ) f o r a d u l t a n i m a l s were h i g h e r t h a n t h o s e o f t h e r e s p e c t i v e organs i n t h e young. Most n o t a b l e i s t h e d i f f e r e n c e i n t h e k i d n e y - t o - b l o o d r a t i o of 10.3 f o r a d u l t s as compared t o 5.4 f o r young, i n d i c a t i n g the greater sequestering of carbofuran-derived r a d i o a c t i v i t y i n a d u l t s t h a n i n young. 1

y

1

p

1

1

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

_

t

F

b

b

c

b

b

1

1

6.38 ( 9 ) b 0.72 3.03 11.35 (16) 40.44 (57) 70.94° Kp = 0.674 h ~

K

b

14.18 ( 9 ) 1.31 5.93 25.21 (16) 89.82 (57) 154.58 = 0.239 h "

±

V (mL) Q ±

(mL/min)

K

K

A

A = 0.00548 h "

17.74 3.195.41 4.861.91 1.421.28 2.242.38

A = 0.00173 h "

39.4010.29 5.782.47 9.491.94 3.153.65 4.98

1

1

P a r a m e t e r s Employed i n t h e P h y s i o l o g i c Model f o r C a r b o f u r a n A b s o r p t i o n and D i s p o s i t i o n i n t h e Young and A d u l t R a t *

from J . T o x i c o l . Environ.

Health.

Observed.

c

* Reprinted

P e r c e n t o f body w e i g h t g i v e n i n p a r e n t h e s e s .

b

22:207-223, 1987 w i t h p e r m i s s i o n o f p u b l i s h e r .

V^, compartmental volume; Q^ b l o o d f l o w r a t e t o t h e o r g a n s ; R^, e q u i l i b r i u m t i s s u e t o b l o o d r a t i o ; Ky, r a t e c o n s t a n t f o r u r i n a r y e x c r e t i o n ; Kp, r a t e c o n s t a n t f o r f e c a l e x c r e t i o n ; K , r a t e c o n s t a n t f o r dermal a b s o r p t i o n .

a

1

Blood Kidneys Liver Skin M u s c l e and f a t Body Ky = 36.06h"

Young

1

Blood Kidneys Liver Skin M u s c l e and f a t Body ky = 26.09 h "

Organ

Adult

Rat

TABLE V I I .

r—

GO

o

w W

3

H

3

O

2

O

o

P

5

8

gg

14.

183

Absorption and Pharmacokinetics

SHAH ET AL.

A s t e a d y s t a t e was r e a c h e d r a p i d l y . The model p r e d i c t e d o r g a n c o n t e n t t o be maximum a t 6 h, f o l l o w e d by a d e c r e a s e i n c o n t e n t as a r e s u l t o f t h e d e c l i n e i n t h e amount t h a t was a v a i l a b l e f o r p e n e t r a t i o n . C o n t i n u o u s e x p o s u r e was s i m u l a t e d w i t h t h e model by m a i n t a i n i n g a u n i t c o n s t a n t amount on t h e s k i n . E q u i l i b r i u m l e v e l s c a l c u l a t e d i n t h e o r g a n s as f r a c t i o n s o f t h e amount on t h e s k i n f o r young and a d u l t , r e s p e c t i v e l y , were 1.5 X 4

10" ,

and

liver, 10~

4

9.4

0.65

X 10~

X 10"

4

4

and

f o r kidney, 4.7

f o r t h e t o t a l body.

h) f o r young and 4

X

10~

4

X 10""

and

f o r s k i n , and

Corresponding

a d u l t s were 52 X 1

4

3.7

10" 4

4

1.4

X 10~

81 X 1 0 ~

4

excretion rates and

17 X 1 0 ~

4

h"

4

and

for 40

X

(fraction/ 1

for urine

1

and 2.5 X 1 0 ~ h " and 0.34 X 1 0 ~ h " f o r f e c e s . From t h i s t e m p o r a l e x p e r i m e n t w i t h a s i n g l e a p p l i e d dose o f c a r b o f u r a n , t h e l i n e a r compartmental f l o A more complete v a l i d a t i o sequence o f doses as w e l l as d a t a from m u l t i p l e dose e x p e r i m e n t s . F o r c h l o r d e c o n e , a model d e v e l o p e d from i n t r a v e n o u s ( i v ) d o s i n g was used b u t m o d i f i e d t o accommodate t h e dermal a b s o r p t i o n pathway. A t i m e - v a r y i n g dermal a b s o r p t i o n r a t e c o n s t a n t was f o u n d t o enhance t h e model f i t . The u r i n a r y e x c r e t i o n r a t e c o n s t a n t was a l s o time-dependent. E x c r e t i o n began a t about 0.75 h"^ and f i n i s h e d a t about 0.01 a t 120 h. Equilibrium tissue/blood r a t i o s a g r e e d w i t h t h o s e f o u n d by i v d o s i n g e x c e p t f o r s k i n . Skin was f o u n d t o have an e q u i l i b r i u m t i s s u e / b l o o d r a t i o o f about 3.5 i n s t e a d o f 6 as d e t e r m i n e d by i v d o s i n g . T h i s was t h e l o w e s t r a t i o f o r any t i s s u e . The h i g h e s t r a t i o was 10 w h i c h o c c u r r e d f o r liver. Dermal a b s o r p t i o n f o r d i n o s e b was b i p h a s i c r e q u i r i n g two f i r s t o r d e r compartments t o a d e q u a t e l y s i m u l a t e t h e o b s e r v e d data. T i s s u e / b l o o d r a t i o s were l o w e s t i n muscle, about 0.15, and h i g h e s t i n k i d n e y , about 0.55. S i n c e a l l r a t i o s were l e s s t h a n 1.0, no s e q u e s t e r i n g o f d i n o s e b r a d i o a c t i v i t y i s i n d i c a t e d . The u r i n a r y pathway was t h e p r e f e r r e d r o u t e o f e x c r e t i o n by about a 4:1 r a t i o as compared t o t h e f e c a l r o u t e . H e x a c h l o r o b i p h e n y l was modeled w i t h s e p a r a t e compartments f o r p a r e n t and m e t a b o l i t e as d e t e r m i n e d from p u b l i s h e d i v e x p e r i m e n t s (14). Dermal a b s o r p t i o n was d e s c r i b e d by a s i n g l e f i r s t o r d e r compartment b u t d e l a y e d by a b o u t 1 h i n young r a t s and 4 h i n adult rats. The t i s s u e / b l o o d r a t i o f o r a d i p o s e f o r t h e p a r e n t compound was f o u n d t o be i n t h e 100's o r 1000's i n d i c a t i n g a v e r y s m a l l r e l e a s e o f p a r e n t from t h e a d i p o s e compartment. M u s c l e had t h e l o w e s t t i s s u e / b l o o d r a t i o f o r p a r e n t o f about 4. The m e t a b o l i s m c o n s t a n t f o r l i v e r was about 0.02 mL/min which i s q u i t e slow. The m e t a b o l i t e s a r e r e a d i l y e x c r e t e d and p r o d u c e d t i s s u e b l o o d r a t i o s o f o n l y 1 t o 4. DISCUSSION: A number o f s t u d i e s have s u g g e s t e d h i g h e r s k i n p e r m e a b i l i t y i n premature and f u l l - t e r m i n f a n t s compared t o a d u l t s . B e h l e t a l . (4) and Maibach and B o i s i t s (16) have r e v i e w e d t h e l i t e r a t u r e on

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

184

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

a g e - r e l a t e d p e r c u t a n e o u s a b s o r p t i o n . B e h l e t a l . (4,17) r e p o r t e d i n v i t r o s k i n p e n e t r a t i o n o f w a t e r , methanol, e t h a n o l , b u t a n o l , h e x a n o l , and o c t a n o l i n male h a i r l e s s mice o f d i f f e r e n t ages. They o b s e r v e d i n c r e a s e d s k i n p e r m e a b i l i t i e s r e a c h i n g maximum v a l u e s around 25 days o f age, t h e n d e c l i n i n g s h a r p l y up t o about 60 days o f age, and r e m a i n i n g more o r l e s s i n v a r i a n t t h e r e a f t e r . In t h e p r e s e n t s t u d y , a g e - r e l a t e d d i f f e r e n c e s i n p e r c u t a n e o u s a b s o r p t i o n o f some o f t h e p e s t i c i d e s t e s t e d were o b s e r v e d . I n many c a s e s s i g n i f i c a n t i n f l u e n c e o f methods employed t o d e t e r m i n e t h e s k i n a b s o r p t i o n o f t h e s e p e s t i c i d e s was a l s o o b s e r v e d . R e s u l t s a t 72 h f r o m t h e i n v i v o method showed a g e - r e l a t e d d i f f e r e n c e s i n s k i n p e n e t r a t i o n f o r c a r b o f u r a n , d i n o s e b , and hexachlorobiphenyl. The f l o w method showed s t a t i s t i c a l l y s i g n i f i c a n t s k i n p e n e t r a t i o n d i f f e r e n c e s f o r dinoseb, c a r b a r y l , c a r b o f u r a n and h e x a c h l o r o b i p h e n y l . The s t a t i c method showed s i g n i f i c a n t young/adult p e n e t r a t i o n d i f f e r e n c e s only f o r c a r b o f u r a n and h e x a c h l o r o b i p h e n y l demonstrates t h a t s i g n i f i c a n v a l u e s a r i s e from t h e d i f f e r e n t methods used. A number o f s t u d i e s have shown good c o r r e l a t i o n between i n v i v o and i n v i t r o s k i n p e r m e a b i l i t y v a l u e s f o r a number o f c h e m i c a l s and a n i m a l s p e c i e s because s k i n a b s o r p t i o n has been c o n s i d e r e d as t h e p a s s i v e d i f f u s i o n p r o c e s s t h r o u g h a r a t e l i m i t i n g , n o n l i v i n g b a r r i e r , s t r a t u m corneum. F r a n z (18) compared t h e p e r c u t a n e o u s a b s o r p t i o n ( i n v i t r o ) o f 12 o r g a n i c compounds w i t h i n v i v o r e s u l t s r e p o r t e d by Feldmann and Maibach ( 1 9 ) . He r e p o r t e d e x c e l l e n t q u a l i t a t i v e agreement between i n v i t r o and i n v i v o methods. He f u r t h e r r e p o r t e d t h e d i s c r e p a n c i e s i n t h e q u a n t i t a t i v e agreement between t h e two s e t s o f d a t a . L a t e r on F r a n z (20) r e p o r t e d t h a t d i s c r e p a n c i e s i n q u a n t i t a t i v e agreement was due s i m p l y t o e x p e r i m e n t a l v a r i a t i o n based on a d d i t i o n a l i n v i t r o e x p e r i m e n t s t o s i m u l a t e i n v i v o c o n d i t i o n s by w a s h i n g s k i n a f t e r 24 h. Bronaugh (21) r e p o r t e d i d e n t i c a l i n v i v o and i n v i t r o rat s k i n absorption values f o r benzoic a c i d , a c e t y l s a l i c y l i c a c i d and u r e a . The two i n v i t r o methods u t i l i z e d h e r e , namely, t h e s t a t i c and f l o w - t h r o u g h d i f f u s i o n c e l l s , have been r e c e n t l y compared by Bronaugh and S t e w a r t ( 2 2 ) . The a u t h o r s (22) r e p o r t e d t h e c o m p a r a t i v e s k i n a b s o r p t i o n v a l u e s f o r w a t e r , c o r t i s o n e , and b e n z o i c a c i d t h r o u g h r a t s k i n as d e t e r m i n e d by s t a t i c and f l o w through d i f f u s i o n c e l l s . They r e p o r t e d s i m i l a r a b s o r p t i o n p r o f i l e s and q u a n t i t a t i v e v a l u e s between t h e two d i f f u s i o n systems f o r w a t e r , c o r t i s o n e , and b e n z o i c a c i d under t h e e x p e r i m e n t a l conditions described. However, d i s c r e p a n c i e s i n a b s o r p t i o n v a l u e s were o b s e r v e d f o r a h y d r o p h o b i c compound, [3-phenyl-2-propenyl-2aminobenzoate ( c i n n a m y l a n t h r a n i l a t e ) ] between t h e two d i f f u s i o n c e l l systems; t h e f l o w - t h r o u g h d i f f u s i o n c e l l system gave enhanced a b s o r p t i o n v a l u e f o r c i n n a m y l a n t h r a n i l a t e compared t o t h e s t a t i c c e l l system. They have f u r t h e r r e p o r t e d good agreement between i n v i v o and i n v i t r o ( s t a t i c as w e l l as f l o w - t h r o u g h d i f f u s i o n c e l l s ) s k i n p e r m e a t i o n f o r c o r t i s o n e and b e n z o i c a c i d when each was applied i n petrolatum v e h i c l e . We have r e p o r t e d d i s c r e p a n c i e s i n t h e s k i n a b s o r p t i o n v a l u e s as d e t e r m i n e d by t h e two i n v i t r o methods. We a l s o o b s e r v e d p o o r

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

14.

SHAH ET AL.

Absorption and Pharmacokinetics

185

c o r r e l a t i o n between i n v i v o and i n v i t r o r e s u l t s f o r t h e s e h i g h l y complex p e s t i c i d a l c h e m i c a l s . There a r e a number o f f a c t o r s t h a t can i n f l u e n c e t h e s k i n a b s o r p t i o n measurements. The i n f l u e n c e o f r e c e p t o r f l u i d f o r i n v i t r o s k i n a b s o r p t i o n measurements has been w i d e l y documented. F o r example, t h e i n v i t r o a b s o r p t i o n o f c h l o r d e c o n e and h e x a c h l o r o b i p h e n y l i n t h e p r e s e n t s t u d y n e v e r exceeded more t h a n t h a n 2.5% i n b o t h d i f f u s i o n c e l l systems. These two c h e m i c a l s have p o o r w a t e r s o l u b i l i t y (0.9 ppb f o r h e x a c h l o r o b i p h e n y l ) and t h e r e c e p t o r f l u i d u t i l i z e d i n t h i s i n v e s t i g a t i o n may n o t be s u f f i c i e n t l y l i p o h i l i c t o remove t h e c h e m i c a l from t h e d e r m i s ( s p l i t - t h i c k n e s s s k i n ) . Bronaugh and S t e w a r t (23) s u g g e s t e d t h a t t h e l a c k o f s o l u b i l i t y o f t h e h y d r o p h o b i c p e n e t r a n t i n t o t h e r e c e p t o r f l u i d f o r i n v i t r o methods c o u l d be a c o n t r i b u t i n g f a c t o r a c c o u n t i n g f o r i n v i v o and i n v i t r o d i s c r e p a n c i e s i n s k i n a b s o r p t i o n v a l u e s . Bronaugh and S t e w a r t (23) r e p o r t e d maximum a b s o r p t i o n o f c i n n a m y l a n t h r a n i l a t e by u s i n g 6% PEG-20 o l e y l e t h e r r e c e p t o c e l l system. R e c e n t l y , S c o t t and Ramsey (24) r e p o r t e d t h a t t h e h i g h e s t a b s o r p t i o n o f c y p e r m e t h r i n was a c h i e v e d when 50% aqueous e t h a n o l was used as t h e r e c e p t o r f l u i d w h i c h was i n agreement w i t h i n v i v o a b s o r p t i o n t h r o u g h t h e r a t s k i n . They f u r t h e r r e p o r t e d t h a t t h e a b s o r p t i o n o f c y p e r m e t h r i n measured w i t h two o t h e r r e c e p t o r f l u i d s (6% V o l p o 20 and 20% f e t a l c a l f serum) was v e r y s i m i l a r t o 50% aqueous e t h a n o l as t h e r e c e p t o r f l u i d . Thus t h e above mentioned study suggests t h a t , the s o l u b i l i t y of the penetrant i n t o r e c e p t o r f l u i d , r a t h e r than the d i f f u s i o n of penetrant through the e p i d e r m a l membrane, i s t h e r a t e l i m i t i n g s t e p . I t appears t h a t t h e r e s u l t s o b t a i n e d by d i f f e r e n t methods have t o be c a r e f u l l y e v a l u a t e d w i t h t h e e x p e r i m e n t a l c o n d i t i o n s prescribed. I t i s a l s o c l e a r from t h e l i t e r a t u r e t h a t t h e i n v i t r o r e s u l t s can be i n f l u e n c e d by a number o f f a c t o r s . Perhaps more c o m p a r a t i v e s t u d i e s may improve t h e u n d e r s t a n d i n g o f t h e i n v i t r o and i n v i v o c o r r e l a t i o n . V a l i d a t i o n o f r e c e n t l y recommended g u i d e l i n e s f o r i n v i t r o p e r c u t a n e o u s p e n e t r a t i o n s t u d i e s (25) w i t h a number o f c h e m i c a l s may h e l p i n u n d e r s t a n d i n g t h e i n v i t r o s k i n absorption process. U t i l i t y o f p h a r m a c o k i n e t i c models has been e x p l o r e d f o r e x t r a p o l a t i o n t o man, a n i m a l s p e c i e s s c a l i n g , e x t r a p o l a t i o n o f r e s u l t s beyond t h e o b s e r v e d t i m e p o i n t , s i m u l a t i o n o f c o n t i n u o u s or m u l t i p l e exposure s i t u a t i o n , t i s s u e dose e x t r a p o l a t i o n , i m p r o v i n g e x p e r i m e n t a l d e s i g n , and o v e r a l l r e d u c t i o n o f a n i m a l usage. P h y s i o l o g i c a l models have been shown t o be adequate f o r o t h e r c h e m i c a l s (26,27). The a p p l i c a t i o n s o f p h y s i o l o g i c a l l y based m o d e l i n g and i t s t e c h n i q u e has been r e v i e w e d by a number o f i n v e s t i g a t o r s (26,28,29). I n t h e p r e s e n t s t u d y , p h y s i o l o g i c a l l y based p h a r m a c o k i n e t i c models were used t o d e c r i b e dermal a b s o r p t i o n and d i s p o s i t i o n p r o c e s s e s f o r t h e c h e m i c a l s s t u d i e d . Rate c o n s t a n t s f o r dermal a b s o r p t i o n , and u r i n a r y and f e c a l e x c r e t i o n , as w e l l as t i s s u e t o b l o o d steady s t a t e r a t i o s were determined. A p h y s i o l o g i c a l model d e s c r i b i n g c a r b a r y l r a d i o a c t i v i t y was f i t t e d t o t h e t i s s u e d i s t r i b u t i o n d a t a . The dermal a b s o r p t i o n was f o u n d t o be d e s c r i b e d by a s i n g l e f i r s t o r d e r compartment f o r b o t h

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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young and a d u l t r a t s o v e r t h e e x p e r i m e n t a l p e r i o d . The o r d e r o f t h e e q u i l i b r i u m t i s s u e / b l o o d r a t i o s was t h e same i n young and a d u l t r a t s . The t i s s u e w i t h t h e l a r g e s t t i s s u e / b l o o d r a t i o o f about 6 o r 7 was k i d n e y . The u n t r e a t e d s k i n has t h e l o w e s t e q u i l i b r i u m t i s s u e / b l o o d r a t i o o f about 0.8 t o 1.0. The r a t e c o n s t a n t f o r k i d n e y t o u r i n e e x c r e t i o n was l a r g e a t 15 t o 20 h"^. A more complex model t o i n c l u d e m e t a b o l i s m , b l o o d c l e a r a n c e , r e l e a s e o f chemical from s o l v e n t t o s k i n , i n c o r p o r a t i o n o f v i a b l e l a y e r , and m e t a b o l i s m d u r i n g a b s o r p t i o n , e t c . , may a i d i n i m p r o v i n g t h e model's u t i l i t y . CONCLUSIONS; P e r c u t a n e o u s a b s o r p t i o n was compared i n young and a d u l t F i s h e r 344 r a t s f o r f i v e p e s t i c i d e s u t i l i z i n g b o t h i n v i v o and i n v i t r o methods. I n v i v o p e n e t r a t i o n o f t h e p e s t i c i d e s d u r i n g a 72 h p e r i o d , ranged from a p p r o x i m a t e l (dinoseb, a d u l t ) , dependin Three p e s t i c i d e s , ( c a r b o f u r a n , h e x a c h l o r o b i p h e n y l , and c h l o r d e c o n e ) showed h i g h e r s k i n p e n e t r a t i o n v a l u e s i n young compared t o a d u l t . D i f f e r e n c e s i n s k i n p e n e t r a t i o n v a l u e s were o b s e r v e d among p e s t i c i d e s due t o methods o f measurement. Two i n v i t r o methods, namely, s t a t i c c e l l and f l o w - t h r o u g h d i f f u s i o n c e l l systems gave d i f f e r e n t p e n e t r a t i o n v a l u e s . A p h y s i o l o g i c a l compartmental model a p p e a r s t o be good f o r s i m u l a t i n g dermal a b s o r p t i o n and d i s p o s i t i o n o f t h e p e s t i c i d e s s t u d i e d . ACKNOWLEDGMENTS: T h i s work was c o n d u c t e d a t N o r t h r o p E n v i r o n m e n t a l Sciences, Research T r i a n g l e P a r k , NC. The r e s e a r c h d e s c r i b e d i n t h i s a r t i c l e has been r e v i e w e d by t h e O f f i c e o f T o x i c S u b s t a n c e s , U.S. E n v i r o n m e n t a l P r o t e c t i o n Agency, and approved f o r p u b l i c a t i o n . A p p r o v a l does n o t s i g n i f y t h a t t h e c o n t e n t s n e c e s s a r i l y r e f l e c t t h e v i e w s and p o l i c i e s o f t h e Agency, n o r does mention o f t r a d e names o r c o m m e r c i a l p r o d u c t s c o n s t i t u t e endorsement o r recommendation f o r u s e .

Literature Cited 1. 2. 3. 4.

5. 6. 7.

Synthetic Organic Chemicals , United States International Trade Commission, 1985, pp 226-241. Durham, W.F.; Wolfe, H.R. Bull. WHO 1962, 26 , 75-91. Wolfe, H.R.; Durham, W.F.; Armstrong, J.F.Arch.Environ. Health 1976, 14 , 622-633. Behl, C.R.; Bellantone, N.H.; Flynn, G.L. In Percutaneous Absorption Mechanisms - Methodology - Drug Delivery . Bronaugh, R.L., Maibach, H.I.; Eds.; Dekker: New York, 1985; pp 183-212. Nachman, R.L.; Esterly, N.B.J. Pediatr . 1971, 79 , 628-632. Wester, R.C.; Noonan, P.K.; Cole, M.P.; Maibach, H.I. Pediatr. Res . 1977, 11 , 737-739. Knaak, J.B.; Yee, K., Ackerman, CR., Zweig, G., Wilson, B.W. Toxicol. Appl. Pharmacol . 1984, 72 , 406-416.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

14. SHAHETAL.

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8. Shah, P.V.; Fisher, H.L.; Sumler, M.R; Chernoff, N.; Hall, L.L. J. Toxicol. Environ. Health 1987, 21 , 353-366. 9. Shah, P.V.; Fisher, H.L.; Month, N.J.; Sumler, M.R.; Hall, L.L. J. Toxicol. Environ. Health 1987, 22 ; 207-223. 10. SAS Institute Inc. SAS User's Guide: Statistics Version-5 Edition, SAS Institute, Inc., Cary, NC 1985. 11. Bungay, P.M.; Dedrick, R.L.; Matthews, H.B.Ann. N.Y. Acad. Sci . 1979, 320 , 257-270. 12. Bungay, P.M., Dedrick, R.L., Matthews, H.B. J. Pharmacokinet. Biopharm . 1981, 309-341. 13. Berman, M., Weiss, M.F. SAAM Manual. (Simulation, Analysis and Modeling) , Version: SAAM 27. Department of Health, Education, and Welfare, Publication (NIH) 78-180, 1978. 14. Lutz, R.J.; Dedrick, R.L.; Matthews, H.B.; Eling, T.E; Anderson, M.W. Drug. Metab. Dispos . 1977, 54 , 386-397. 15. Bischoff, K.B., Dedrick R.L.; Zaharko D.S.; Longstreth J.A.J. Pharm. Sci 16. Maibach, H.I.; Boisits Function ; Dekker: New York, 1982. 17. Behl, C.R.; Flynn, G.L.; Kurihara, T.; Smith, W.M.; Bellantone, N.H., Gatmaitan, O.; Pierson, C.L.; Higuchi, W.I.; HO, N.F.H. J. Soc. Cosmet. Chem. 1984, 35 , 237-252. 18. Franz, T.J. J. Invest. Dermatol . 1975, 64 , 190-195. 19. Feldmann, R.J.; Maibach, H.I. J. Invest. Dermatol . 1970, 54 , 399-404. 20. Franz, T.J. Curr. Probl. Dermatol . 1978, 7, 58-68. 21. Bronaugh, R.L.; Stewart, R.F.; Congdon, E.R. Toxicol. Appl. Pharmacol. 1982, 62 , 481-488. 22. Bronaugh, R.L.; Stewart, R.F.J. Pharm. Sci . 1985, 74 , 6467. 23. Bronaugh, R.L.; Stewart, R.F. J. Pharm. Sci. 1984,73, 12551257. 24. Scott, R.C.; Ramsey, J.D.J. Invest. Dermatol . 1987,89, 142146. 25. Skelly, J.P.; Shah, V.P.; Maibach, H.I.; Guy R.H.; Wester, R.C.; Flynn, G.; Yacobi, A. Pharm. Res . 1987, 4 , 265-267. 26. Anderson, M.E.; Keller, W.C. InCutaneous Toxicity. Target Organ Toxicology Series ; Drill, V.A.,; Lazar, P., Eds., Raven Press, New York, 1982. p 9-27. 27. McDougal, J.N.; Jepson, G.W.; Clewell III, H.J.; MacNaughton, M.G.; Anderson, M.E. Toxicol. Appl Pharmacol . 1986, 85 , 286-294. 28. Lutz, R.J.; Dedrick, R.L.; Zaharko, D.S. Pharmacol. Ther . 1980, 11, 559-592. 29. Gerlowski, L.E.; Jain, R.K.J. Pharm. Sci . 1983, 72 , 11031127. RECEIVED July 5,1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 15

Biological Monitoring Techniques for Humans Exposed to Pesticides Use, Development, and Recommended Refinements T. L. Lavy and J. D. Mattice Altheimer Laboratory, University of Arkansas, Fayetteville, AR 72703

Observations and major conclusions on the design of human applicator exposure studie based 10 studie are presented. An is suitable for measuring an absorbed dose of a compound i s given along with a general approach for developing an ana­ lytical technique. Whether one uses excretion of the intact pesticide or a known urinary metabolite as the test c r i t e r i a , it i s important to have quantitative human absorption and excretion data for each pesticide to be able to provide accurate meaningful quantitative risk assessment information. Pesticides benefit us in helping to produce food and fiber, but they also have the potential to harm us. The need for protection for those applying pesticides or those associated with their production is a concept that has been readily accepted for over 20 years. Although some specific information exists for a pesticide regarding its toxicity to rats or other test animals, there i s no uniform agreement among researchers regarding the extent of protection needed by humans in contact with the pesticide, how to achieve the desired level of protection, how to quantify the exposures occurring nor how to evaluate the postulated health effects from pesticides entering the body from contaminated clothing or dermal surfaces. In casual conversation the words "exposure" and "dose" are often used for the same concept. In our context they have specific meanings. Exposure is being accessible to something, such as a pesticide. If i t i s on our skin or in the air we breathe, we are exposed. Dose i s defined as the amount that gets inside our bodies. In this paper we plan to address some of the problems associated with applicator exposure data obtained by analyzing patches attached to clothing of workers exposed to pesticides, and absorbed dose data obtained by measuring urinary excretion of the pesticide. Important facts to be considered in designing studies which will provide quan­ titative absorbed dose data while at the same time allowing collec­ tion of patch data will also be discussed. The latter portions of 0097-6156/89/0382-0192$06.00/0 1989 American Chemical Society c

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

15.

LAVY AND

MATTICE

Biological Monitoring Techniques

193

the paper w i l l o u t l i n e the steps i n v o l v e d with developing the c r i ­ t e r i a and t e c h n i q u e s f o r u s i n g human u r i n e as a b i o l o g i c a l m o n i ­ toring tool. F o r t h e p a s t n i n e y e a r s we have been c o n d u c t i n g s t u d i e s m e a s u r i n g d o s e and e x p o s u r e f o r p e o p l e w o r k i n g w i t h 11 d i f f e r e n t compounds ( T a b l e I ) . D u r i n g our s t u d i e s i n t h e l a t e 1970's on w o r k e r s a p p l y i n g 2,4,5-T and 2,4-D i n t h e f o r e s t ( 1 , 2 ) , c o n s i d e r a b l e e f f o r t was d e v o t e d t o c o l l e c t i n g both p a t c h d a t a and d a t a f r o m u r i n e a n a l y s e s o f e x p o s e d w o r k e r s . One o f t h e g o a l s o f t h o s e s t u d i e s was t o f i n d i f a h i g h c o r r e l a t i o n e x i s t e d between t h e h e r b i c i d e d e p o ­ s i t e d on p a t c h e s and t h e a b s o r b e d h e r b i c i d e d o s e as d e t e r m i n e d f r o m u r i n e a n a l y s i s . E a r l i e r , S a u e r h o f f (3) had shown r a p i d and n e a r l y q u a n t i t a t i v e e x c r e t i o n o f phenoxy h e r F i c i d e s w h e t h e r t h e y e n t e r e d t h e body o r a l l y o r d e r m a l l y . By p h o t o g r a p h i n g each o f our w o r k e r s i n h i s f i e l d a t t i r e , o b s e r v i n g t h e p h o t o g r a p h , and e m p l o y i n g a v e r a g e human a r e a m e a s u r e m e n t s f o r d i f f e r e n t body p o r t i o n s (40 i t was p o s s i b l e to c a l c u l a t e th A f t e r e x t r a p o l a t i n g the a t t a c h e d t o s e v e r a l p o r t i o n s o f t h e w o r k e r ' s c l o t h i n g near b a r e s k i n a r e a s , i t s h o u l d have been p o s s i b l e t o p r e d i c t t h e amount o f h e r ­ b i c i d e d e p o s i t e d on b a r e dermal s u r f a c e s . We had hoped t h a t r e s u l t s f r o m t h i s c a l c u l a t i o n w o u l d c o r r e l a t e s t r o n g l y w i t h t h e amount o f phenoxy h e r b i c i d e e x c r e t e d i n u r i n e o f t h e e x p o s e d i n d i v i d u a l . U n f o r t u n a t e l y , f r o m t h e s t a n d p o i n t of s i m p l i c i t y o f measurement, a low c o r r e l a t i o n , 0.377 (1_), was f o u n d . Aware o f t h i s low c o r r e l a ­ t i o n , i t t h u s became i m p o r t a n t t o m e a s u r e t h e p a r a m e t e r most l i k e l y t o c o r r e l a t e w i t h p o t e n t i a l h e a l t h e f f e c t s . S i n c e any a d v e r s e human h e a l t h e f f e c t s t h a t may a r i s e due t o p e s t i c i d e e x p o s u r e w i l l be more a f u n c t i o n o f t h e l e v e l s p r e s e n t i n s i d e t h e body r a t h e r t h a n t h e amounts d e p o s i t e d on c l o t h i n g , g l o v e s o r s k i n , t h e m a j o r i t y o f our e f f o r t s a f t e r t h e i n i t i a l two f o r e s t r y s t u d i e s have been d i r e c t e d t o w a r d . o b t a i n i n g q u a n t i f i a b l e a b s o r b e d d o s e i n f o r m a t i o n . We h a v e c h o s e n t o t a l u r i n e c o l l e c t i o n and c o n s e q u e n t u r i n e a n a l y s i s as t h e t o o l t o measure the absorbed dose. A l t h o u g h b l o o d , u r i n e and p e r h a p s o t h e r body f l u i d s o r e x c r e ­ t i o n s c o u l d c o n c e i v a b l y be used as b i o l o g i c a l m o n i t o r i n g mediums, we h a v e used u r i n e a n a l y s e s e x c l u s i v e l y as our b i o l o g i c a l m o n i t o r i n g t o o l . A l t h o u g h w o r k e r s a r e not e a g e r t o c o l l e c t t h e i r t o t a l u r i n e o u t p u t , t h e y much p r e f e r t o c o l l e c t t h e i r u r i n e t h a n t o have t h e i r b l o o d sampled d a i l y . S i n c e a c c u r a t e f i n d i n g s of a w o r k e r e x p o s u r e s t u d y a r e so v i t a l l y d e p e n d e n t upon t h e f u l l c o o p e r a t i o n o f t h e w o r k e r s i n v o l v e d , a l l p o s s i b l e c o m m u n i c a t i o n s and p r e p a r a t i o n s s h o u l d be made s e v e r a l weeks p r i o r t o t h e i n i t i a t i o n o f t h e s t u d y . I t i s i m p o r t a n t t h a t t h e i n v e s t i g a t o r go t o t h e f i e l d , meet t h e w o r k e r s , and d i s c u s s t h e p r o t o c o l w i t h them a t l e a s t o n c e p r i o r t o t h e s t u d y . R e s u l t s o f our e a r l i e r s t u d i e s have shown t h a t r e p r o d u c i b l e q u a n t i f i a b l e p e s t i c i d e e x c r e t i o n d a t a can o n l y be o b t a i n e d by making t o t a l u r i n e c o l l e c t i o n s o v e r t h e measurement p e r i o d . In our i n i t i a l 2,4,5-T s t u d y {1) when u r i n e was c o l l e c t e d o v e r c o n s e c u t i v e 2 4 - h o u r p e r i o d s t h e r e was much l e s s v a r i a b i l i t y i n t h e c o n c e n t r a t i o n s o f 2,4,5-T e x c r e t e d i n u r i n e when compared t o c o n s e c u t i v e 1 2 - h o u r s a m p l e s . A l t h o u g h 1500-1700 mis o f u r i n e i s an a v e r a g e d a i l y u r i ­ n a r y e x c r e t i o n volume used by some r e s e a r c h e r s , our s t u d i e s i n d i c a t e 9

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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T a b l e I . Human E x p o s u r e S t u d i e s U t i l i z i n g B i o l o g i c a l M o n i t o r i n g T e c h n i q u e s Completed o r Underway a t t h e A l t h e i m e r L a b o r a t o r y Compound

Urinary metabolite

bayleton

4-chlorophenol

Sensitivity (ppm) 0.05

Dose e x c r e t e d U r i n e Animal Ref (%) 90 30-40

cow, pig rat

a

dog rat

19 19

a

benomyl

MBC, 4-MBC, 5-MBC

0.10

b i f enox

5-(2,4-dichlorophenoxy) -2-nitrobenzoi

0.02

29-53

rat

b

captan

tetrahydrophthalimide

0.03

13

rat

16,17

carbaryl

naphthyl s u l f a t e 1-naphthol

0.20

40-70

rat

c

chlorpyrifos

3,5,6-trichloro-2pyridinol

0.01

70

man

14

2,4-D

2,4-D

0.04

95-100

man

3

2,4-DP

2,4-DP

0.04

95-100

man

5

picloram

picloram

0.01

90-97

22

90

dog, cow man

62

mice

d

man

23

pydrin

4-chloro-(l-methylethyl)benzene acetic acid

2,4,5-T

2,4,5-T

a

b

c

d

e

e

0.04

16 86

95-100

6

T h o r n t o n , J . S., Mobay C h e m i c a l C o r p . , P. 0. Box 4 9 1 3 , Hawthorn Road, K a n s a s C i t y , M0 64120, P e r s o n a l C o m m u n i c a t i o n , 1 9 8 5 . N o r r i s , F . A., R h o n e - P o u l e n c I n c . , P. 0. Box 1 2 5 , B l a c k H o r s e L a n e , Monmouth J u n c t i o n , N . J . 08852, P e r s o n a l C o m m u n i c a t i o n , 1984. H a r r i s o n , S., Union C a r b i d e A g r i c u l t u r a l P r o d u c t s Co. I n c . , P. 0. Box 12014, T. W. A l e x a n d e r D r i v e , R e s e a r c h T r i a n g l e P a r k , NC 27709, Personal Communication, 1985. B i s h o p , J . L . , S h e l l A g r i c u l t u r a l C h e m i c a l Co., P. 0. Box 4248, M o d e s t o , CA 95352, P e r s o n a l C o m m u n i c a t i o n , 1 9 8 6 . T o be d e t e r m i n e d .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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195

t h e use of an average d a i l y u r i n a r y e x c r e t i o n volume i s not s u f f i c i e n t t o o b t a i n good q u a n t i t a t i v e p e s t i c i d e e x c r e t i o n d a t a . Some f i e l d workers r e g u l a r l y e x c r e t e d between 500 and 600 mis per 24 hours w h i l e o t h e r s e x c r e t e d over 6000 mis i n a s i m i l a r 24-hour p e r i o d (5). S i n c e i t i s not s i m p l e or c o n v e n i e n t t o c o l l e c t ones t o t a l urTne output over an extended p e r i o d i t i s i m p e r a t i v e t h a t the worker be t o t a l l y aware of the importance of p r o v i d i n g a l l t h e i r urine. The workers must be informed t h a t t h e i r i n d i v i d u a l p e r f o r mance w i l l d i c t a t e whether the study i s a success or f a i l u r e . An i m p o r t a n t p o r t i o n of t h e expenses a s s o c i a t e d w i t h c o n d u c t i n g an exposure study i n v o l v i n g humans should i n c l u d e payments f o r t h e workers s u p p l y i n g t h e i r t o t a l u r i n e o u t p u t . Although t h e r e s e a r c h e r can not be a b s o l u t e l y c e r t a i n t h a t a l l workers are complying w i t h h i s i n s t r u c t i o n s , some g u i d e l i n e s we have f o l l o w e d i n an attempt t o c o l l e c t or account f o r t h e t o t a l u r i n e output a r e : (a) I f a worker i s aware t h a t a p o r t i o n of the u r i n e sample was i n a d v e r t e n t l y l o s t an e s t i m a t e of t h e volum collected. (Complianc with unusually high loss r e c o r d s ) , (b) C r e a t i n i n e c o n c e n t r a t i o n s a r e run on each u r i n e sample. By m u l t i p l y i n g the c r e a t i n i n e c o n c e n t r a t i o n (mg/1) by t h e amount of u r i n e e x c r e t e d (1) i t i s p o s s i b l e t o c a l c u l a t e the amount of c r e a t i n i n e e x c r e t e d on a d a i l y b a s i s . S i n c e the amount of c r e a t i n i n e e x c r e t e d d a i l y i s r e l a t i v e l y c o n s t a n t , we can determine i f s u b s t a n t i a l amounts of u r i n e have been lost. Knowledge on the p a r t of the worker t h a t we w i l l be a n a l y z i n g each sample f o r c r e a t i n i n e , coupled w i t h the f a c t t h a t t h e d o l l a r s p a i d t o the p a r t i c i p a n t s are not made u n t i l a p e r i o d of time l a t e r ( s u f f i c i e n t t o a l l o w a n a l y s i s of the samples f o r c r e a t i n i n e c o n t e n t ) h e l p s p r o v i d e us w i t h a f a i r degree of assurance of worker c o o p e r a tion. Another f a c t o r of s i g n i f i c a n c e w i t h r e s p e c t t o worker c o o p e r a t i o n i s the f a c t t h a t most workers are t r u l y i n t e r e s t e d i n knowing whether they a r e being exposed t o the p e s t i c i d e s and i f the amount of p e s t i c i d e e n t e r i n g t h e i r body i s l i k e l y t o cause p r e s e n t or f u t u r e h e a l t h p r o b l e m s . The n e g a t i v e p u b l i c i t y which i s o f t e n a s s o c i a t e d w i t h t h e use of p e s t i c i d e s has i n c r e a s e d the l e v e l of concern f o r some of the w o r k e r s . The optimum u r i n e c o l l e c t i o n p e r i o d f o r a c h i e v i n g a c c u r a t e r e p r o d u c i b l e r e s u l t s i s dependent on s e v e r a l f a c t o r s , such a s : rate of e n t r y i n t o body, m e t a b o l i c changes o c c u r r i n g and e x c r e t i o n r a t e from the body. In the case of the phenoxy h e r b i c i d e s , Sauerhoff (3) has i n d i c a t e d t h a t w e l l over 90% of the absorbed dose i s e x c r e t e d w i t h i n the f i r s t 5 days f o l l o w i n g e x p o s u r e . L a b o r a t o r y s t u d i e s by Nolan et a l . (6) have shown t h a t p i c l o r a m i s e x c r e t e d i n u r i n e even more r a p i d l y than the phenoxy compounds. In our study e v a l u a t i n g f o r e s t workers exposed t o h e r b i c i d e s p r a y m i x t u r e s c o n t a i n i n g 4 p a r t s 2 , 4 - D t o 1 p a r t p i c l o r a m , the e l u t i o n p a t t e r n s f o r the two compounds were markedly d i f f e r e n t ( F i g u r e 1). This f i g u r e shows t h a t p i c l o r a m i s e l u t e d much more r a p i d l y than 2 , 4 - D . Comparing F i g u r e 1 w i t h F i g u r e 2 i t i s e v i d e n t t h a t the r a t e of e x c r e t i o n has l i t t l e , i f a n y , r e l a t i o n s h i p t o the amount of herbicide excreted. Feldman and Maibach (7) have shown t h a t 6% of d e r m a l l y a p p l i e d 2 , 4 - D can be absorbed through the s k i n . In l a b o r a t o r y s t u d i e s Nolan et a l . (6) showed t h a t o n l y 0.18% of d e r m a l l y a p p l i e d p i c l o r a m would be a F s o r b e d . The c o m p o s i t i o n of the spray

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

196

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

TYPE OF TREE APPLICATION: HYPOHATCHET

APPLICATION

F i g u r e 1. workers.

•

2,4-D

•

PICLORAM

DAY

Rate of p i c l o r a m and 2 , 4 - D i n u r i n e of exposed

TYPE OF TREE APPLICATION: HYPOHATCHET .024

•

2,4-D PICLORAM

"g .020

o> .016 £ Q LU .012 LU UJ .008 UJ Q O CD .004 GC UJ X I

APPLICATION DAY

2 DAY

F i g u r e 2 . Amount of p i c l o r a m and 2 , 4 - D e x c r e t e d (mg) (exp r e s s e d as mg h e r b i c i d e / k g body w e i g h t ) .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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LAVY AND MATTICE

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Biological Monitoring Techniques

mix i n our study was 4 p a r t s 2,4-D t o 1 p a r t p i c l o r a m . The amount of p i c l o r a m e x c r e t e d i n u r i n e was much l e s s than t h a t of 2 , 4 - D , and was c o n s i d e r a b l y l e s s than the 4 : 1 r a t i o t h a t was p r e s e n t i n the spray mix. Twenty y e a r s ago i f one had been a t t e m p t i n g t o e x p l a i n t h e s e low p i c l o r a m e x c r e t i o n l e v e l s i t might have been suggested t h a t major p o r t i o n s were being r e t a i n e d i n the body. However, our f i n d i n g s of r e l a t i v e l y low l e v e l s of p i c l o r a m i n human u r i n e were p r e d i c t a b l e from N o l a n ' s (6^) l a b o r a t o r y s t u d i e s i n which he found t h a t o n l y 0.18% of d e r m a l l y d e p o s i t e d p i c l o r a m would be a b s o r b e d . A p o p u l a r m i s c o n c e p t i o n perhaps i n i t i a l l y hampered b i o l o g i c a l m o n i t o r i n g of body f l u i d s as a q u a n t i f i a b l e means of measuring the absorbed dose of p e s t i c i d e s i n humans. With the d i s c o v e r y t h a t DDT and o t h e r h i g h l y f a t s o l u b l e c h l o r i n a t e d hydrocarbon i n s e c t i c i d e s a r e s t o r e d i n f a t t y t i s s u e s of mammals, i t was perhaps i n f e r r e d t h a t o n l y a small f r a c t i o n of any p e s t i c i d e e n t e r i n g the body was excreted. Thus, t h e c h e m i s t r y of t h e s e i n s e c t i c i d e s may have i n a d v e r t e n t l y p r o v i d e d a majo monitoring techniques f o banning of some of t h e s e more p e r s i s t e n t c h l o r i n a t e d i n s e c t i c i d e s and the advent of new c h e m i s t r y has i n many i n s t a n c e s r e s u l t e d i n t h e development of compounds which are more p o l a r and h e n c e , l e s s f a t s o l u b l e than the e a r l i e r p e s t i c i d e s . Today, f e e d i n g s t u d i e s i n v o l v i n g small a n i m a l s and l ^ c - l a b e l e d p e s t i c i d e s are used t o determine i f the p e s t i c i d e or i t s m e t a b o l i t e ( s ) i s e x c r e t e d i n u r i n e , f e c e s , or b o t h . Some i n f o r m a t i o n i s a v a i l a b l e t o the r e s e a r c h e r a t t e m p t i n g t o e x t r a p o l a t e between animal t e s t s and a c t u a l human e x c r e t i o n d a t a . However, f o r most newer compounds most i n f o r mation r e g a r d i n g p e s t i c i d e e x c r e t i o n from humans must be e x t r a p o l a t e d from small animal s t u d i e s . F o l l o w i n g are s t e p s we f e e l should be taken when d e v e l o p i n g a procedure f o r u s i n g u r i n e a n a l y s i s as a t o o l f o r b i o l o g i c a l m o n i toring: Determine in Urine

i f the Parent Compound and/or M e t a b o l i t e s are

Excreted

S t u d i e s w i t h t e s t animals u s i n g r a d i o l a b e l e d compounds can help answer t h i s q u e s t i o n . I f most of t h e a c t i v i t y i s r e c o v e r e d i n the u r i n e , then u r i n e i s p o t e n t i a l l y a good c h o i c e f o r a n a l y s i s . However, the absence of a c t i v i t y i n u r i n e does not n e c e s s a r i l y mean t h a t u r i n e would not be a good c h o i c e f o r a n a l y s i s , as w i l l be discussed l a t e r . At the same t i m e , the k i n e t i c s of e x c r e t i o n as a f u n c t i o n of dermal or o r a l d o s i n g can be determined t o e s t a b l i s h the l e n g t h of time d u r i n g which u r i n e w i l l have t o be c o l l e c t e d . Identify

the Excreted

Compounds t h a t Contain the

Activity

Are the compounds e x c r e t e d as parent compound, m e t a b o l i t e s , or both? Are they f r e e or bound t o some o t h e r moiety? Chromatography and mass s p e c t r o m e t r y or i n f r a r e d s p e c t r o s c o p y can be used t o answer these q u e s t i o n s . An example of t h i s would be the work of G a r d i n e r , B r a n t l e y , and Sherman (8) and G a r d i n e r , K i r k l a n d , K l o p p i n g , and Sherman {9} i n i d e n t i f y i n g methyl 5-hydroxy-2-benzimidazolecarbamate (5-MBC) as a m e t a b o l i t e of methyl l-(butylcarbamoyl)-2-

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

b e n z i m i d a z o l e carbamate (benomyl) i n r a t u r i n e . Rats g i v e n an o r a l dose of l ^ C - l a b e l e d benomyl e x c r e t e d most of the a c t i v i t y i n the u r i n e i n 24 h r . A radioautogram of a f r e s h u r i n e sample developed on a s i l i c a t i c p l a t e showed one major and two minor l ^ c - c o n t a i n i n g compounds. A r a d i o a u t o g r a m of a t i c p l a t e developed i n t h e same manner of a u r i n e sample t h a t had been e n z y m a t i c a l l y h y d r o l y z e d w i t h a 3 - g l u c u r o n i d a s e a r y l s u l f a t a s e s o l u t i o n showed one ^ C - c o n t a i n i n g compound which had an Rf d i f f e r e n t from t h a t of the o t h e r t h r e e compounds i n d i c a t i n g t h a t f r e s h u r i n e c o n t a i n e d g l u c u r o n i d e and/or s u l f a t e c o n j u g a t e s of one m e t a b o l i t e . The m e t a b o l i t e had an Rf v a l u e the same as a known 5-MBC sample. A f t e r f u r t h e r c l e a n - u p w i t h t i c the i d e n t i t y of the compound was c o n f i r m e d by o b t a i n i n g both i n f r a r e d s p e c t r a (8) and mass s p e c t r a ( 9 ) . Decide f o r What Compounds the A n a l y s i s W i l l be Done I f s e v e r a l m e t a b o l i t e s ar or more t o g e t h e r and a n a l y z a g a i n as an example, methyl 2 - b e n z i m i d a z o l e c a r b a m a t e (MBC), methyl 4-hydroxy-2-benzimidozolecarbamate (4-MBC), and 5-MBC can a l l be analyzed f o r simultaneously (10). Determine i f t h e Amount of M e t a b o l i t e E x c r e t e d C o r r e l a t e s w i t h A d m i n i s t e r e d Dose

the

I t may be t h a t t h e l e v e l of m e t a b o l i t e i s w e l l c o r r e l a t e d w i t h the amount a d m i n i s t e r e d o r a l l y but may not be w e l l c o r r e l a t e d w i t h what is applied dermally. The r e s e a r c h e r would then need t o know the main r o u t e of e n t r y i n t o the body f o r the s i t u a t i o n being s t u d i e d t o d e c i d e i f the m e t a b o l i t e i n u r i n e would p r o v i d e a good measure of dose. Develop an A n a l y t i c a l

Technique

A s u c c e s s f u l t e c h n i q u e w i l l i n c l u d e e s t a b l i s h i n g l i m i t s of d e t e c t i o n which are low enough t o determine i f a harmful dose has been received. Compounds which have a h i g h e r p o t e n t i a l f o r harm would r e q u i r e lower l i m i t s of d e t e c t i o n . A lower l i m i t of d e t e c t i o n f o r 3 , 5 , 6 - t r i c h l o r o - 2 - p y r i d i n o l ( 3 , 5 , 6 - T C P ) , the p r i n c i p a l u r i n a r y metab o l i t e of c h l o r p y r i f o s (11) which has an acute o r a l LD50 i n r a t s of 97 t o 276 mg/kg ( 1 2 ) , would be needed than f o r b i f e n o x a c i d , the u r i n a r y m e t a b o l i t e of 5 - ( 2 , 4 - d i c h l o r o p h e n o x y ) - 2 - n i t r o b e n z o i c acid ( N o r r i s , F. A . , Rhone-Poulenc I n c . , P. 0 . Box 125, Black Horse L a n e , Monmouth J u n c t i o n , NJ 08852, Personal Communication, 1984) which has an acute o r a l LD50 i n r a t s of over 6400 mg/kg ( 1 2 ) . Recovery and v a r i a t i o n i n r e c o v e r y need t o be e s t a b l i s h e d as a f u n c t i o n of c o n c e n t r a t i o n w i t h one c o n c e n t r a t i o n being near the l i m i t of d e t e c tion. A means of c o n f i r m i n g the i d e n t i t y of the a n a l y t e i n a sample should be e s t a b l i s h e d . This c o u l d be done by comparing the r e t e n t i o n time on two columns having d i f f e r e n t s e l e c t i v i t i e s on GC or HPLC or by f i n g e r p r i n t i n g the suspect sample by o b t a i n i n g a mass s p e c t r a , IR s p e c t r a , or UV s p e c t r a and comparing i t t o a s p e c t r a of a standard. Two d i f f e r e n t a n a l y t i c a l i n s t r u m e n t s c o u l d be used such

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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as GC and HPLC or two d i f f e r e n t means of d e t e c t i o n such as GC w i t h e l e c t r o n c a p t u r e d e t e c t i o n and n i t r o g e n - p h o s p h o r u s d e t e c t i o n . An example of a t y p i c a l a n a l y t i c a l procedure f o l l o w s : The f i r s t s t e p would be h y d r o l y s i s of c o n j u g a t e s . This i s t y p i c a l l y , although not n e c e s s a r i l y , a c c o m p l i s h e d w i t h a c i d . The c o n j u g a t e s of benomyl m e t a b o l i t e s have been h y d r o l y z e d by m i x i n g 25 g of u r i n e w i t h 25 ml of 15N H 3 P O 4 and 1 g of sodium b i s u l f i t e and h e a t i n g on a steam bath f o r 1 hr (1(D). The c o n j u g a t e s of 4 - c h l o r o p h e n o l , the m e t a b o l i t e of b a y l e t o n , have been h y d r o l y z e d by r e f l u x i n g o v e r n i g h t w i t h c o n c e n t r a t e d HC1 ( T h o r n t o n , J . S . , Mobay Chemical C o r p . , P. 0 . Box 4913, Hawthorn Road, Kansas C i t y , MO 64120, Personal Communication, 1985). Conjugates of 3 , 5 , 6 - T C P the m e t a b o l i t e of c h l o r p y r i f o s were h y d r o l y z e d w i t h 2 ml of H 2 S O 4 i n 10 ml of u r i n e at 90°C f o r 1 hr (13). The p r e c e d i n g c o n d i t i o n s are at or near r e f l u x , but t h i s may not always be d e s i r a b l e . In the case of b i f e n o x , m i l d a c i d h y d r o l y s i s (equal volumes of u r i n e and 0 . 4 M H C 1 3 7 ° o v e r n i g h t ) i s t h e p r e f e r r e d method of h y d r o l y s i A g r o c h e m i c a l s , L y f i e l d Road Communication, 1 9 8 6 ) . Once h y d r o l y z e d , any a n a l y t e s h o u l d remain s t a b l e under t h e h y d r o l y s i s c o n d i t i o n s . The second s t e p would t y p i c a l l y be a p a r t i t i o n i n g s t e p . This may i n v o l v e s h a k i n g w i t h an a p p r o p r i a t e o r g a n i c s o l v e n t i n a s e p a r a tory funnel. I f the a n a l y t e has a c i d i c or b a s i c f u n c t i o n a l g r o u p s , pH can be a d j u s t e d t o keep the a n a l y t e i n e i t h e r t h e aqueous or o r g a n i c p h a s e . An a l t e r n a t i v e t o p a r t i t i o n i n g w i t h an o r g a n i c s o l v e n t i s t o pass the aqueous sample through a s o l i d phase e x t r a c t i o n c o l u m n . Columns are a v a i l a b l e from s e v e r a l chemical supply companies and v a r y from nonpolar (analogous t o p a r t i t i o n i n g w i t h a nonpolar s o l v e n t ) to p o l a r . A f t e r p a r t i t i o n i n g , the samples may be f u r t h e r c l e a n e d up w i t h l i q u i d - s o l i d chromatography. T h i s may i n v o l v e the use of commercial columns mentioned above or columns prepared i n the l a b u s i n g s i l i c a or f l o r i s i l . S i l i c a or f l o r i s i l f u l l y a c t i v a t e d may r e t a i n t h e compounds too s t r o n g l y . To p a r t i a l l y d e a c t i v a t e them t h e y can be f i r s t a c t i v a t e d t o a r e p r o d u c i b l e s t a r t i n g p o i n t by h e a t i n g f o r 1 hr at 1 0 5 ° f o r s i l i c a and at l e a s t 5 h r at 1 3 0 ° f o r f l o r i s i l ( 1 4 ) . A measured amount of water i s then added, the c o n t a i n e r s s e c u r e l y s e a l e d , and the c o n t e n t s a l l o w e d t o e q u i l i b r a t e o v e r n i g h t . The e l u t i o n of the a n a l y t e i s c h a r a c t e r i z e d u s i n g a s t a n d a r d and v a r i o u s s o l v e n t s or s o l v e n t m i x t u r e s . Two s o l v e n t s o f t e n used f o r e l u t i o n from s i l i c a or f l o r i s i l a r e d i e t h y l e t h e r and c h l o r o f o r m . The d i e t h y l e t h e r s h o u l d be anhydrous. A n a l y t i c a l grade e t h e r c o n t a i n s a p p r o x i m a t e l y 0 . 5 % water which can a f f e c t the chromatography and w i l l not be c o n s i s t e n t from one batch of e t h e r t o the n e x t . C h l o r o f o r m i s s t a b i l i z e d w i t h e i t h e r a nonpolar hydrocarbon (an a l k e n e ) or a p p r o x i m a t e l y 0 . 5 t o 1% e t h a n o l . Chloroform s t a b i l i z e d w i t h the nonpolar hydrocarbon should be used f o r the cleanup chromatography. E t h a n o l , b e i n g much more p o l a r than c h l o r o f o r m , w i l l cause the same problems as water does i n e t h e r . An example of t h i s i s the e l u t i o n of 4 - n i t r o p h e n o l and phenol u s i n g n o n s t a b i l i z e d c h l o r o f o r m and c h l o r o f o r m s t a b i l i z e d w i t h 3/4% e t h a n o l . The r e t e n t i o n time of 4 - n i t r o p h e n o l r e l a t i v e t o phenol was 1 . 6 w i t h s t a b i l i z e d c h l o r o f o r m but 3 . 8 w i t h n o n s t a b i 1 i z e d c h l o r o f o r m ( 1 5 ) . In some cases d e r i v a t i z a t i o n may be n e c e s s a r y . Derivatization may be used t o i n c r e a s e the v o l a t i l i t y of a compound f o r GC a n a l y s i s

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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or t o improve peak s h a p e . E s t e r i f i c a t i o n of a c i d s would be an example of t h i s u s e . D e r i v a t i z a t i o n may a l s o be used t o s e p a r a t e t h e a n a l y t e from i n t e r f e r e n c e s . The methyl e s t e r of 2,4-D e l u t e s w i t h i n t e r f e r i n g compounds i n u r i n e , but the b u t y l e s t e r e l u t e s l a t e r and can be q u a n t i f i e d (2). D e r i v a t i z a t i o n can a l s o c r e a t e or enhance s e n s i t i v i t y f o r a s p e c i f i c type of d e t e c t i o n . In a l a r g e study i t w i l l not be p o s s i b l e t o a n a l y z e a l l samples immediately. F o r t i f i e d samples need t o be i n t e r s p e r s e d w i t h the unknown samples t o monitor s t o r a g e s t a b i l i t y . I d e a l l y , the samples would be f o r t i f i e d w i t h the same chemical compound t h a t i s i n the unknown s a m p l e s . However, the c o n j u g a t e s are u s u a l l y not a v a i l a b l e and f o r t i f i c a t i o n w i l l need t o be done w i t h the nonconjugated a n a lyte. Potential Studies

Problems i n R e l a t i n g Animal U r i n a r y E x c r e t i o n

t o Human

One of the d i f f i c u l t i e i s t h a t i n o r d e r t o r e l a t e what i s found i n u r i n e t o what has e n t e r e d the body, we u s u a l l y need t o r e l y on e x c r e t i o n data from t e s t a n i m a l s . Below a r e concerns t o be aware of when i n t e r p r e t i n g urine analysis r e s u l t s . The E x c r e t i o n Route Can Depend on the Type of Test A n i m a l . As shown b e l o w , 90% of o r a l l y a d m i n i s t e r e d b a y l e t o n was e x c r e t e d as u r i n a r y m e t a b o l i t e s from cows and p i g s as compared t o 30 t o 40% f o r r a t s ( T h o r n t o n , J . S . , Mobay Chemical C o r p . , P. 0 . Box 4913, Hawthorn Road, Kansas C i t y , MO 64120, Personal Communication, 1 9 8 5 ) . The d i s t r i b u t i o n of benomyl m e t a b o l i t e s i n u r i n e and f e c e s was almost r e v e r s e d f o r dogs as opposed t o r a t s (9). Compound

Animal

Bayleton

cow & p i g rat dog rat

Benomyl

Distribution Uri ne 90 30-40 16 86

(%) Feces

83 13

The E x c r e t i o n Route May Depend on Sex. For a group of f i v e male r a t s o r a l l y dosed w i t h l a b e l e d b i f e n o x , 29.1% of the dose was i n u r i n e and 63.4% i n f e c e s . For a group of f i v e female r a t s , 52.6% was i n u r i n e and 46.1% was i n f e c e s ( N o r r i s , F. A . , Rhone-Poulenc, I n c . , P. 0 . Box 125, Black Horse Lane, Monmouth J u n c t i o n , NJ 08852, P e r s o n a l Communication, 1 9 8 4 ) . This may or may not be s i g n i f i c a n t s i n c e gene p o o l s from a h i g h l y i n b r e d s p e c i e s of r a t may be so narrow t h a t a sex d i f f e r e n c e would show whereas human gene p o o l s are so v a r i e d t h a t no d i f f e r e n c e i n metabolism and e x c r e t i o n would e x i s t as a f u n c t i o n of s e x . R e s u l t s Using a T r a c e r May Depend on the L o c a t i o n of the L a b e l . S t u d i e s have been done u s i n g Captan l a b e l e d i n the c a r b o n y l carbon and the t r i c h l o r o m e t h y l carbon ( 1 6 - 1 8 ) . The d i s t r i b u t i o n of

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

15.

LAVY AND MATTICE

201

Biological Monitoring Techniques

N-S-eci: a c t i v i t y as a f u n c t i o n of the l o c a t i o n of the l a b e l f o r o r a l doses g i v e n t o r a t s . Label

U n ne

*CC13 *C=0

52 85

D i s t r i b u t i o n (%) Expired a i r

i s shown below

Feces 16 12

23 0

The f o l l o w i n g h y p o t h e t i c a i l l u s t r a t i o n s of how r e s u l t be m i s l e a d i n g . G i v e n : Compound AB i s m e t a b o l i z e d w i t h m e t a b o l i t e A being e x c r e t e d i n u r i n e and m e t a b o l i t e B being e x c r e t e d i n f e c e s . AB

A(urine) + B(feces)

I f the l a b e l were i n A, the r e s e a r c h e r would r e c o v e r 100% of the a c t i v i t y i n the u r i n e and would d i s c o v e r t h a t A i s a good m e t a b o l i t e t o a n a l y z e f o r t o measure AB. If the l a b e l were i n B, no a c t i v i t y would be r e c o v e r e d i n the u r i n e , and the r e s e a r c h e r may c o n c l u d e t h a t t h e r e i s no m e t a b o l i t e i n u r i n e . A second example would be i f AB were m e t a b o l i z e d t o A and B, both of which are i n u r i n e . However, B i s f u r t h e r m e t a b o l i z e d t o C, D, E, F, G, and H.

AB

all

A + B-

in urine

^H I f the l a o e l were i n A , the r e s e a r c h e r would r e c o v e r a l l of the a c t i v i t y i n one u r i n a r y m e t a b o l i t e and conclude t h a t m e t a b o l i t e i s a good measure f o r AB. If the l a b e l had been i n B i t would be f r a c t i o n a t e d i n t o the components C, D, E, F, G, and H. The r e s e a r c h e r may c o n c l u d e t h a t t h e r e i s no m e t a b o l i t e t h a t r e p r e s e n t s more than 10 to 20% of the dose when, i n f a c t , m e t a b o l i t e A, undetected by t r a c e r t e c h n i q u e s , r e p r e s e n t s 100% of the d o s e . Thus, a l t h o u g h the use of r a d i o l a b e l s i s h i g h l y b e n e f i c i a l f o r i d e n t i f y i n g some p e s t i c i d e m e t a b o l i t e s under c e r t a i n c o n d i t i o n s , t h e i r use may at times be misleading.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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The R e s u l t s May Depend on t h e Avenue of E n t r y I n t o t h e Body. DeBaun i n v e s t i g a t e d t h e d i s t r i b u t i o n of m e t a b o l i t e s of Captan ( 1 6 ) . Three of t h e m e t a b o l i t e s a r e l i s t e d below a l o n g w i t h t h e i r d i s t r i b u t i o n i n r a t u r i n e as a f u n c t i o n of o r a l or i n t e r p e r i t o n e a l e n t r y i n t o t h e body. The r e s u l t s from t h e o r a l study i n d i c a t e t h a t A would be a good a n a l y t e t o use as a measure of C a p t a n . (A)

(B) 0

HO3SCH2SSCH2SO3H

HO3SCH2SSCH2SO3H

dithiobis acid)

d i s u l f i d e monoxide d e r i v a t i v e of A

(methanesulfonic (C)

CH2-CH-CO2H

I

I

S

NH

\/ C

thiazolidine-2-thione4-carboxylic acid Compound

Oral

(A) (B) (C)

54 14 19

% Recovered Interperitoneal 0 0 major

However, i f exposure were not o r a l , (A) might not be a good measure. Some s t u d i e s have shown t h a t , f o r a p p l i c a t o r s at l e a s t , dermal expos u r e i s t h e main s o u r c e of dose ( 1 9 - 2 1 ) . For consumers e a t i n g p r o d u c e , r e s u l t s from f e e d i n g s t u d i e s would be a p p r o p r i a t e . In most c a s e s , t h e r e a r e no data on e x c r e t i o n of compounds from humans as a f u n c t i o n of dermal or o r a l e n t r y , and thus we must e x t r a p o l a t e from animal s t u d i e s as best we c a n . A f t e r t h e a n a l y s i s has been p e r f o r m e d , t h e dose needs t o be determined. Below a r e sample c a l c u l a t i o n s showing how t h i s would be done w i t h b a y l e t o n and i t s m e t a b o l i t e 4 - c h l o r o p h e n o l as an example. Data T o t a l u r i n e e x c r e t e d f o r 4 days - 4738 ml Humans body w t . - 75 kg 4 - c h l o r o p h e n o l c o n c e n t r a t i o n i n u r i n e - 0 . 2 7 ug/ml Recovery of 4 - c h l o r o p h e n o l from u r i n e - 80%

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

15.

LAVY AND MATTICE Amount of urine Molecular

203

Biological Monitoring Techniques

o r a l dose of b a y l e t o n e x c r e t e d as 4 - c h l o r o p h e n o l 35% ( r a t ) 90% ( p i g ) weight - 4 - c h l o r o p h e n o l 128.5 g/mole - b a y l e t o n 2 9 4 . 5 g/mole

in

Calculations mg of 4 - c h l o r o p h e n o l (4738 ml)

excreted

(0.27

ug/ml)

(10-3 mg/ug)/0.80

= 1.60 mg

1.60 mg of 4 - c h l o r o p h e n o l r e p r e s e n t s 1.60 mg (294.5) = 3.67 mg (T20")

bayleton

absorbed amoun = 3 . 9 2 mg/0.9 = 4 . 3 6 mg ( p i g ) dose = 10.49 mg/75 kg = 0.140 mg/kg

(rat)

= 4 . 3 6 mg/75 kg = 0 . 0 5 8 mg/kg ( p i g ) A n a l y s i s of u r i n e can p r o v i d e meaningful i n f o r m a t i o n r e g a r d i n g t h e dose r e c e i v e d by i n d i v i d u a l s f o r c e r t a i n compounds. The goal i s to i d e n t i f y a d e t e c t a b l e m e t a b o l i t e t h a t can be r e l a t e d back t o dose of the parent compound. If no p r e v i o u s work has been done w i t h the compound i n q u e s t i o n , i n i t i a l work w i l l p r o b a b l y r e q u i r e use of a r a d i o l a b e l e d compound. Care needs to be used i n e v a l u a t i n g the r e s u l t s of t r a c e r s t u d i e s s i n c e the observed r e s u l t s may depend on t h e l o c a t i o n of the l a b e l , on the s p e c i e s of t e s t a n i m a l , or on the method of d o s i n g ( o r a l v s . d e r m a l ) . M i n i m i z i n g Absorbed Dose Some s p e c i f i c g u i d e l i n e s f o r l i m i t i n g the absorbed dose of humans who handle or come i n t o c o n t a c t w i t h p e s t i c i d e s b e i n g a p p l i e d follow: 1. Read and obey the l a b e l . 2. Be aware t h a t a user can d e c r e a s e h i s e x p o s u r e . 3. Change c l o t h i n g ( s h i r t , t r o u s e r s , b o o t s , and g l o v e s ) a f t e r a p p l i c a t i o n and launder contaminated c l o t h i n g . 4. Avoid e a t i n g , or use of tobacco when hands are c o n t a m i n a t e d . 5. Wash hands immediately a f t e r e x p o s u r e . 6. Shower as soon as p o s s i b l e a f t e r p o t e n t i a l e x p o s u r e . F u t u r e Needs Some of the major o b s t a c l e s r e m a i n i n g b e f o r e we have a f u l l u n d e r s t a n d i n g of p e s t i c i d e s and t h e i r f a t e i n humans a r e : a) What f a c t o r s r e g u l a t e the amount and r a t e of p e s t i c i d e t h a t p e n e t r a t e s the s k i n ? b) What are the e x c r e t i o n r o u t e s and r a t e s f o r p e s t i c i d e s or t h e i r m e t a b o l i t e s ? c) What are the " s a f e " l e v e l s t h a t a human can absorb w i t h o u t the o c c u r r e n c e of a h e a l t h e f f e c t - now or at some time i n the f u t u r e ?

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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B e f o r e r e s e a r c h e r s can be expected t o make new, s i g n i f i c a n t , and i n t e l l i g e n t s c i e n t i f i c d e c i s i o n s r e g a r d i n g t h e p h a r m a c o k i n e t i c s of p e s t i c i d e s i n humans, more s t u d i e s i n v o l v i n g human s u b j e c t s a r e necessary. Our c u r r e n t knowledge of t h e t o x i c o l o g i c a l p r o p e r t i e s of p e s t i c i d e s , coupled w i t h small animal f e e d i n g or d o s i n g s t u d i e s c o v e r i n g a wide c o n c e n t r a t i o n r a n g e , should a l l o w us t o choose a s a f e human range thousands of f o l d below any a n t i c i p a t e d No Observed E f f e c t Level (NOEL). Informed human v o l u n t e e r s c o u l d then be dosed w i t h a p e s t i c i d e c o n c e n t r a t i o n f a r lower than t h e l e v e l s commonly r e c e i v e d by f i e l d workers m i x i n g or a p p l y i n g t h e commercial p r o d u c t . I n f o r m a t i o n gained from these c o n t r o l l e d absorbed dose s t u d i e s c o u l d be used i n e f f o r t s t o improve p r o t e c t i v e c l o t h i n g , g l o v e s , masks, hand w a s h i n g , and o t h e r p r o c e d u r e s , f o r f i e l d workers who a r e exposed t o much h i g h e r l e v e l s . Legal concerns w i l l probably prevent i n i t i a t i o n of t h i s proposed p l a n , but i t i s t h e l o g i c a l p l a n t o adopt. I f i t i s wrong t o g i v e a v o l u n t e e r a dose 100-1000 times l e s s than t h e amount w j o b s , then i t i s wrong t which causes t h e worker t o be r e c e i v i n g t h e h i g h e r d o s e . It i s h y p o c r i t i c a l t o say t h a t i t i s dangerous and wrong t o g i v e t h e v o l u n t e e r t h e low d o s e , but i t i s not dangerous or wrong t o a l l o w t h e worker t o r e c e i v e a dose 100-1000 times h i g h e r . The development of proper experiments u s i n g l a b o r a t o r y animals f o r e s t a b l i s h i n g an a p p r o p r i a t e NOEL i s e s s e n t i a l . Both s h o r t - and l o n g - t e r m animal t r i a l s t u d i e s a r e n e c e s s a r y b e f o r e we w i l l be i n a p o s i t i o n t o make v a l i d s c i e n t i f i c d e c i s i o n s w i t h r e s p e c t t o t h e s a f e use of p e s t i c i d e s . S i n c e a n a l y t i c a l methodologies now a l l o w us t o a n a l y z e down t o t h e ppb and ppt l e v e l , we must base our t o x i c o l o g i c a l d e c i s i o n s r e g a r d i n g r i s k assessments on a p p r o p r i a t e NOEL c o n c e n t r a t i o n s r a t h e r than t h e mere presence or absence of t h e pesticide in question. I t appears t h a t not a l l f a c e t s of s c i e n c e develop at t h e same r a t e . U n t i l s o c i e t y i s prepared t o accept u s i n g humans as t e s t s u b j e c t s , we w i l l be f o r c e d t o accept t h e l e s s a c c u r a t e answers found from animal s t u d i e s .

Literature Cited 1. Lavy, T. L. Project Completion Report to National Forest Products Association, August 30 to October, 1978. 2. Lavy, T. L. Project Completion Report to National Forest Products Association, 1980. 3. Sauerhoff, M. W.; Braun, W.; Glau, G.; Gehring, P. Toxicology 1977, 8:3-11. 4. Durham, W. F.; Wolfe, H. Bulletin of the World Health 5. Lavy, T. L.; Norris, L.; Mattice, J. Project Completion Report to National Forest Products Association, 1984. 6. Nolan, R. J.; Freshour, N.; Kastl; P.; Saunders, J. Toxicol. Appl. Pharmacol. 1984, 76:264-69. 7. Feldman, R. J.; Maibach, H. J. Toxicol. Appl. Pharmacol. 1974, 28:126-32. 8. Gardiner, J. A.; Brantley, R.; Sherman, H. J. Agric. Food Chem. 1968, 16(6):1050-52. 9. Gardiner, J. A.; Kirkland, K.; Klopping, H.; Sherman, H. J. Agric. Food Chem. 1974, 22(3):419-27.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

15. LAVY AND MATTICE

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

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205

Kirkland, J. J. J. Agric. Food Chem. 1973, 21(2):171-77. Smith, G. N.; Watson, B.; Fischer, F. J. Agric. Food Chem. 1967, 15:132-38. Farm Chemicals Handbook, Meister Publishing Co., Willoughby, OH, 1981. Nolan, R. J.; Rick, D.; Fresham, N.; Saunders, J. Toxicol. Appl. Pharmacol. 1984, 73:8-15. Official Methods of Analysis of the Association of Official Analytical Chemists; William Horowitz, Ed.; 1980, 26.027, 29.002, 34.011, 13th ed. Waters Associates Liquid Chromatography School. Captan Position Document 2/3, Office of Pesticide Programs, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington,D.C.,June 1985. Hoffman, L. J.; DeBaun, J.; Kuan, J.; Mann, J. Western Research Center, Stauffer Chemical Co. 1973 DeBaun, J. R.; Miaullis J. Xenobiotica 19 Libich, S.; To, J.; Frank, R.; Sirons, G. Am. Ind. Hyg. Assoc. J. 1984, 45:56-62. Lavy, T. L.; Shepard, J.; Mattice, J. J. Agric. Food Chem. 1980, 28:626-30. Lavy, T. L.; Walstad, J.; Flynn, R.; Mattice, J. J. Agric. Food Chem. 1982, 30:375-81. Picloram: The effects of its use as a herbicide on environmen­ tal quality. National Research Council of Canada, Ottawa, Gehring, P. J.; Kramer, C.; Schwetz, B.; Rose, J.; Rowe, V. Toxicol. Appl. Pharmacol. 1973, 26:352-61.

RECEIVED December 16, 1987

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter

16

New Approaches to Analysis of Organophosphate Metabolites in the Urine of Field Workers C. P. Weisskopf and J. N. Seiber Department of Environmental Toxicology, University of California, Davis, CA 95616 A procedure is describe and thiophosphate metabolite large bore capillary gas chromatography (GC) columns, and the methylating reagent trimethylanilinium hydroxide. Although field worker monitoring for organophosphate pesticide exposure by measurement of urinary metabolites can have several advantages, alternate methods are frequently favored, in part due to difficulties in the analysis of these compounds. The described method overcomes many of the obstacles of conventional approaches to urinary metabolite analysis. Metabolite extraction is rapid, requiring at most 15 minutes per sample. Derivatiza­ tion takes place in the GC injector block, and capillary columns lead to improved separations for all compounds. Five urinary metabolites are detectable at concentrations of 2 to 10 ng/ml (ppb) urine. There has been a long-standing interest in the estimation of human exposure to organophosphates, particularly since these compounds are widely used and are the most frequent cause of pesticide related occupational illnesses. Upon entering the body, most organophosphorus pesticides may be metabolized to yield one or more of the six common dialkyl phosphates shown in Table I. Quantities of metabolites excreted in the urine have been found to correspond to the pesticide dose (1-3). Measurement of these urinary metabolites can thus provide a basis for comparison of exposure in a study population. Measurement of intact pesticides or metabolites in biological samples may give a better estimation of the amount of material actually entering the body than external measurement methods. Analysis for principle organophosphate pesticide alkyl or aryl leaving groups can yield information on the identity of the pesticide to which the subject was exposed, since few pesticides share the same leaving group. However, this requires the development of an analytical method specific for each compound, and is thus not amenable to a multi-residue approach. The dialkyl phosphates in Table I represent possible metabolites of approximately 75% of the organophosphate pesticides in the EPA's Pesticides and Industrial Chemicals Repository. 0097-6156/89/0382-0206$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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207

Table I. Common dialkyl phosphates

DMP DEP DMTP DETP DMDTP DEDTP

0,0-Dimethyl phosphate 0,0-Diethyl phosphate 0,0-Dimethyl phosphorothionate 0,0-Diethyl phosphorothionate 0,0-Dimethyl phosphorodithioate 0,0-Diethyl phosphorodithioate

The primary disadvantage to the measurement of urinary dialkyl phosphates lies i n the difficulty of the analysis. These compounds are highly water soluble, ionized i n urine, and must be derivatized to be sufficiently volatile for gas chromatographic (GC) analysis. Although there are many published procedures for extraction and analysis of urinar dialkyl phosphate (3-8) th method remai tedious and cumbersome. Th metabolites, derivatization wit column GC. Metabolite extraction is often incomplete or inconsistent (4), and it has been difficult to get adequate chromatographic resolution of all metabolites both from each other and from other constituents of urine (5-7). Diazoalkanes have frequently been used for alkylation of dialkyl phosphates and thiophosphates prior to G C analysis. Diazoalkane reagents such as diazoethane and diazopentane give an isomer mixture of thionate and thiolate esters, formed i n irregular proportions, when the metabolites D M T P or D E T P are derivatized (8). Analysis of both isomers may be required, resulting i n increased analysis time and poorer detection limits. When coupled with the hazards of the preparation and use of diazoalkanes, this makes the use of an alternative derivatizing reagent desirable (4). Trimethylanilinium hydroxide ( T M A H ) is a methylating reagent frequently used in clinical tests for barbiturates. A t elevated temperatures, provided i n this case by the G C injector block, T M A H is able to methylate dialkyl phosphates. Dale et al. (9) first reported the use of T M A H for the hydrolysis of intact organophosphates and methylation of hydrolysis products. Miles and Dale (10) followed with a study that included the use of T M A H for the injector block methylation of DMDTP. Use of T M A H for the methylation of D M T P in the extracts of urine of exposed workers was reported by Moody et al. (U_). The present study was designed to overcome some of the complexities of previous methods by developing a simpler and faster analytical procedure through the use of disposable extraction cartridges, injector block derivatization, and quantification using large bore capillary G C columns. Experimental Section Solvents and Reagents. Trimethylanilinium hydroxide ( T M A H ) was obtained from Pierce Chemical Co, Rockford, IL, under the trade name MethElute, a 0.2 M solution of T M A H i n methanol. Handeling precautions for this mixture are the same as those for methanol. Solvents were pesticide residue grade from J. T. Baker, Phillipsburg, NJ. Salts and acids were analytical reagent grade from Mallinckrodt, Inc., Paris, K Y . Extraction Apparatus. Disposable cyclohexyl extraction cartridges with a 6 ml reservoir containing 1 g sorbent were used (Analytichem International, Harbor City, CA). A ten-place vacuum manifold was used for cartridge elution (J. T. Baker Co, Phillipsburg, NJ). Sufficient vacuum (approximately 100 mm Hg) was

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applied to provide for a flow rate of 5 ml/min of eluting solution through the cartridges. Preparation of Standard Solutions. Analytical grade standards of the six metabolites in Table I were obtained from the EPA's Pesticides and Industrial Chemicals Repository. Stock 1 mg/ml solutions of the individual dialkyl phosphates in methanol were prepared and stored at 4°C in the dark. Analytical mixed standards of all six metabolites in acetone (0.015 to 1.5 ng//il) were prepared daily in volumetric flasks, and brought to final volume after the addition of T M A H immediately prior to G C injection. T M A H was added to the standard solutions in the proportion of 50 /il TMAH/ml solution. Chromatographic Conditions. Apparatus: The instrument used was a Tracor model MT 220 equipped with a flame photometric detector (FPD) operated in the phosphorus mode. The column was a 30 m x 0.5 mm ID large bore DB-1701 capillary column, with an 86% dimethyl and 14% cyanopropylphenyl polysiloxane bonded stationary phase an CA). Gas flows were: heliu 40 ml/min; air 90 ml/min; and hydrogen 60 ml/min. Operating temperatures were: injector, 300°C.; column, 110°C.; and FPD and FPD transfer line, 170°C. Quantification of the methylated metabolites was performed by external standard calculations based on peak areas using a Hewlett Packard 3390 A electronic integrator. 1-3 /il aliquots of the mixed standard solutions or urine extracts were injected. Standard curves were prepared from duplicate injections of mixed standards at a minimum of three concentrations. Determination of Dialkyl Phosphates in Urine. The preparation of samples for analysis is outlined in Figure 1. An aliquot (4 ml) of urine was pipetted into a 15 ml test tube. Granular ammonium sulfate (ca 1.8 g) and 4 drops 70% acetic acid were added, and the mixture vortexed for 30 sec. The cyclohexyl extraction cartridges were prewashed with one column volume (ca 6 ml) each of acetone, water, and ammonium sulfate-saturated water. The urine sample was then passed through the cartridge. The cartridges were washed with 2 ml of a solution of 20% acetone in hexane, and the wash discarded. The vacuum was increased to about 600 mm Hg, and the cartridges were aspirated for 5 minutes. The cartridges were then placed into 15 ml graduated centrifuge tubes and eluted with acetone to a volume of 1.4 ml by gravity flow or the application of positive pressure to the extraction cartridge using a pipette bulb. Immediately prior to GC injection ca 0.4 g powdered anhydrous sodium sulfate and 100 /xl T M A H was added to the acetone eluate followed by brief vortexing. Results and Discussion The procedure reported here was capable of analyzing DEP, DMTP, DETP, DMDTP, and DEDTP. It is unsuitable for DMP due to interferences caused by the presence of inorganic phosphate. The effect of various salts on the recovery of dialkyl phosphate metabolites was tested in developing this procedure. Salts were added to urine at amounts slightly in excess of that needed to produce a saturated solution, allowing for reproducible results without expending much time in weighing. Salt addition usually improved recovery of metabolites from fortified urine samples, with sulfate salts giving better results than either acetate or chloride salts (Table II). A saturated solution of ammonium sulfate was found to be the best of those tested.

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16.

WEISSKOPF AND SEIBER

Organophosphate Metabolites in Urine

4 ml Urine add:

CH Cartridge ca 1. 4 drops 70% HOAc

water salt saturated water

Vortex 30 seconds Add Urine to Washed Cartridge wash: 2 ml 20% acetone in hexane aspirate: 5 minutes Column + Retained Metabolites acetone elution to 1.4 ml Acetone Eluate + Metabolites add:

ca 0.4 g Na^O, 100 Ml TMAH

Gas Chromatography Figure 1. Flow diagram for the analysis of dialkyl phosphates in urine.

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Table II. Effect of salt addition on metabolite recovery, normalized for ammonium sulfate

grams added

salt

none sodium sulfate sodium chloride ammonium sulfate ammonium acetate

-

1.1 1.4 1.7 4.0

DEP

0.1 0.9 0.4 1.0 0.0

normalized recovery DMTP DMDTP DETP DEDTP

0.1 1.0 0.7 1.0 0.0

0.1 0.8 0.7 1.0 0.2

0.2 1.0 0.8 1.0 0.4

0.5 0.9 1.0 1.0 0.5

The p H of the urine was also found to have a large effect on recoveries (Table III). Recoveries droppe of 5 of the unmodified urine adjust the p H to approximately 4, and declined when hydrochloric acid was used to reduce the p H further. Acetic acid was used routinely as the p H modifier. Table III. Effect of p H modification on metabolite recovery, normalized for acetic acid

modifier

sodium hydroxide none acetic acid hydrochloric acid hydrochloric acid

pH

DEP

7 5 4 3.5 2.5

0.5 0.9 1.0 0.4 0.2

normalized recovery DMTP DMDTP DETP DEDTP

0.5 0.8 1.0 0.8 0.6

0.6 0.9 1.0 0.7 0.5

0.6 0.9 1.0 0.8 0.4

0.6 0.9 1.0 0.8 0.4

A hexane/acetone wash followed by aspiration was found to eliminate most of the water held by the cartridges, as well as removing some interfering compounds that reduced the efficiency of metabolite derivatization. Subsequent elution with 1.4 ml acetone removed more than 9 5 % of the metabolites retained on the cartridges. Samples could then be analyzed directly, eliminating the possibility of evaporative loss of the compounds or expenditure of time in reducing solvent volumes, both drawbacks of prior methods which used solvent extraction rather than extraction with solid phase cartridges. The efficiency of T M A H as a methylation reagent was tested using a variety of solvents, sample extracts, and injector temperatures. Derivatization yields were estimated by comparing molar phosphorus response of the methylated metabolites on the flame photometric detector with the response of a trimethyl phosphate standard. Derivatization yields were highest when the solvent was acetone and the injector block of the G C was above 250°C. The methylation yield of D M P could not be determined due to interference from inorganic phosphate. Both compounds are methylated to produce trimethyl phosphate (Figure 2), making T M A H unsuitable for analysis of D M P without prior removal of inorganic phosphate. Approximately 6 0 % of the D E P and 9 5 % of the four other metabolites in acetone are methylated. When the solvent was an acetone extract

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

16.

WEISSKOPF AND SEIBER

I

0

1 2

1 4

1 6

Organophosphate Metabolites in Urine

1 1 1 r 8 10 12 14

i

0

1 2

1 4

1 6

1 1 1 r8 10 12 14

TIME (minutes)

Figure 2. Gas chromatogram of dialkyl phosphates using FPD detection, a) reagent blank; b) dialkyl phosphate standards, 0.015 ng each except DEP, 0.03 ng; c) human urine with no detectable metabolites; d) human urine fortified at 10 ppb each metabolite.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

of urine, DEP derivatization was reduced to 40%; the other compounds were unaffected. The FPD response for injections ranging from 0.015 to 3 ng was linear with r > 0.999 for all five dialkyl phosphates tested. The minimum detectable level per injection of DEP was 30 pg, and 15 pg for DMTP, DMDTP, DETP, and DEDTP. The recovery data in Table IV was obtained by analysis of urine samples fortified at several levels. The same blank urine sample was used for all fortifications. The low recovery of DEP was the result of both incomplete retention of the metabolite on the extraction cartridges and reduced methylation efficiency in the urine matrix. Minimum levels of detection of the metabolites in urine was 10 ppb for DEP, and 2 ppb for the remaining four metabolites. 2

Table IV. Recovery of dialkyl phosphates from fortified urine samples

ppb

2 10 40 100 200

% recovery DEP

a

n.d. 8±1 34 ± 7 31 ± 1 27 ± 4

78 90 106 93 84

± ± ± ± ±

23 5 5 5 6

83 83 113 104 96

± ± ± ± ±

98 131 109 96 103

8 4 6 7 7

± ± ± ± ±

9 14 4 9 3

48 79 92 87 87

± ± ± ± ±

7 4 4 6 5

a. n.d. = none detected

Three urine samples of different concentrations were fortified at levels of 20 ppb to test the variability of metabolite recovery from one urine sample to another in our method (Table V). Creatinine content was used as the measure of urine concentration, with 0.4 mg creatinine per ml urine indicating a sample that is quite dilute, and 3.4 mg/ml one that is quite concentrated. These values encompass the range of urine concentrations that might be encountered in a typical study. Average metabolite recovery was similar to that shown in Table IV, with little difference in standard deviation, indicating no effect of urine concentration. Table V. Variation of recovery with urine concentration

creatinine (mg/ml urine)

DEP

0.4 1.1 3.4 avg (%) std. dev. (%)

23 43 25 30 11

% recovery (fortified at 20 ppb) DMTP DMDTP DETP

102 113 104 106 6

95 86 75 85 10

DEDTP

113 97 106 105 8

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

91 85 86 87 3

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The performance of this method on authentic urine samples was examined by analyzing a group of 143 urine samples that were made available from a larger study on farm worker exposure. Concentrations of DMTP, DMDTP, DETP, and DEDTP were determined. Average metabolite concentrations for all compounds were less than 35 ppb (Table VI). Only 16% of the samples tested had no detectable metabolites. This large set of samples did not have a detrimental effect on column or instrument performance. Retention times over the course of the study varied by no more than 3%, and peak areas for standard injections by less than 15%. There was no sign of a general decrease in column, instrument, or detector performance, a problem in some previous procedures (11). Table VI. Results of the analysis of 143 human urine samples

avg (ppb) range (ppb) % positive

DMTP

DMDTP

DETP

DEDTP

2 - 27 55%

2 - 14 56%

2 - 31 41%

2-4 6%

Recoveries and limits of detection using the procedure presented here compares favorably with existing methods for DMTP, DETP, DMDTP, and DEDTP. One commonly used method, that of Shafik et al. (6), can detect the 6 common metabolites at levels above 5 ppb. Recoveries from urine fortified at 100 ppb were greater than 95% for all 6 metabolites with this technique. However, several researchers (1.4.5.11) reported poor recoveries and excessive variation in results, indicating that the method may be dependent on the familiarity of the operator with the procedure. Another potential drawback is that only 25-30 samples can be analyzed per week, although this was a significant improvement over existing procedures at the time when this method was developed. The method of Lores and Bradway (8) detects DMP, DEP, DMTP, and DETP at concentrations in urine greater than 10 ppb, and 8-10 samples can be analyzed per day. Recoveries ranged from 40 to 100% when urine was fortified at 500 ppb. Variation in recovery of metabolites from one urine sample to the next averaged 15%. All 6 common dialkyl phosphates may be detected by the method of Reed and Watts (5). The average minimum level of detection in urine is 60 ppb, with metabolite recoveries greater than 90% at fortification levels of 800 ppb. The use of the method described here can result in more rapid analysis of samples compared to existing methods. The time necessary to prepare a single sample for G C analysis is about 15 minutes. Ten samples, the number of cartridges that can be held by our vacuum manifold, can be prepared in forty minutes. Using duplicate standard and sample injections and a three point standard curve, 12 samples can be chromatographed per day. This method is ideally suited to the use of a G C autoinjector, since the chromatographic step is the most time consuming. A single analyst using an autoinjector could easily complete 30-40 samples per day. Existing methods for the analysis of dialkyl phosphates have generally proven inadequate due to long sample preparation and analysis time, poor or variable metabolite recoveries, or lack of sensitivity. Our method is free of these defects in the analysis of DMTP, DETP, DMDTP, and DEDTP. Although DEP recoveries were relatively low, they were consistent enough to yield valid results at urine concentrations over 10 ppb. This method is unsuitable for the analysis of DMP.

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We are seeking to extend this method to the analysis of DMP, and to improve DEP analysis by increasing both the retention of DEP on the extraction cartridges and the derivatization yield. An injector block propylating reagent that produces a unique derivative for inorganic phosphate and each metabolite is being tested and appears promising. Dimethyl propyl phosphate produced upon derivatization of DMP is easily distinguished from the tripropyl derivative of inorganic phosphate. Continuing work on salt addition, pH effects, and solvents for cartridge washing and sample elution should result in better retention of metabolites on the extraction cartridges and cleaner samples. Both effects can improve the efficiency of the method. In summary, we have developed a method for the analysis of urinary dialkyl phosphates that is both rapid and simple. It works well for the four common sulfur containing dialkyl phosphates, and adequately for DEP. DMP cannot be analyzed by this method in its present form. Preliminary work indicates that remaining problems can be overcome without a significant increase in extraction or analysis time.

References 1. Morgan, D. P.; Hetzler, H. L.; Slach, E. F.; Lin, L. I. Arch. Environ. Contam. Toxicol. 1977, 6, 159-173. 2. Franklin, C. A.; Fenske, R. A.; Greenhalgh, R.; Mathieu, L.; Denley, H. V.; Leffingwell, J. T.; Spear, R. C. J. Toxicol. Environ. Health 1981, 7, 715731. 3. Bradway, D. E.; Shafik, T. M.; Lores, E. M. J. Agric. Food Chem. 1977, 25, 1353-1358. 4. Bradway, D. E.; Moseman, R.; May, R. Bull. Environ. Contam. Toxicol. 1981, 26, 520-523. 5. Reid, S. J.; Watts, R. R. J. Anal. Toxicol. 1981, 5, 126-132. 6. Shafik, T.; Bradway, D. E.; Enos, H. F.; Yobs, A. R. J. Agric. Food Chem. 1973, 21, 625-629. 7. Blair, D.; Roderick, H. R. J. Agric. Food Chem. 1976, 24, 1221-1223. 8. Lores, E. M.; Bradway, D. E. J. Agric. Food Chem. 1977, 25, 75-79. 9. Dale, W. E.; Miles, J. W.; Churchill, F. C. J. AOAC 1976, 59, 1088-1093. 10. Miles, J. W.; Dale, W. E. J Agric. Food Chem. 1978, 26, 480-482. 11. Moody, R. P.; Franklin, C. A.; Riedel, D.; Muir, N. I.; Greenhalgh, R.; Hladka, A. J Agric. Food Chem. 1985, 33, 464-467. RECEIVED April 19, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter

17

Pilot Study for Biological Monitoring of 1,3-Dichloropropene 1,2

3

3

2

John D. Osterloh , Rhoda Wang , Linda O'Connell , Peyton Jacob III , and Keith T. Maddy 3

1

Departments of Laboratory Medicine and Medicine, University of California, San Francisco, CA 94143 Division of Clinical Pharmacology, University of California, San Francisco, CA 94143 Worker Health and Safety Branch, State of California Department of Food and

2

3

A pilot study wa performe tion patterns of the metabolite of dichloropropene (DCP), N-acetyl-S-cis-3-chloroprop-2enyl)-cysteine (3CNAC). Urine from 3 applica­ tors was collected every 2-16 hours for 3 days, while personnel air concentrations of DCP were determined by EC-GC. 3CNAC in urine was extrac­ ted, derivatized and measured on capillary gas chromatograph-mass spectrometry by monitoring the two abundant ions 117 and 176 m/e. Sensit­ ivity was 0.1 ug/mL of urine. Air concentra­ tions of DCP ranged from 0-10.3 ppm. Urine concentrations of 3CNAC ranged 0.9-17.1 ug/mL. Excretion of peak concentrations of metabolite followed peak exposure by variable periods of time (0-16 hours). Cumulative daily excretion of 3CNAC increased in proportion to cumulative daily air exposure. 1,3-Dicrdoropropene (DCP) is an agricultural fumigant applied by pressure injection into soil for pre-implantation eradication of nematodes. Commercial preparations contain 98% of approximately equal parts of cis and trans DCP. Because of its vapor pressure (28 mmHg), the primary route of exposure to human workers is suspected to be pulmonary. Low ppm air concentrations have been documented during loading, mixing and applications processes (1,2). Both isomers are metabolized to their respective mercapturic acids in rats (3-5). We have demonstrated previously that monitoring of human applicators is possible by measuring the metabolite N-acetyl-S-(cis-3chloroprcp-2-enyl)-cysteine (3CNAC) (2). Protection and biological monitoring of workers is of concern since DCP has produced forestomach squamous and urinary bladder transitional cell cancers following chronic oral gavage studies in rats and mice (6). Subchronic inhalational studies in rats and mice at c

0097-6156/89/0382-0215$06.00/0 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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90 or 150 ppm have demonstrated hyperplasia respiratory epithelium, degeneration of nasal mucosa and hyperplasia of transitional c e l l epithelium in the urinary bladder (W.T. Stott, Dow Chemical Co., personal communication). Chronic inhalation studies are not published, but increased respiratory neoplasia are suggested (W.T. Stott, personal communication). DCP is an indirect mutagen (7-11) which binds (8,9) and depletes sulfhydryl groups in the liver and kidney (12). Earlier studies indicated that the rat kidney may show toxic effects in the renal tubular epithelium following inhaled concentrations as low as 3 ppm after chronic inhalation (13). This effect has not been confirmed in two subsequent subchronic animal studies following inhalation of concentrations as high as 150 ppm (14,15). However, a kinetic study by one of these groups has shown that a 3 hour exposure to 90 ppm reduces kidney sulfhydryl content 31% (12). While differences in studies may be attribute or species differences chemistry assays used in chronic studies may not have the sensitivity to demonstrate such subtle renal effects. Safe air concentrations of DCP which protect workers are not known. Estimates of possible carcinogenic risk based on possible air concentrations and animal studies do not yield real or specific thresholds. Twenty-four hour cumulative urinary excretion of 3CNAC increases in rough proportion to increasing air concentrations " time products (2). Single random determinations of urinary 3CNAC only indicate that some systemic exposure has occurred, but are not indicative of known exposure thresholds or toxic effects. It i s also not known how quickly excretion of 3CNAC follows pulmonary exposure. Knowledge of timing and toxic effects would aid in determining appropriate urine collection procedures. As a pilot to a larger study, we evaluated three workers by measuring personal air concentrations of DCP urinary excretion of 3CNAC and of Nacetyl-glucoseamidase (NAG). The latter is an enzyme released from injured tubular epithelium in the kidney. It has been studied as a more sensitive indicator of renal injury following a wide variety of nephrotoxics (16-18). Analytical concerns in measuring 3CNAC w i l l also be discussed. Materials and Methods Protocol. Three male applicators of DCP were studied for 2-3 days. Field collections were done under the auspices of the Worker Health and Safety Branch of California Department of Food and Agriculture. Application rates of DCP were 12 gallons per acre. The s o i l was sandy and dry with air temperatures exceeding 100°F for most of the day. The work periods of potential exposure to DCP varied from 3.5-10 hours/day. Workers wore light cotton clothes during application of DCP. During loading, chemical resistant gloves, apron, boots, and NIOSH-approved respirators were worn. Dermal exposure from

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Monitoring of 1,3-Dichloropropene

small spills during these closed loading operations may have occurred, but none were recorded. The extent of the contribution of dermal exposure to urinary 3CNAC excretion i s unknown. Applicators were chosen only on a f i r s t available basis in the locale where DCP was being applied. No attempt was made to exclude a subject with prior exposure to DCP in this pilot study. At the field site, prior to beginning work, the applicator was attached with a personal a i r sampling device and instructed as to collection of urinary specimens. Operator breathing zone a i r samples were drawn by personal a i r sampling pumps (MSA Fixt Flo) through charcoal adsorbent tubes (SKC #226-09, 400/200 mg) via tygon tubing. Tube openings were position down across the chest and did not interfere with work habits. Pumps were calibrated to 1 l/min using a Kurz 540S mass flow calibrator. Sorben hours or when there wa (loading vs. repair) intervals. These tubes were capped and stored on dry ice, then refrigerated (-20°) until analysis. Procedures, flow rates and breakthrough volumes are given elsewhere (19,20). Urines were collected by instructing the applicators to urinate into a separate 500 mL opaque polyethylene container for each voiding. Each container was frozen on dry ice and then -70°C until analysis. Urine collection occurred only when the applicator would empty his bladder. Thus, collection intervals were variable and spanned 2-16 hours. Urine collection periods did not coincide with intervals for air collections. Analysis of air concentrations of DCP. Amounts of DCP in sorbent tubes were determined in accord with established procedures (1,19,20) that includes elution of tubes with carbon disulfide mixed with an internal standard and direct injection onto electron capture gas chromatograph. Sensitivity and precision of these determinations is 0.1 ug collection (column). Urinary NAG Determinations. Urinary activities of NAG were determined by the manual method of Piagen et al (1978) with modifications incorporated by Al-Bander et a l (1986). This i s a fluorometric method which measures the hydrolysis of 4methylumelliferyl-Beta-D-glucosaminide. Calculated activity in nanomoles/hr/mL of urine is expressed in terms of milligrams of creatinine excreted (nanomoles/hr/mg), since this corrects for variability due to urine flow rates (23). Creatinine concentrations were determined by the Jaffe reaction (24). Intraassay coefficients of variation were <5% at 50 nmol/hour/mg of creatinine. The normal reference interval i s <100 nmol/hour/mg.

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Urinary 3CNAC Determinations. Synthesis of the major metabolite, N-acetyl-S-cis-3-chloroprop-2-enyl)-cysteine (3CNAC), and the isomeric internal standard, N-acetyl-S-2chlorcprcp-2-enyl) -cysteine (2CNAC), from N-acetyl cysteine and the appropriate dichloropropene has been described elsewhere (2,4). Purification procedures included multiple recrystallizations with verification of the product and i t s purity by thin layer chromatography, flame ionization gas chromatography, electron capture gas chromatography (EC-GC), melting points, infrared spectra, nuclear magnetic resonance spectra and GC-MS. Analytic grade solvents were purchased from either Mallinkrodt, Burdick and Jackson or Aldrich. Stock standards of the 3CNAC and 2CNAC were prepared in methanol or acetone at concentrations of 1.0 mg/ml for use in chromatography and spiking blank urine solutions as standards. Prior to developing quantitation, other ga employed, including packed column chromatography with flame ionization, nitrogen phosphorus and mass selective detectors; and also direct injection into various capillary columns with electron capture detection. Most of these other chromatographic techniques were unsuitable for analysis of urines containing 3CNAC due to endogenous interferences or non­ symmetrical peak shapes. On-column injection into wide bore (0.5 mm) columns of methyl silicone or methyl phenyl silicone were generally unsuitable with EC-GC instrumentation due to poor peak symmetry and sensitivity. However, with split injection into narrow bore methyl silicone columns, acceptable sensitivity (0.5 ug/mL) has been achieved, but endogenous unknown urinary constituents limit the u t i l i t y of this method. Previously, extraction of 3CNAC from acidified urine relied on a diethyl ether extraction (2). To improve cleanup and yield, different extraction techniques were applied. Urine or water (5.0 mL) containing 100 ug/mL of 3CNAC were acidified to pH 1 with addition of 0.6 mL 6N HC1. Ten mL of each solvent was added, gently shaken for 5 minutes and centrifuged at 2000 g for 10 minutes. Eight mL of solvent phase was evaporated at 45°C under nitrogen. Following diazomethane derivatization and quantitation on EC-GC, peak areas were compared to 3CNAC standards. Blank unspiked urines were carried through to estimate interferences at the retention time of 3CNAC. Each solvent gave the following results (% recovery, % urinary interference): dichloromethane (16%, 15%), diethyl ether (10%, 100%), chloroform (30%, unknown), t-butyl methyl ether (80%, <10%), ethylacetate with saturated NaCl in aqueous phase (<10%, unknown). Ion pair extractions (tetramethylammonium salts in various solvents) and ion exchange column separation (bonded quarternary amnonium) gave poor recoveries of the 3CNAC carboxylic anion. Many different derivatization techniques following i n i t i a l extraction were investigated. However, only dimethylformamide dipropyl acetal (Propyl-8, Pierce Chemical Co.), or diazomethane (generated from Diazald, Aldrich Chemical

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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OSTERLOH ET AL.

Monitoring of 1,3-Dichloropropene

Company) yielded 100% propyl and methyl ester derivatives, respectively. Following extraction and derivatization, an extraction-wash was employed to further clean up the sample. Chlorobutane partitioned with water gave better results than hexane, toluene or more polar solvents. Procedure. Frozen urine i s thawed to room temperature and 1.0 ml i s mixed with 0.5 ml of 6 N HC1 and 10 uL of the stock internal standard (2CNAC, 1 mg/ml) in a 13 x 100 mm screw top borosilicate tube. Standards are prepared by spiking urine with appropriate quantities of the stock 3CNAC methanolic solution. Standard concentrations were 0, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0 ug/mL of urine. Two ml of t-butyl methyl ether (TBME) is added to each tube, capped and extracted for 10 minutes while shaking. A l l samples are centrifuged for 10 minutes at 2000 g. The TBME phase (1.5 mL) i s transferred to a new labeled tube and evaporate ice bath, 200 uL of an at 5°C and capped. Following a reaction time of 30 minutes, the ether and excess diazomethane are evaporated to dryness under nitrogen flow at room temperature. Two ml of n-butyl chloride i s added, mixed, followed by 1 ml of water. This extraction/wash i s shaken for 5 minutes and then centrifuged for 10 minutes at 2000 g. The n-butyl chloride (1.5 mL) i s evaporated under nitrogen at room temperature. Toluene (300600 ul) is added to the residue for GC-MS analysis. Capillary GC-MS instrumentation included a Hewlett-Packard 5970B quadrapole mass selective detector connected to a 5890 gas chromatograph. The fused s i l i c a column was 12 m x 0.2 mm and coated with cross-linked methylsilicone. Helium carrier gas flow was 1 ml/min. The injector was heated to 200°C and operated in the splitless mode. The column was heated at 70°C for 4 minutes, then increased at 20°C/min to 225°C for a total program time of 12 minutes. The quadrapole was calibrated with perfluorotributylamine at masses of 69, 219, and 502. The electron multiplier voltage was 1600 volts and ionization voltage was 70 keV. The quadrapole was run in a low resolution mode with mass peak widths at half height (resolution) were 0.5 AMU. The ions monitored were 117 and 176 with a dwell time of 50 msec. One uL of the toluene sample was injected by a Hewlett-Packard 7673 automatic sampler. After standards calibration, a l l samples are determined in the same run. Controls were run every 8-10 samples. Quantitation was performed by comparing peak height ratios of 3CNAC methyl ester/2CNAC methyl ester for the ion monitored (either 117 or 176) to a standard curve of peak height ratios for the standards. Results Urinary 3CNAC Procedure. Figure 1 shows electron capture gas chromatogram of a blank urine containing no added 3CNAC and a urine of a subject containing 10 ug/mL of 2CNAC and 20 ug/mL of

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w w 53 o W w « 05

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MINUTES

Figure 1. Electron capture gas chromatcgram of a blank urine extraction (described in procedure) and a workers urine containing 10 ug/mL of 2CNAC (Rt=3.3) and 20 ug/mL of 3CNAC (Rt=3.8). Conditions were as follows. Injector: split ratio l:50 Injector temperature: 300°C, Column: 0.25 mm x 15 m fused s i l i c a coated with methyl silicone, Column oven temperature program: 180° isothermal, Detector temperature: 300°C, Carrier gas flow: 2.3 ml/min. f

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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3CNAC. Figure 2 shows the selected ion chromatogram of a subject exposed to DCP. The retention time of 2CNAC methyl ester is 10.33 minutes and 3CNAC methyl ester is 10.58 minutes. While not quantitated in these studies, small amounts of trans 3CNAC methyl ester may appear at 10.75 min. Figure 3 shows the mass spectrum of 3CNAC methyl ester. The 2CNAC methyl ester has similar abundances for ions 117 and 176 used in quantitation. Table I shows the operating characteristics of this assay by capillary GC-MS as compared to the previously used packed column GC-MS (2).

Table I. Operating characteristics of GC-MS Methods for Urinary 3C-NAC Determination

Linearity Regression Coefficient Sensitivity Specificity Coefficient of variation at 10 ug/mL

117 ion 176 ion 117 ion 176 ion

Packed

Capillary

0-30 ug/mL 0.999

0-30 ug/mL 0.999

0.3 ug/mL 0.3 ug/mL good excellent 4%

0.1 ug/mL 0.2 good excellent 8%

Biological monitoring. Air concentrations of DCP, urinary concentrations of 3CNAC and urinary activity of NAG are given for each subject in Figures 4, 5 and 6. Air concentrations of DCP varied from 0-10.3 ppm (0-47 mg/m ). The application of DCP accounted for >80% of the time of exposure during work hours. Mixing/loading, repair and break periods accounting for the remainder of the time. 3

Urinary concentrations of 3CNAC tended to rise and peak during or shortly after exposure. Lags between peak exposure and peak excretion were 0-16 hours. Urinary NAG activity did not seem to rise and f a l l with exposure to DCP or urinary excretion of 3CNAC, though i t was abnormally elevated in two of three subjects at least once during the course of this pilot study. Figure 7 shows that the 24 hour excretion of 3CNAC (mg) increased with rises in the daily cumulative cone'time product for DCP. Also, the concentration of 3CNAC in the urine on the morning following exposure increases in rough proportion to the cumulative conc'time product for DCP from the previous days(Fig. 8). (The correlations for evening urine concentration or excretion on the morning after (ug/mg C) were not as precise.)

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

222 Ion

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In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

17. OSTERLOH ET AL.

Monitoring of 1,3-Dichloropropene

DAY ONE

DAY TWO

DAY THREE

TIME O F EXPOSURE (hrs)

Figure 4. Subject #1. Air concentrations of DCP, urinary concentrations and excretion of 3CNAC, and urinary activity of NAG are given. mgC = milligram of creatinine. Conc'time product i s computed for each preceding exposure interval.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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n

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DAY TWO

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Figure 5. Subject #2. Air concentrations of DCP urinary concentrations and excretion of 3CNAC, and urinary activity of NAG are given. mgC = milligrams of creatinine. Gone "time product i s computed for each preceding exposure interval. Note change in scale for some measures as compared to Figures 4 and 6. r

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

OSTERLOH ET AL.

Monitoring ofl,3-Dichloropropene

II

DAY ONE

DAY TWO

DAY THREE

TIME O F EXPOSURE (hrs)

Figure 6. Subject #3. Air concentrations of DCP urinary concentrations and excretion of 3CNAC and urinary activity of NAG are given. mgC = milligrams of creatinine. Gonc'tiine product i s computed for each preceding exposure interval. Note change in scale for some measures as compared to previous Figures 4 and 5. f

f

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Figure 7. Plot of 24 hour excretion of 3CNAC versus cumulative 24 hr conc'time product for DCP air exposure. Data from subjects #1 and 2. • = 1984 study of five 24 hr urine collections on five persons (from Ref. No. 2). o = Five 24 hr urine collections in this study from two persons.

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Figure 8. Plot of urinary concentration of 3CNAC from morning urine versus cumulative 24 hr conc'time product for DCP air exposure on preceding day.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

17. OSTERLOH ET AL.

Monitoring ofl,3-Dichloropropene

Discussion Urinary 3CNAC Procedures. Our prior attempts at capillary ECGC methods have been several-fold less sensitive than GC-MS procedures in determining 3CNAC in urine samples. There are two likely reasons for this: 1) 3CNAC is polar and difficult to isolate with selectivity from other polar urinary acids (intermediary acids of metabolism and similar amino acids such as methionine); 2) the single chloro group on a polar molecule only slightly enhances the electron capture signal strength over other polar molecules. Recently, Onkenhout et a l (1986) has used other detectors (nitrogen phosphorus, sulphur selective and mass selective) to detect the urinary metabolites in rats. We find that with capillary nitrogen selective gas chromatography of the 3CNAC in urine, the sensitivity is again limited due to endogenous urinary components Onkenhout et a l used different columns separate cis and trans cis and trans-3CNAC on methyl silicone columns under GC-MS conditions, but have not used trans 3CNAC as a marker for exposure since earlier animal studies indicated less was excreted in the urine (3). From qualitative observation of past chromatograms on humans, relatively l i t t l e trans isomer i s excreted compared to cis-3CNAC. Our GC-MS approach has been to monitor both ions 117 and 176 m/e. An occasional human sample will have an interfering substance near the 117 ion peak, whereas the 176 ion gives routinely clean peaks. It would not seem likely that the 176 ion is more selective, since this ion is characteristic of any mercapturic conjugate i f at that specific retention time. Monitoring more specific ions of lower abundance (e.g. molecular ion, 251) reduces sensitivity accordingly. While intraassay precision was slightly worse when compared to the older packed column GC-MS (2), this i s attributable to more complicated extraction in the capillary GC-MS procedure (3 steps vs. 6). This might be improved by omitting the evaporation - chlorbutane/water wash steps and choosing the above-mentioned more selective ions. Biological monitoring. In field studies of an individual, replicate measures of personal air concentrations are d i f f i c u l t . Data from single personal a i r samplings are intrinsically "time averaged" for the period of collection. Therefore, a single high a i r concentration for a short period may bias the overall collection. In this pilot study of human exposure in the field, such a i r concentrations represent f i r s t approximations of potential exposure. The urinary concentrations of a metabolite of a pesticide are considered an estimate of actual and individual exposure. However, this i s only true when relationships between absorption, distribution, metabolism and excretion are known with some certainty. Usually, inherent biovariability between individuals precludes

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establishing explicit relationships or limits. General relationships between exposure and excretion, exposure and effect or excretion and effect can be approached. In our pilot study, we have not established any certain relationships, but have uncovered those to investigate. Several practical considerations are evident as well. In a larger study to follow, we intend to examine the relation between various expressions of exposure (peak concentrations, time weighted exposures and cumulative exposure) and the measures of excretion (ug/mL, ug/hour, ug/mg creatinine, total ug excreted). In this study, i t can only be noted that excretion of metabolite does increase in response to DCP air exposure. This rise in metabolite excretion lags behind DCP peak exposure for a variable period of 0-16 hours. The few data points preclude determination of a precise relationship or pharmacokineti metabolite does not appea start to decrease just before the next exposure. If urinary excretion of 3CNAC increases in proportion to what appears to be primarily a pulmonary exposure, then this may affirm the pulmonary route as the major route of exposure in workers. However, in unprotected workers dermal exposure and biologic variability would tend to confound this relationship. Stott and Kostl (1986) have shown that pulmonary uptake of DCP is 82% at 80 ppm in rats. Quantitative dermal absorption studies are not available. In actual work practice, milligrams of DCP can easily come into direct contact with the skin during repair and loading operations. Since urinary excretion of a metabolite such as 3CNAC is derived from a l l routes of absorption, urinary excretion may be more closely related to effects than air concentrations of DCP. Renal injury in animals following 3 ppm chronic inhalation exposure (13) is controversial since others have not seen alterations in histology or kidney weight following inhalation studies of 1-24 months at higher concentrations (50-90 ppm) (14,15). In part, this may be due to purer preparations of DCP used in recent experiments. Indeed, impurities causing direct mutagenicity can be removed from DCP preparations (8). These form in DCP preparations by autooxidation and may be a source of injury to humans, in which case DCP air measurements or urinary metabolite determinations would only serve as a marker for the impurity. However, DCP is an indirect mutagen following mono-oxigenase activation (8-11 and others). Thiols are protective of DCP mutagenicity in vitro (8,9). DCP will bind and deplete thiol stores in liver and kidney (12). Perhaps a subtle measure of DCP kidney toxicity, the significance of thiol depletion requires further investigation. In this pilot, MAG enzyme activity was chosen as a possible indicator of renal injury, should any occur at a l l with DCP exposure. NAG i s a lysosomal enzyme released from renal

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

17. OSTERLOH ET AL.

Monitoring of 1,3-Dichloropropene

tubular epithelial cells only during the period of acute injury. Price (1982) has reviewed i t s clinical relevancy. In rat models of transient renal tubular injury produced by maleic acid, NAG urinary activities begin to decrease within a few hours after injury has occurred (22). NAG excretion i s considered to be increased only briefly after injury. We saw no clear correspondence between increased NAG urinary activity and excretion of metabolite in these three subjects. However, the fact that two of the three subjects had greater than normal (>100 nmol/hr/mg of creatinine) urinary activity at one point in our surveillance, requires that further investigation be made. It is reported that NAG urinary activity is constant for an individual (21). The interval to interval variability in these subjects suggests some external factor at work. It should be made clear that other unmeasured effects may contribute to elevated NAG activity such as underlying disease (other renal disease, hypertension) (gentamicin, salicylates nephrotoxins such as heavy metals (see review by Price). Such variables were unknown in these subjects. Of fundamental and practical concern are lessons learned from experience. Communication between worker and investigator should be clear. Missed portions of urine collections on subject 3 in this study precluded expression of urinary amounts of 3CNAC (total ug), but rather only creatinine corrected excretion. Subjects #1 and 2 excreted a total of 21.5 and 3.2 mg during the study period. But these amounts are misleading in this limited study, since pre-study exposure was not excluded. From the Figures (4-6), i t i s evident that subjects #2 and 3 were s t i l l excreting 3CNAC from a prior exposure at the beginning of the study. Baseline measurements are helpful when determining the magnitude of new metabolite excretion. Creatinine determinations should always be made in studies involving sequential or 24 hour urine collection. This allows not only to correct for urine flow dependency, but to assess the completeness of the collection. Intervals of collection should coincide as much as possible with worker change in activity (end of day, end of application, lunch, repairs). Also, urine collections and air tube changes should coincide. These two recommendations enable easier data analysis. From this pilot study, i t was learned that NAG excretion during DCP exposure requires investigation and that 3CNAC excretion is not exactly concurrent with exposure. Collection of urine for sometime during and after (<24 hours) worker exposures may be representative of that earlier exposure. Literature Cited 1. Maddy, K. T. Publication No. HS-686. California Department of Food and Agriculture. 1979.

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2. Osterloh, J. D.; Cohen, B.; Poppendorf, W.; Pond, S. M. Arch. Environ. Health 1984, 39, 271-275. 3. Hutson, D. H.; Moss, J. A.; Pickering, B. A. Fd. Cosmet. Toxicol. 1971, 9, 677-680. 4. Climie, I. J. G.; Hutson, D. H.; Morrison, B. J.; Stoydin, G. Xenobiotica 1969, 9, 149-156. 5. Onkenhout, W.; Mulder, P. P.; Boogaard, P.J.; Buijs, W.; Vermeulen, N. P. Arch. Toxicol. 1986, 59, 235-241. 6. Yang, R. S. H.; Huff, J. E.; Boorman, G. A.; Haseman, J. K.; Kornreich, M.; Stookey, J. L. J. Toxicol. Environ. Health 1986, 18, 377-392. 7. Talcott, R. E.; King, J. JNCI 1984, 72, 113-116. 8. Watson, W. P.; Brooks, T. M.; Auckle, K. R.; Hutson, D. H.; Lang, K. L.; Smith, R. J.; Wright, A. S. Chem. Biol. Interact. 1987, 61, 17-30. 9. Creedy, C. L.; Brooks T M.; Dean B J.; Hutson D H.; Wright, A. S. Chem 10. Van der Hude, W.; Scheutwinkel B.; Basler, A. Environ. Mutagen. 1987, 9, 401-410. 11. Valencia, R.; Mason, J. M.; Woodruff, R.C.;Zimmering, S. Environ. Mutagen. 1985, 7, 325-348. 12. Stott, W. T.; Kastl, P. E. Toxicol. App. Pharmacol. 1986, 85, 332-341. 13. Torkelson, T. R.; Oyen, F. Am. Ind. Hygiene Assoc. J. 1977, 38, 217-223. 14. Parker, C. M.; Coate, W. B.; Voelker, R. W., J. Toxicol. Environ. Health 1982, 9, 899-910. 15. Stott, W. T.; Young, J. T.; Calhoun, L. L.; Battjes, J. E. Fund. Appl. Toxicol. In submission. 16. Price, R. G. Toxicology 1982, 23, 99-134. 17. Kunin, C. M.; Chesney, R. W.; Craig, W. A.; England, A. C.; DeAngelis, C. Pediatrics 1978, 62, 751-760. 18. Lauwerys, R. R.; Bernard, A. Am. J. Ind. Med. 1987, 11, 275-285. 19. NIOSH Manual of Analytical Methods (3rd ed.) Vol. 1, 1984, U.S. Dept. Health and Human Services, Publication No. 84100. Methods 1003 and 1013 (also 1982 ed Methods S-104). 20. Leiber, M. A.; Berk, H. C. Anal. Chem. 1984, 56, 21342137. 21. Piagen, K.; Peterson, J. J. Clin. Invest. 1978, 61, 751762. 22. Al-Bander, H. A.; Mock, D. M.; Etheredge, S. B.; Paubert, T. T.; Humphreys, M. H.; Morris, R. C. Kidney International 1986, 30, 804-812. 23. Jung, K.; Schulze, G.; Reinholdt, C. Clin Chem. 1986, 32, 529-532. 24. Fundamentals of Clinical Chemistry, Tietz, N.W. (ed); W.B. Saunders Company, Philadelphia, 1976, p 995-998. RECEIVED May 24, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 18

Determination of Chlordimeform Residues as 4-Chloro-o-Toluidine in Human Urine Using High-Performance Liquid Chromatography M . W. Cheung, R. A. Kahrs, W. B. Nixon, J. A. Ross, and B. G. Tweedy Biochemistry Department, Agricultural Division, Ciba-Geigy Corporation, Greensboro NC 27419

A rapid and specifi oped for the monitoring of worker exposure to chlor­ dimeform (Galecron). The method was developed prior to the second year (1979) reintroduction program of Galecron for use on cotton. Residues of chlordimeform and metabolites i n urine from field workers were hydrolyzed in a test tube under basic conditions to 4-chloro-o-toluidine which was partitioned into n-hexane. Residues of 4-chloro-o-toluidine i n the organic phase were then analyzed using an automated high performance liquid chromatograph equipped with a μ-Porisil column and UV detection at 254 nm. The limit of detection is 0.05 ppm and the average procedural recovery is 99 ± 10% (n=105). Chlordimeform residues in urine stored under ambient laboratory conditions are stable for at least 195 days. The method has been validated and accounts for 35-40% of the total radio­ active residues i n rat urine as 4-chloro-o-toluidine. The method utilized disposable culture tubes which reduced cost and eliminated glassware contamination. The method generated reliable results which were rapidly transmitted to field personnel for the purpose of monitoring and minimizing worker exposure. f

Chlordimeform, N-(2-methyl-4-chlorophenyl)-N ,N'-dimethylformamidine, t h e a c t i v e i n g r e d i e n t i n G a l e c r o n 4E, i s an e f f e c t i v e a c a r i c i d e / i n s e c t i c i d e f o r insect control i n cotton. The a n a l y t i c a l method p u b l i s h e d i n the P e s t i c i d e A n a l y t i c a l Manual (1) was n o t s u i t a b l e as a m o n i t o r i n g method. A gas chromatographic method was d e v e l o p e d i n 1978 p r i o r t o t h e f i r s t y e a r r e i n t r o d u c t i o n program o f G a l e c r o n f o r use i n c o t t o n c u l t u r e . As p a r t o f t h e second y e a r (1979) r e i n t r o d u c t i o n program o f G a l e c r o n , a r a p i d and s p e c i f i c HPLC a n a l y t i c a l method was d e v e l o p e d t o m o n i t o r worker exposure i n

c

0097-6156/89/0382-0231$06.00/0 1989 American Chemical Society

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o r d e r t o ensure s a f e h a n d l i n g o f t h e c h e m i c a l i n f i e l d a p p l i c a t i o n s . F i e l d workers which i n c l u d e d m i x e r s , l o a d e r s , a p p l i c a t o r s , p i l o t s and o t h e r f i e l d p e r s o n n e l were r e q u i r e d t o submit d a i l y u r i n e samples which were a n a l y z e d f o r r e s i d u e s o f c h l o r d i m e f o r m and m e t a b o l i t e ( t o t a l c h l o r d i m e f o r m r e s i d u e s ) . Residues o f c h l o r dimeform and m e t a b o l i t e s were h y d r o l y z e d under b a s i c c o n d i t i o n s t o 4 - c h l o r o - o - t o l u i d i n e . The a n a l y t e was p a r t i t i o n e d i n t o hexane and t h e r e s i d u e s were determined u s i n g normal-phase HPLC and a f i x e d wavelength U V - d e t e c t o r a t 254 nm. The method i s r a p i d , s p e c i f i c and c o s t e f f e c t i v e . Cleanup o f t h e samples o t h e r t h a n p a r t i t i o n i n g i s unnecessary. The d e t a i l s o f t h e a n a l y t i c a l methodology a r e described i n t h i s report. Chlordimeform ( I ) N-(2-methy1-4-chloropheny1)-N',N dimethylformamidine 1

M.W.

= 196.6

4-Chloro-o- t o l u i d i n e ( I I ) Code No.: CGA-41657 C H C1N M.W. 141.6 7

8

MATERIALS AND METHODS MATERIALS. Chlordimeform ( I ) and 4 - c h l o r o - o - t o l u i d i n e ( I I ) a n a l y t i c a l s t a n d a r d s were s u p p l i e d by t h e P r o d u c t i o n T e c h n i c a l A n a l y t i c a l S e c t i o n , A g r i c u l t u r a l D i v i s i o n , CIBA-GEIGY C o r p o r a t i o n . The p u r i t i e s f o r ( I ) and ( I I ) were >99% and >98%, r e s p e c t i v e l y . HPLC grade hexane, 2 , 2 , 4 - t r i m e t h y l p e n t a n e ( i s o o c t a n e ) and 2-propanol were from B u r d i c k & J a c k s o n o r Baker C h e m i c a l s . Reagent grade e t h y l a l c o h o l was from F i s h e r S c i e n t i f i c ( C a t a l o g No. A407-500). Reagent grade sodium h y d r o x i d e , 50% (w/w) was from Baker C h e m i c a l s . Borosilicate g l a s s c u l t u r e tubes (16 x 125 mm) were from C o r n i n g ( C a t a l o g No. 99447). B l a c k p l a s t i c b o t t l e caps w i t h P o l y - S e a l p o l y e t h y l e n e cap l i n e r s , s i z e no. 15 were from F i s c h e r S c i e n t i f i c ( C a t a l o g No. 02-883-5A). Multi-Temp B l o c k Heaters (Model No. 2093 f o r 36 c u l t u r e tubes o r No. 2097-6 f o r 72 c u l t u r e tubes) w i t h module b l o c k t o f i t 16-mm o.d. c u l t u r e tubes were from L a b - L i n e I n s t r u m e n t s , I n c . A u t o m a t i c t r a n s f e r p i p e t t e s f o r d i s p e n s i n g u r i n e were O x f o r d MacroSet T r a n s f e r p i p e t t e s w i t h d i s p o s a b l e p l a s t i c t i p s ( F i s h e r S c i e n t i f i c C a t a l o g No. 21-195). Reagent d i s p e n s e r p i p e t t e s , O x f o r d A u t o m a t i c P i p e t t o r Model S-A, were from F i s h e r S c i e n t i f i c ( C a t a l o g No. 13-687-7A). V o r t e x - G e n i e hand m i x e r s were from S c i e n t i f i c I n d u s t r i e s , I n c . HPLC v i a l s were from F i s c h e r S c i e n t i f i c , ( C a t a l o g No. 06-406AA). The components o f t h e HPLC system a r e d e s c r i b e d i n Table I . PREPARATION OF STANDARDS. S t a n d a r d s o l u t i o n s o f 1 mg/mL o f ( I ) and ( I I ) were p r e p a r e d by d i s s o l v i n g 100 mg o f each s e p a r a t e l y i n 100 mL o f e t h y l a l c o h o l i n 100-mL v o l u m e t r i c f l a s k s . S t a n d a r d ( I ) was d i l u t e d w i t h e t h y l a l c o h o l and was used o n l y f o r f o r t i f i c a t i o n

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p u r p o s e s . Standard ( I I ) was s e r i a l l y d i l u t e d w i t h i s o o c t a n e t o a w o r k i n g range o f 0.02-1.25 ng/mL. A s t a n d a r d curve u s i n g t h r e e o r more i n j e c t i o n s o f d i f f e r e n t q u a n t i t i e s o f ( I I ) c o u l d be p r e p a r e d . However, t h e d e t e r m i n a t i o n o f ( I I ) u s i n g HPLC and a UV fixed-wavel e n g t h d e t e c t o r a t 254 nm has been found t o be l i n e a r from 0.40 ng t o 200 ng and t o i n t e r s e c t t h e o r i g i n u s i n g t h e c o n d i t i o n d e s c r i b e d i n t h i s method. T h e r e f o r e , f o r r o u t i n e r e s i d u e d e t e r m i n a t i o n , o n l y one c a l i b r a t i o n p o i n t , n o r m a l l y 25 ng o f ( I I ) , and t h e o r i g i n were required. ANALYTICAL PROCEDURES. A 5.0-mL a l i q u o t o f u r i n e was t r a n s f e r r e d t o a c u l t u r e tube (16 x 125 mm) u s i n g an automatic t r a n s f e r p i p e t t e . Recovery samples were o b t a i n e d u s i n g c o n t r o l u r i n e f o r t i f i e d w i t h t h e a p p r o p r i a t e amount o f ( I ) . An a l i q u o t o f 1.0 mL o f sodium h y d r o x i d e (50%, w/w) s o l u t i o n was added w i t h an a u t o m a t i c d i s p e n s i n g p i p e t t e . The c u l t u r e tube was t h e n capped w i t h a P o l y S e a l cap and mixed w e l c u l t u r e tube was t h e n p l a c e m i n u t e s . The h e a t i n g b l o c k was m a i n t a i n e d a t 125°C. ( A l t h o u g h t h e h e a t i n g b l o c k temperature may drop t o a p p r o x i m a t e l y 110°C i n i t i a l l y i f many samples were heated a t once, t h i s had no adverse e f f e c t on the a n a l y t i c a l r e s u l t s . ) A f t e r t h e 15-30 minutes o f r e a c t i o n t i m e , the c u l t u r e tube was removed, t r a n s f e r r e d t o a t e s t - t u b e r a c k and c o o l e d i n a water b a t h t o 30-35°C. A l t e r n a t i v e l y , t h e t e s t tube may be c o o l e d i n t h e a i r t o ambient temperature. A f t e r c o o l i n g , an a l i q u o t o f 5.0 mL o f hexane was d i s p e n s e d i n t o each tube u s i n g an a u t o m a t i c d i s p e n s i n g p i p e t t e . The m i x t u r e was s t i r r e d f o r 10 seconds u s i n g a V o r t e x hand mixer. The l a y e r s were t h e n a l l o w e d t o separate. I f an e m u l s i o n was formed, t h e samples were kept a t 30-35°C and immersed f o r s e v e r a l seconds i n an u l t r a s o n i c b a t h . T h i s p r o c e d u r e h e l p e d t o s e p a r a t e t h e phases. An a l i q u o t o f t h e s u p e r n a t a n t hexane l a y e r was t r a n s f e r r e d t o an i n j e c t i o n v i a l f o r t o t a l c h l o r d i m e f o r m r e s i d u e a n a l y s i s as 4 - c h l o r o - o - t o l u i d i n e . CHEMICAL ANALYSIS USING HPLC. The hexane e x t r a c t c o n t a i n i n g ( I I ) was a n a l y z e d by HPLC u s i n g a s i l i c a g e l column ( u - P o r i s i l ) and UV d e t e c t i o n a t 254 nm. The HPLC system was s t a n d a r d i z e d by i n j e c t i n g 25 ng o f 4 - c h l o r o - o - t o l u i d i n e (20 uL o f t h e 1.25 ng/uL s t a n d a r d s o l u t i o n ) . The mobile phase was 10% 2-propanol:90% i s o o c t a n e . D e t a i l s f o r t h e HPLC a n a l y s i s a r e g i v e n i n T a b l e I . Although a standard curve u s i n g s e v e r a l i n j e c t i o n s o f standard at d i f f e r e n t c o n c e n t r a t i o n s c o u l d be p r e p a r e d , t h e UV-detector response f o r ( I I ) has been found t o be l i n e a r , i n t e r s e c t i n g t h e o r i g i n , using the c o n d i t i o n s described. T y p i c a l c a l i b r a t i o n data o f s t a n d a r d ( I I ) a r e p r e s e n t e d i n Table I I . L e a s t squares l i n e a r r e g r e s s i o n a n a l y s i s o f t h e s e d a t a produced c o r r e l a t i o n c o e f f i c i e n t b e t t e r t h a n 0.999 and t h e c a l i b r a t i o n curve i n t e r s e c t e d t h e o r i g i n o r v e r y c l o s e t o i t . T h e r e f o r e , o n l y one c a l i b r a t i o n p o i n t was r e q u i r e d . A 20-uL a l i q u o t o f hexane e x t r a c t from t h e u r i n e sample was i n j e c t e d i n t o t h e HPLC system u s i n g t h e c o n d i t i o n s d e s c r i b e d i n T a b l e I . An a u t o a n a l y z e r , such as t h e Waters WISP 710 o r 712B, c o u l d be used f o r sample i n j e c t i o n .

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Table I .

HPLC C o n d i t i o n s f o r t h e A n a l y s i s o f 4 - C h l o r o - c a r T o l u i d i n e R e s i d u e s i n Human U r i n e

Instrument:

Waters A s s o c i a t e s Model 6000A S o l v e n t D e l i v e r y System, or K r a t o s S p e c t r o f l o w Model 400 pump; Waters Model 440 Absorbance D e t e c t o r o r K r a t o s S p e c t r o f l o w Model 783 V a r i a b l e Wavelength UV d e t e c t o r , and Waters I n t e l l i g e n t Sample P r o c e s s o r WISP 710 o r 712B D e t e c t o r Wavelength: 254 nm Column: S i l i c a G e l , 10 m i c r o n p a r t i c l e s i z e 4.6-mm i d x 25-cm length eg: Rheodyne, I n c . , No. SI 10A [ L i C h r o s o r b SI 100 (E. Merck)] o r Waters, No. 27477, u P o r a s i l Column Temperature: Ambient M o b i l e Phase: 10% 2-propanol i n i s o o c t a n e Flow R a t e : 3.0 mL/min S o l v e n t Temperature: Ambien R e t e n t i o n Time: 2.7 min. L i m i t of S e n s i t i v i t y : 0.4 ng

Table I I .

L e a s t Squares L i n e a r R e g r e s s i o n A n a l y s i s o f T y p i c a l C a l i b r a t i o n Data o f 4 - C h l o r o - o - T o l u i d i n e

Nanograms of ( I I ) 10.0 5.0 5.0 2.0 1.0 0.5 0.5

Peak H e i g h t (cm)

Correlation Coefficient

Slope

Intercept

0.5963

0.0894

16.80 8.05 8.05 3.10 1.60 0.80 0.80 0.9997

DETERMINATION OF SAMPLE RESIDUES. An a l i q u o t o f 20 uL o f t h e hexane e x t r a c t e q u i v a l e n t t o 20 mg o f u r i n e was i n j e c t e d i n t o t h e HPLC u s i n g an a u t o m a t i c i n j e c t o r . The peak h e i g h t o f ( I I ) was measured and t h e ng o f ( I I ) c a l c u l a t e d by E q u a t i o n 1. peak h e i g h t i n sample ng o f ( I I ) =

X 25 ng

(1)

peak h e i g h t o f 25 ng ( I I ) Standard A l t e r n a t i v e l y , a computer d a t a system c o u l d be used t o i n t e g r a t e peak a r e a o f compound ( I I ) . I f a s t a n d a r d curve was c o n s t r u c t e d , t h e d a t a c o u l d be i n p u t t e d i n t o e i t h e r a computer

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d a t a system, o r an e l e c t r o n i c c a l c u l a t o r such as H e w l e t t - P a c k a r d Model 11C c a l c u l a t o r u s i n g l e a s t squares l i n e a r r e g r e s s i o n p l o t t o o b t a i n the ng p r e s e n t i n the i n j e c t e d a l i q u o t . The ppm of c h l o r d i m e f o r m u s i n g E q u a t i o n 2.

e q u i v a l e n t s i n u r i n e was c a l c u l a t e d

ng ( I I ) found ppm chlordimeform

=

X 1.39 mg u r i n e i n j e c t e d

1 X — R

(2)

The f a c t o r 1.39 i s the m o l e c u l a r weight r a t i o of ( I ) t o ( I I ) w h i c h c o n v e r t s the ppm ( I I ) t o ppm e q u i v a l e n t s of ( I ) , and R i s the p e r c e n t r e c o v e r y f a c t o r e x p r e s s e d as a d e c i m a l ( e . g . , 100% = 1.0, 85% = 0.85). The mg e q u i v a l e n t s E q u a t i o n 3. 20 uL i n j e c t e d 5,000 mg u r i n e X

= 20 mg

(3)

5,000 uL t o t a l hexane volume A f l o w diagram of the a n a l y t i c a l p r o c e d u r e s i s p r e s e n t e d i n F i g u r e 1. R e p r e s e n t a t i v e HPLC chromatograms of s t a n d a r d s , c o n t r o l , s p i k e d and f i e l d worker u r i n e samples are shown i n F i g u r e 2. RESULTS AND

DISCUSSION

The v a l i d i t y o f the method has been i n v e s t i g a t e d by a n a l y z i n g u r i n e from r a t s o r a l l y dosed w i t h C - c h l o r d i m e f o r m . The a c c o u n t a b i l i t y of the method as ( I I ) i s 35-40% of the t o t a l C r e s i d u e i n the urine. The r e s i d u e c o n c e n t r a t i o n s of ( I I ) were d e t e r m i n e d by u s i n g automated HPLC normal-phase chromatography and f i x e d - w a v e l e n g t h UV d e t e c t o r a t 254 nm. The a b s o r p t i o n spectrum of 4 - c h l o r o - o t o l u i d i n e w i t h an a b s o r p t i o n maximum a t 235 nm i s shown i n F i g u r e 3. T h i s s p e c t r a l c h a r a c t e r i s t i c rendered m i n i m a l t o no m a t r i x i n t e r f e r e n c e s from u r i n e . However, some i n t e r f e r e n c e from i n t a k e o f drugs such as Darvon and T o l i n a s e and some a n t i b i o t i c s has been o b s e r v e d . I f a v a r i a b l e wavelength UV d e t e c t o r i s a v a i l a b l e , d e t e c t i o n a t 235 nm can be an advantageous a l t e r n a t i v e . U r i n e samples s u b m i t t e d by f i e l d p e r s o n n e l were c o n t a i n e d i n Nalgene b o t t l e s under ambient c o n d i t i o n s . The s t o r a g e s t a b i l i t y o f t o t a l c h l o r d i m e f o r m r e s i d u e s determined as 4 - c h l o r o - o - t o l u i d i n e was i n v e s t i g a t e d . Three l a r g e composite u r i n e samples, each a p p r o x i m a t e l y one l i t e r , were o b t a i n e d by combining s e v e r a l f i e l d w o r k e r s u r i n e samples which c o n t a i n e d t o t a l c h l o r d i m e f o r m r e s i d u e s . These samples were a n a l y z e d f o r compound ( I I ) a t v a r i o u s i n t e r v a l s up t o s i x and o n e - h a l f months (195 days) w h i l e kept i n Nalgene b o t t l e s under ambient l a b o r a t o r y c o n d i t i o n s . No d e g r a d a t i o n of t o t a l c h l o r d i m e f o r m r e s i d u e s was observed. An average of 102 ± 3% (n=3) o f the zero-day v a l u e s was r e c o v e r e d a f t e r s i x and o n e - h a l f months o f s t o r a g e . R e s u l t s are shown i n T a b l e I I I . 14

14

1

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

5.0 mL Urine 1.0 mL 50% NaOH

4 Heating Block (125°C)

I Cool

Add 5.0 mL Hexane, Vortex

Aqueous (Discard)

Hexan | I Automated HPLC (254 nm)

Total Time: F i g u r e 1.

F i g u r e 2.

25 Samples/ 2 Hours

Flow Diagram o f t h e M i n i a t u r i z e d HPLC Procedures

Analytical

R e p r e s e n t a t i v e HPLC Chromatograms o f S t a n d a r d ( I I ) , C o n t r o l , S p i k e d and F i e l d Worker U r i n e Samples (A: 1.0 ng o f ( I I ) ; B: 5.0 ng o f ( I I ) ; C: C o n t r o l U r i n e , <0.05 ppm; D: S p i k e d C o n t r o l a t 0.05 ppm, 105%; E: F i e l d Worker U r i n e , <0.05 ppm)

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Determination ofChlordimeform Residues

T a b l e I I I . Storage S t a b i l i t y R e s u l t s o f T o t a l Residues i n U r i n e

Chlordimeform

P e r c e n t Recovered Day Day 58 132

Day 0

Day 1

Composite Sample No. 1 (from S i t e 702)

100

103

N/A

97.8

103

Composite Sample No. 2 (from S i t e 702)

100

106

88.9

87.3

98.4

Composite Sample No. 3 (from s i t e 901)

100

93.2

88.6

84.1

105

Sample Code

Day 195

Samples were composited from f i e l d p e r s o n n e l t o o b t a i n homogeneous s t o r a g e samples. U r i n e samples were s t o r e d i n Nalgene b o t t l e under ambient l a b o r a t o r y c o n d i t i o n s . An a l i q u o t was a n a l y z e d a t v a r i o u s intervals. N/A = Not A n a l y z e d

A second s t o r a g e s t a b i l i t y s t u d y u s i n g c o n t r o l u r i n e s p i k e d w i t h ( I ) , s t o r e d i n a Nalgene b o t t l e and a n a l y z e d f o r ( I I ) was a l s o performed. R e s u l t s showed t h a t 103% o f t h e r e s i d u e s were accounted f o r as compound ( I I ) a f t e r f o u r weeks o f s t o r a g e i n Nalgene b o t t l e s under ambient l a b o r a t o r y c o n d i t i o n s . R e s u l t s a r e c o n t a i n e d i n T a b l e IV. The v a l i d i t y o f t h e method was f u r t h e r demonstrated by proced u r a l r e c o v e r y experiments conducted d u r i n g m o n i t o r i n g programs i n 1979 and 1980. U r i n e samples were s p i k e d w i t h ( I ) and a n a l y z e d as ( I I ) . The o v e r a l l average r e c o v e r y over t h e f o r t i f i c a t i o n range o f

T a b l e IV.

Storage S t a b i l i t y o f Chlordimeform Residues i n Human U r i n e Determined as 4 - C h l o r o - o - T o l u i d i n e Under Ambient Laboratory Conditions

Sample Code S p i k e d Sample

Day 0 100

P e r c e n t Recovered Day 7 Day 14 99.6

Day 28

103.3

100.3

A l i q u o t s were withdrawn and a n a l y z e d a t v a r i o u s i n t e r v a l s . Average v a l u e s o f d u p l i c a t e a n a l y s e s o f c o n t r o l u r i n e samples s p i k e d w i t h 0.50 ppm o f c h l o r d i m e f o r m .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

238

BIOLOGICAL MONITORING FOR PESTICIDE

EXPOSURE

LAB INFORMATION Fl QW

MEMPHIS, LAB

TN

GREENSBORO,

NC

LAB

ARDSLEY,

NY,

IBM

370

GREENSBORO

FIELD

F i g u r e 3.

SITES

U V - V i s i b l e A b s o r p t i o n Spectrum o f 4-Chloro-o-Toluidine

380 WAVELENGTH

F i g u r e 4.

300 (NM)

I n f o r m a t i o n Flow Between A n a l y t i c a l L a b o r a t o r i e s and F i e l d C o o r d i n a t o r s V i a IBM Computer

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

18. CHEUNG ETAL.

239

Determination of Chlordimeform Residues

0.05 ppm t o 10 ppm was 99 ± 10% (n=105). A summary o f the r e c o v e r y d a t a i s shown i n T a b l e V. Another f e a t u r e o f the method d e s c r i b e d i n t h i s r e p o r t was t h e i n c o r p o r a t i o n o f a computer system ( H e w l e t t - P a c k a r d HP-1000 compu­ t e r ) i n t h i s m o n i t o r i n g program. The H e w l e t t - P a c k a r d computer c o l l e c t e d HPLC a n a l y t i c a l d a t a g e n e r a t e d i n l a b o r a t o r i e s i n Memphis, Tennessee, and G r e e n s b o r o , N o r t h C a r o l i n a , p e r f o r m e d t h e q u a n t i t a t i o n o f r e s i d u e l e v e l s , and t r a n s f e r r e d t h e d a t a t o an IBM 370 mainframe computer i n A r d s l e y , New York. R e s i d u e r e s u l t s were t r a n s m i t t e d t o f i e l d p e r s o n n e l t y p i c a l l y w i t h i n 24 hours a f t e r sample r e c e i p t . The computer system e n a b l e d r a p i d t r a n s m i t t a l o f r e s u l t s to f i e l d personnel. F i g u r e 4 shows a g r a p h i c a l r e p r e s e n t a ­ t i o n o f the d a t a and i n f o r m a t i o n t r a n s m i t t e d . D u r i n g 1979-1980, over 40,000 u r i n e samples were a n a l y z e d u s i n g t h i s HPLC method.

T a b l e V.

Procedural Recoverie 4-Chloro-o-Toluidin

Fortifi­ cation Levels (ppm)

Range o f Recovery

0.05-10.0

Average Procedural Recovery

(%)

(%)

72-116

99

Standard Deviation N

(%) ±

10

105

In summary, a r a p i d , s i m p l e , c o s t e f f e c t i v e and s p e c i f i c u r i n e m o n i t o r i n g method was d e v e l o p e d i n c o n j u n c t i o n w i t h t h e r e i n t r o d u c ­ t i o n o f c h l o r d i m e f o r m ( G a l e c r o n ) f o r use on c o t t o n u s i n g v e r s a t i l e HPLC t e c h n i q u e s . Under normal c o n d i t i o n s , 2 5 samples can be a n a l y z e d i n two h o u r s . By v i r t u e o f t h e s p e c t r a l c h a r a c t e r i s t i c s of 4 - c h l o r o - o - t o l u i d i n e , s p e c i f i c r e s i d u e d e t e r m i n a t i o n u s i n g a f i x e d - w a v e l e n g t h UV d e t e c t o r a t 2 5 4 nm was a c c o m p l i s h e d . Inexpen­ s i v e , d i s p o s a b l e c u l t u r e tubes r e d u c e d c o s t and e l i m i n a t e d g l a s s ­ ware c o n t a m i n a t i o n . R e l i a b l e m o n i t o r i n g r e s u l t s were g e n e r a t e d and r a p i d l y communicated t o f i e l d p e r s o n n e l . LITERATURE CITED

1.

FDA Pesticide Analytical Manual, Vol. II, U. S. Department of Commerce, National Technical Information Service, Pesticide Reg.

Sec.

180.285 ( 1 1 / 1 / 7 3 ) .

RECEIVED June 23, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 19

Biological Monitoring Technology for Measurement of Applicator Exposure S. Dubelman and J. E. Cowell Monsanto Agricultural Company, Monsanto Company, St. Louis, MO 63198

The measurement of a pesticide and its metabolites in urine is a direct and accurate means of determining actual applicato Biological monitorin studies with alachlo techniques necessary g y mining body dose. Animal metabolism and pharmacokinetic studies provided data about the nature of the metabo­ l i t e s , the elimination pathway (urine/feces) and the kinetics of excretion. Urine from applicators using alachlor was collected for a 5 day period following application. Twelve-hour urine composites were analyzed by GCMS or HPLC for DEA and HEEA, the chemophores derived from alachlor and its metabolites. Method sensitivity was 5.0 ppb (μg/L) for the total measured residue. Appropriate correction factors derived from animal studies were applied to the measured urinary excretion to determine the body dose. Background A number o f t e c h n i q u e s a r e c u r r e n t l y used t o m o n i t o r a p p l i c a t o r exposure t o p e s t i c i d e s . These i n c l u d e p a s s i v e d o s i m e t r y , b i o l o g i c a l m o n i t o r i n g and f l u o r e s c e n c e v i d e o imaging. T a b l e I shows a comparison o f the d i s t i n c t i v e f e a t u r e s o f each t e c h n i q u e . The main advantage o f p a s s i v e d o s i m e t r y i s t h e ease w i t h which samples can be o b t a i n e d and a n a l y z e d . Analysis i s usually f o r the p a r e n t p e s t i c i d e o n l y , and no metabolism i n f o r m a t i o n i s necessary. T h i s method produces data t h a t i s s u i t a b l e f o r i n c l u s i o n i n g e n e r i c databases c o n t a i n i n g l a r g e numbers o f s t u d i e s w i t h d i f f e r e n t p e s t i c i d e s . T h i s t e c h n i q u e can be used t o measure expo0097-6156/89/0382-0240$06.00/0 © 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

19.

DUBELMAN AND COWELL

Table I. Technique

Biological Monitoring Technology

241

Comparison o f Exposure Measurement Techniques Measurement

Advantage/Best

Passive Dosimetry

Dermal D e p o s i t i o n (Patches) Inhalation (Air Sampling) Parent P e s t i c i d e Analysis

D i s c r e t e Work Activities Most V u l n e r a b l e Body Areas Simple A n a l y s i s S e p a r a t e Dermal Inhalation Values

Fluorescence/ Imaging

Dermal D e p o s i t i o n (Video Imaging)

Most V u l n e r a b l e Body Areas

Use

Education/Training Biological Monitoring

Excreted P o r t i o n of Body Dose ( U r i n e ) Pesticide + Metabolite Analysis

I n t e g r a t e d Exposure: I n h a l a t i o n + Dermal + Ingestion No Need f o r Dermal Absorption Factor No C l o t h i n g P e n e t r a t i o n Assumption C l o s e s t Estimate of T o t a l Body Dose

sures r e c e i v e d d u r i n g d i s c r e t e work a c t i v i t i e s w i t h i n a workday, which makes i t v a l u a b l e i n e v a l u a t i n g exposure r e d u c t i o n p r a c tices. There are s e v e r a l d i s a d v a n t a g e s i n u s i n g p a s s i v e d o s i m e t r y f o r c a l c u l a t i o n o f body dose. One i s the need t o measure the amount of c h e m i c a l absorbed t h r o u g h the s k i n and l u n g s , o r t o assume 100% a b s o r p t i o n o f the compound. The l a t t e r assumption i s not supported by a number o f r e c e n t s t u d i e s . A l a r g e source o f e r r o r i s a l s o i n h e r e n t i n the e x t r a p o l a t i o n of r e s i d u e s found i n r e l a t i v e l y s m a l l t r a p p i n g d e v i c e s ( e . g . , gauze p a t c h e s ) t o e n t i r e body s u r f a c e a r e a s . S i n c e the p a t c h e s g e n e r a l l y cover 6% o r l e s s o f the body, exposure c o u l d be g r o s s l y over- o r u n d e r - e s t i m a t e d i f p e s t i c i d e d r o p l e t s h i t o r miss the p a t c h . An a d d i t i o n a l assumption l e a d i n g t o over- o r u n d e r - e s t i m a t i o n i n v o l v e s the amount o f p e s t i c i d e i n t e r c e p t e d by c l o t h i n g d u r i n g p e s t i c i d e a p p l i c a t i o n . Scenari o s u s i n g no p r o t e c t i o n (nude a p p l i c a t o r ) , 50%, 80%, o r 100% c l o t h i n g p r o t e c t i o n have been used by EPA i n c a l c u l a t i n g body doses from gauze p a t c h data f o r a number o f p e s t i c i d e s . E s t i m a t e s o f body dose can range s e v e r a l o r d e r s o f magnitude depending on how much body s u r f a c e i s assumed t o be p r o t e c t e d by c l o t h i n g . V i d e o imaging w i t h f l u o r e s c e n t t r a c e r s i s not y e t w i d e l y used. An o b s t a c l e t o w i d e s p r e a d use i s the c u r r e n t u n a v a i l a b i l i t y o f n o n t o x i c f l u o r e s c e n t t r a c e r s w h i c h would be c o m p a t i b l e w i t h a wide

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

242

range o f a g r i c u l t u r a l f o r m u l a t i o n s . The t e c h n i q u e q u a n t i f i e s dermal d e p o s i t i o n , which i s o f t e n t h e major component o f t h e exposure, b u t does n o t account f o r t h e p o r t i o n o f t h e dose r e s u l t i n g from i n h a l a t i o n o r i n g e s t i o n . E s t i m a t e s o f dermal p e n e t r a t i o n are s t i l l n e c e s s a r y f o r c a l c u l a t i o n o f body dose. An i m p o r t a n t advantage o f t h e t e c h n i q u e i s i t s a b i l i t y t o p r o v i d e a v i s u a l q u a l i t a t i v e assessment o f t h e a f f e c t e d body a r e a s . T h i s knowledge can be used t o d e s i g n exposure r e d u c t i o n measures and t o educate a g r i c u l t u r a l workers. The p r i m a r y advantage o f b i o l o g i c a l m o n i t o r i n g i s t h a t t h e a c t u a l body dose can be d i r e c t l y determined by measuring t h e concentration of the p e s t i c i d e or i t s metabolites i n the urine. T h i s dose i n c l u d e s exposure from a l l s o u r c e s : dermal, i n h a l a t i o n and i n g e s t i o n . There i s no need t o determine dermal a b s o r p t i o n o r c l o t h i n g p e n e t r a t i o n f a c t o r s , s i n c e a l l these a r e i n t r i n s i c and accounted f o r i n t h e m o n i t o r i n g The main disadvantage o f t h e technique i s t h a t d e t a i l e k i n e t i c s i s needed b e f o r I n a d d i t i o n , t h i s t e c h n i q u e i s a p p l i c a b l e o n l y t o those chemicals where u r i n e i s t h e major r o u t e o f e x c r e t i o n . N e i t h e r b l o o d n o r f e c e s a r e p r a c t i c a l m a t r i x e s f o r t h e measurement o f t o t a l exposure. Alachlor Studies:

M e t a b o l i s m , E x c r e t i o n and Methods

M e t a b o l i s m and e x c r e t i o n k i n e t i c s s h o u l d be known b e f o r e t h e b i o l o g i c a l m o n i t o r i n g study a r e conducted. R e s u l t s o f these studies f o r a l a c h l o r , 2-chloro-2 ,6 -diethyl-N-(methoxymethyl) a c e t a n i l i d e , t h e a c t i v e i n g r e d i e n t i n Lasso h e r b i c i d e (Monsanto Co.), a r e shown i n T a b l e I I . The r a t metabolism s t u d y ( o r a l d o s i n g ) , r e q u i r e d f o r r e g i s t r a t i o n under FIFRA, p r o v i d e d m e t a b o l i t e i d e n t i f i c a t i o n data w h i c h s e r v e d as a b a s i s f o r d e v e l o p i n g a n a l y t i c a l methods f o r use i n t h e a p p l i c a t o r exposure s t u d y . 1

TABLE I I .

1

R e s u l t s o f M e t a b o l i s m and E x c r e t i o n S t u d i e s on A l a c h l o r

Study Oral Dosing C-Rats

Results I d e n t i f i c a t i o n o f Many M e t a b o l i t e s Y i e l d i n g E i t h e r DEA o r HEEA Upon Hydrolysis

O r a l Dosing C-Mice

I d e n t i f i c a t i o n of Metabolites Containing DEA and HEEA; S p e c i e s Comparison

Intravenous Dosing C-Monkeys

97% Dose Recovered i n U r i n e + Feces 87% Dose E x c r e t e d i n U r i n e 80% Dose E x c r e t e d i n 48 Hours Q u a n t i t a t i o n o f DEA/HEEA R a t i o

Dermal A b s o r p t i o n C-Monkeys

8.5% o f Dose Absorbed Through S k i n 88% o f Dose i n U r i n e

1 4

1 4

14

14

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

19.

DUBELMAN AND COWELL

Biological Monitoring Technology

243

F o r a l a c h l o r , the mouse metabolism study d i d not p r o v i d e much a d d i t i o n a l knowledge a p p l i c a b l e t o the exposure s t u d y , except f o r c o r r o b o r a t i o n o f m e t a b o l i t e p r o f i l e s and a d d i t i o n a l s p e c i e s compari s o n o f e x c r e t i o n pathways. The i n t r a v e n o u s i n j e c t i o n o f C - l a b e l e d p e s t i c i d e t o monkeys was used t o determine e x c r e t o r y r e c o v e r y and d i s t r i b u t i o n o f the t o t a l amount o f c h e m i c a l between u r i n e and f e c e s . T h i s s t u d y p r o v i d e d c o r r e c t i o n f a c t o r s f o r human e x c r e t o r y r e c o v e r y . Dermal a p p l i c a t i o n o f C - l a b e l e d a l a c h l o r t o monkeys p r o v i d e d e x c r e t i o n data t o determine the l e n g t h o f time f o r u r i n e c o l l e c t i o n by humans. The i n t r a v e n o u s and dermal monkey s t u d i e s showed t h a t 87-88% of the a d m i n i s t e r e d dose was e x c r e t e d i n the monkey u r i n e , and e x c r e t i o n was e s s e n t i a l l y complete f o u r days a f t e r dose a d m i n i s t r a t i o n . T h i s s e r v e d as a b a s i s f o r d e t e r m i n i n g t h a t u r i n e samples would be c o l l e c t e d f o r 5 days i n the a p p l i c a t o r exposure s t u d i e s and a n a l y t i c a l r e s u l t s woul f a c t o r i n order to a r r i v was i n a d d i t i o n t o a n a l y t i c a l c o r r e c t i o n s n o r m a l l y a p p l i e d f o r l o s s e s i n the r e s i d u e method. A s p e c i e s comparison f o r e x c r e t o r y r e c o v e r y and m e t a b o l i t e c l a s s i f i c a t i o n i s g i v e n i n Table I I I , w h i c h showed the m e t a b o l i t e p a t t e r n i n human and monkey u r i n e t o be n e a r l y i d e n t i c a l . 1 4

1 4

Table I I I . Comparison o f E x c r e t i o n P r o f i l e s and M e t a b o l i t e C l a s s i f i c a t i o n i n Various Species

% 1 4 c -- E x c r e t i o n Urine Feces

% Metabolite Class HEEA DEA

Rat

50

50

35

65

Mouse

35

65

40

60

Monkey

88

12

80

20

Human

—

—

80

20

The rodent and monkey metabolism s t u d i e s i n d i c a t e d t h a t a l a c h l o r was r a p i d l y t r a n s f o r m e d t o a number o f m e t a b o l i t e s t h a t c o n t a i n e d d i e t h y l - a n i l i n e (DEA) and 2 - ( l - h y d r o x y e t h y l ) - e t h y l a n i l i n e (HEEA) m o i e t i e s . An a n a l y t i c a l method f o r measuring these metabol i t e s i n the u r i n e of a p p l i c a t o r s was then developed based on h y d r o l y t i c c o n v e r s i o n o f a l l m e t a b o l i t e s t o DEA and HEEA, as shown i n F i g u r e 1. The l i b e r a t e d a n i l i n e s were t h e n q u a n t i t a t e d by n e g a t i v e c h e m i c a l i o n i z a t i o n GCMS ( I n d i a n a and Canada s t u d i e s ) a f t e r convers i o n t o the c o r r e s p o n d i n g h e p t a f l u o r o b u t y r i c a n h y d r i d e (HFBA) d e r i v a t i v e s , o r d i r e c t l y by HPLC w i t h e l e c t r o c h e m i c a l detection (Missouri studies).

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

244

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

The s e n s i t i v i t y o f the methods was each m e t a b o l i t e c l a s s .

5.0

PPB,

o r 2.5

ppb

for

F i e l d Procedures Based on the monkey e x c r e t i o n s t u d i e s , a p e r i o d o f 5 days was e s t a b l i s h e d f o r c o l l e c t i o n o f a p p l i c a t o r u r i n e . Male a p p l i c a t o r s c o l l e c t e d each u r i n e v o i d i n a s e p a r a t e g l a s s b o t t l e and d e l i v e r e d the c o n t a i n e r s d a i l y t o the study a d m i n i s t r a t o r . The study admini s t r a t o r combined the v a r i o u s u r i n e v o i d s i n t o 12-hour composites f o r each a p p l i c a t o r and r e c o r d e d the d a i l y volumes. A l l samples were l a b e l e d w i t h name, date and time o f c o l l e c t i o n and s t o r e d frozen u n t i l analyzed. C o n t r o l o r b a s e l i n e u r i n e samples were c o l l e c t e d from each a p p l i c a t o r p r i o r t o the exposure study. C o n t r o l u r i n e was f o r t i f i e d i n the f i e l d w i t h r e p r e s e n t a t i v e m e t a b o l i t e s and h a n d l e d e x a c t l y as the "exposure" samples These samples p r o v i d e d r e c o v e r f o r any l o s s e s d u r i n g t r a n s p o r t F i e l d i n f o r m a t i o n was c o l l e c t e d on s p e c i a l l y d e s i g n e d f i e l d s u r v e y forms. Data c o l l e c t e d d e s c r i b e the c o n d i t i o n s o f each a p p l i c a t i o n , and i n c l u d e d weather, a p p l i c a t i o n d e t a i l s , p e r s o n a l c l o t h i n g and h y g i e n e , wind speed and d i r e c t i o n , h u m i d i t y , type o f equipment, amount o f a l a c h l o r a p p l i e d , p r o t e c t i v e equipment ( g l o v e s , g o g g l e s ) , body w e i g h t and a number of o t h e r i t e m s . Photographs and c o n t i n u o u s v i d e o t a p e s p r o v i d e d a d d i t i o n a l documentation. F o r the M i s s o u r i and Canadian s t u d i e s , an attempt was made t o c o r r e l a t e the b i o l o g i c a l m o n i t o r i n g r e s u l t s w i t h r e s u l t s from passive dosimeters. F o r t h a t p u r p o s e , 6 gauze p a t c h e s , 10 X 10 cm were a t t a c h e d t o the c l o t h i n g n e a r the forehead ( c a p ) , back ( s h i r t ) , c h e s t ( s h i r t ) , c h e s t ( u n d e r s h i r t ) , forearm ( s l e e v e on dominant arm) and t h i g h (pant l e g ) . Care was t a k e n t o l o c a t e t h e s e so as not t o b l o c k exposed s k i n s u r f a c e s . No gauze pads were used i n the I n d i a n a study. R e s u l t s and

Discussion

R e s u l t s from r e c o v e r y experiments o f l a b o r a t o r y and f i e l d f o r t i f i c a t i o n s are shown i n T a b l e s IV ( u r i n e ) and V (gauze p a d s ) . These were used t o c o r r e c t the v a l u e s o b t a i n e d i n each c o r r e s p o n d i n g study. I n g e n e r a l , t h e r e was r e a s o n a b l e agreement between l a b o r a t o r y and f i e l d r e c o v e r i e s , i n d i c a t i n g t h a t l o s s e s were l a r g e l y due t o a n a l y t i c a l p r o c e d u r e s , not t r a n s p o r t o r s t o r a g e s t a b i l i t y factors. A summary o f data o b t a i n e d f o r a l l a p p l i c a t o r s i s shown i n T a b l e V I . The body dose i s e x p r e s s e d i n micrograms per k i l o g r a m body w e i g h t per pound a c t i v e i n g r e d i e n t a p p l i e d ( p g / k g / l b ) . This i s a c o n v e n i e n t u n i t t h a t a l l o w s e x t r a p o l a t i o n t o any number of c o n d i t i o n s when the data i s l a t e r used f o r r i s k assessment. Any one o f the b i o l o g i c a l m o n i t o r i n g v a l u e s i n T a b l e VI can be c a l c u l a t e d as f o l l o w s : The t o t a l amount o f a l a c h l o r m e t a b o l i t e s e x c r e t e d by the a p p l i c a t o r d u r i n g the days f o l l o w i n g the a p p l i c a t i o n i s o b t a i n e d by a d d i n g the v a l u e s found i n each u r i n e sample

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

96.2

15.3

Recov. Mean (%)

Std.

1985 = M i s s o u r i

19841 = I n d i a n a

1984C = Canada

HPLC/EC

8

95

72

2.52500

LAB 19841

GC/MS

34

No. o f S p i k e s

Dev.

10200

Fortification Range (ppb)

1984C

14.3

85.9

143

2.550

1985

20.3

18

95

48

24

100

1000

FIELD 19841

10200

1984C

8.1

85.1

18

2.525

1985

72

67

34

65.6

16

2.52500

10200

13.2

LAB 19841

1984C

11.6

87.2

141

2.550

1985

8.9

67.8

16

64

48

51000

10200

24

FIELD 19841

1984C

HEEA - Y i e l d i n g M e t a b o l i t e s

A l a c h l o r Recovery S t u d i e s - U r i n e

DEA - Y i e l d i n g M e t a b o l i e s

TABLE IV.

7.5

74.1

18

2.5250

1985

246

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

H

{

HYDROLYSIS

-N

H

DEA YIELDING ALACHLOR METABOLITES

HYDROLYSIS

HEEA YIELDING ALACHLOR METABOLITES F i g u r e 1. a n d HEEA.

Hydrolysis

TABLE V.

of Alachlor Metabolites

t o DEA

A l a c h l o r Recovery S t u d i e s - Gauze Pads

LAB

FIELD 1984 ( c ) 1985

1984 ( c )

1985

Fortification Range (pg)

0.01-600

0.5-5000

0.01-40

0.5-5000

No. o f S p i k e s

12

27

16

56

89.7

69.6

89.2

7.3

24.5

11.2

Recovery Mean (%)

S t d . Dev.

104

11.5

1984 = Canadian Study 1985 = M i s s o u r i Study

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

19. DUBELMAN AND COWELL

TABLE V I .

Biological Monitoring Technology

247

A l a c h l o r Body Dose E s t i m a t e s

Study/ A p p l i c a t o r Formul. Body Dose ( f j g / k g / l b ) A p p l i c a t o r No. Weight(kg) Oper.(1) B i o l . M o n i t . ( 2 ) P a s s i v e Dos.(3) IND IND IND IND CAN CAN

01 03 05 07 01 05

85 80 86 84 70. ,5 81. ,8

EC/MLA EC/MLA EC/MLA EC/MLA EC/MLA EC/MLA

0. 00526 0. 00574 0 0. 000676 0. 0270 0. 0199

IND IND IND IND CAN CAN MO MO MO MO

02 04 06 08 03 04 05 07 09 10

86 59 89 64 95. , 79. ,5 68. ,1 77. .2 63. .6 90, .8

MT/MLA MT/MLA MT/MLA

0. 0218 0. 0227 0 00425

MT/MLA MT/MLA MT/MLA MT/MLA MT/MLA

0.,0405 0.,000722 0..00123 0 0..00149

0.0813 0.000153 0.000566 0.00135 0.000201

MO MO MO MO

06 08 11 12

95. .3 88. .5 79, .5 86, .3

WDG/MLA WDG/MLA WDG/MLA WDG/MLA

0..00158 0.,00231 0..000559 0

0.000268 0.000354 0.000332 0.000927

MO MO MO MO

27 28 29 30

70, .4 74, .9 74, .9 70, .4

EC/ML EC/ML EC/ML EC/ML

0 0 0,.00269 0

0 0.0000127 0.0000224 0

MO MO MO MO

25 26 31 32

77, .2 84, .0 81 .7 77 .2

MT/ML MT/ML MT/ML MT/ML

0,.000559 0,.000135 0 .000372 0 .00130

0.0000702 0.0000112 0.000184 0.000403

MO MO MO MO

33 34 35 36

81 .7 77 .2 102 .0 72 .6

WDG/ML WDG/ML WDG/ML WDG/ML

0 .000294 0 .000300 0 .000398 0 .000228

0.000118 0.000389 0.000102 0.000310

MLA = M i x e r / L o a d e r & A p p l i c a t i o n ML = M i x e r / L o a d e r Only

(1) (2) (3)

-

0.0151 0.128

-

IND = I n d i a n a CAN = Canada MO = M i s s o u r i

A l l US a p p l i c a t o r s a p p l i e d 80 l b a l a c h l o r t o 20 a c r e s ; Canadi a n s a p p l i e d 160 l b t o 40 a c r e s (EC) o r 150 t o 37 a c r e s (MT). C o r r e c t e d f o r 8 8 % u r i n a r y e x c r e t i o n (l4C/monkey e x c r e t i o n ) A d j u s t e d f o r 8.5% dermal a b s o r p t i o n (l4C/monkey dermal s t u d y )

American Chemical Society Library 1155 16th St. f N.VY.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; Washington, O.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

248

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

c o l l e c t e d from t h e a p p l i c a t o r . F o r example, Table V I I shows t h a t M i s s o u r i A p p l i c a t o r #10 e x c r e t e d a t o t a l o f 9.48 Mg a l a c h l o r a f t e r a p p l i c a t i o n o f 80 l b a l a c h l o r t o a 20 a c r e p l o t (4 l b / a c r e ) . T h i s v a l u e i s then c o r r e c t e d f o r t h e 88% e x c r e t o r y r e c o v e r y o b t a i n e d i n t h e C-monkey s t u d i e s , and n o r m a l i z e d f o r body weight and pounds o f a c t i v e i n g r e d i e n t a p p l i e d : 14

9.48 Mg 0.88

1

x

1

x

90 kg body weight

0.00149 Mg/kg/lb

=

80 l b a p p l i e d

The v a l u e 0.00149 Mg/kg/lb i s shown i n t h e body dose column ( b i o l . m o n i t o r i n g ) o f Table VI under a p p l i c a t o r "MO 10". The v a l u e s i n t h e n e x t column o f Table V I , i . e . , body dose from t h e p a s s i v e d o s i m e t r y s t u d i e s , a r e c a l c u l a t e d from gauze pads by m u l t i p l y i n g t h e pad v a l u e by t h e exposed body area w h i c h i t represents: 2

Body dose (Mg/kg/lb) = measured ( g / c m ) X H

body weight

(kg)

X

exposed s k i n

2

(cm )

l b applied

2

2

Areas o f exposed s k i n were 650 cm f o r t h e f a c e , 150 cm f o r the f r o n t o f t h e neck and 110 cm f o r t h e back o f t h e neck (Davies 1980). A l l o t h e r areas were assumed t o be p r o t e c t e d by the c l o t h i n g . The hands were p r o t e c t e d by rubber g l o v e s , as p e r l a b e l d i r e c t i o n s . The measured v a l u e s were c o r r e c t e d f o r t h e 8.5% dermal a b s o r p t i o n o b t a i n e d i n t h e C - d e r m a l monkey s t u d i e s , p r o d u c i n g t h e body dose v a l u e s shown i n T a b l e V I . I n h a l a t i o n was n o t measured, but was p r e v i o u s l y shown t o be n e g l i g i b l e i n comparison t o dermal 2

14

TABLE V I I .

E x c r e t i o n P r o f i l e f o r M i s s o u r i A p p l i c a t o r #10 2

1

Hours A f t e r Application 0-12 12-24 24-36 36-48 48-60 60-72 72-84 84-96 96-108 108-120

Combined* Pesticide/ Metabolite Residue (Mg/mL) 0.01414 0.00499 0.00280 N.D. N.D. N.D. N.D. N.D. N.D. N.D.

3

Volume of Urine (mL) 434 451 388 691 1214 270 809 875 1042 531

Total excreted

4

Total* Mg 6.14 2.25 1.09 0 0 0 0 0 0 0 9.48

Mg

* Results corrected f o r a n a l y t i c a l / f i e l d f o r t i f i c a t i o n recovery N.D. = n o t d e t e c t e d (LOD = 0.0025 Mg/mL) ± T o t a l Mg o b t a i n e d by m u l t i p l y i n g t h e v a l u e i n column 2 w i t h t h e c o r r e s p o n d i n g v a l u e i n column 3. i s

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

19.

DUBELMAN AND COWELL

249

Biological Monitoring Technology

deposition. (Lauer and A r r a s , 1982; L i b i c h , e t a l . , 1984; Lavy, 1978). There appears t o be no d i f f e r e n c e i n exposure f o r the t h r e e f o r m u l a t i o n s t e s t e d , j u d g i n g by the v a l u e s o b t a i n e d f o r m i x e r l o a d e r s ( a p p l i c a t o r s M025-M036) a p p l y i n g the e m u l s i f i a b l e concent r a t e (Lasso), microencapsulated (Lasso MT), o r water d i s p e r s i b l e g r a n u l e (Lasso WDG) f o r m u l a t i o n s . V a r i a b i l i t y w i t h i n f o r m u l a t i o n s overshadows any p o t e n t i a l d i f f e r e n c e s between f o r m u l a t i o n s , as shown i n F i g u r e 2. S i n c e each o f the s t u d i e s was performed under a d i f f e r e n t s e t of c o n d i t i o n s (open v e r s u s c l o s e d - c a b t r a c t o r , e t c . ) , average body doses were c a l c u l a t e d f o r each s e p a r a t e study. V a l u e s are shown i n Table V I I I . These v a l u e s can be used f o r r i s k assessment c a l c u l a t i o n s under any number o f assumed farm s i z e s o r c o n d i t i o n s . I t s h o u l d be n o t e d , however, t h a t t h e s e v a l u e s are a p p l i c a b l e o n l y t o a p p l i c a tors using small containers nant i n s m a l l farms (<30 a p p l i c a t o r s use m a i n l y b u l k or m i n i - b u l k c o n t a i n e r s , such as the Monsanto S h u t t l e , w h i c h d e l i v e r s the f o r m u l a t i o n i n t o the s p r a y e r i n a " c l o s e d - s y s t e m " f a s h i o n . Body doses f o r such systems have been r e p o r t e d elsewhere ( C o w e l l , 1987) and average 1.8 X 10 Mg/kg/lb, s e v e r a l f o l d lower t h a n the s m a l l c o n t a i n e r s t u d i e s described i n Table V I I I . 4

Lasso MT

Figure

2.

Exposure

Lasso (EC)

Levels

Lasso

of

WDG

Mixer/Loaders.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

250

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Table V I I I .

Average Body Dose i n B i o l o g i c a l M o n i t o r i n g

Study

Studies

Avg. Body Dose (Mg/kg/lb)

Conditions

Indiana (8 A p p l i c a t o r s )

Closed-Cab T r a c t o r S o i l Incorporated

7..8 X 1 0 ~

3

Canada (4 A p p l i c a t o r s )

Open T r a c t o r Small Sprayer Surface Applied

5..0 X 10~

2

Missouri (20 A p p l i c a t o r s )

Large C l o s e d Farme

8,.1 X 10~

4

Tractor

Conclusion B i o l o g i c a l m o n i t o r i n g o f u r i n a r y m e t a b o l i t e s i s a d i r e c t and e f f e c t i v e way o f d e t e r m i n i n g a c t u a l a p p l i c a t o r exposure. Knowledge o f animal m e t a b o l i s m and e x c r e t i o n k i n e t i c s i s n e c e s s a r y f o r c a l c u l a t i o n o f body dose. S t u d i e s w i t h a l a c h l o r showed t h a t body dose averages can range from 5 X 10~~ ng/kg/lb f ° farmers u s i n g s m a l l open t r a c t o r s and s m a l l c o n t a i n e r s , t o 1.8 X 10 |Jg/kg/lb f o r l a r g e farmers o r commercial a p p l i c a t o r s u s i n g l a r g e equipment and b u l k c o n t a i n e r s . 2

r

4

Literature Cited 1. 2. 3. 4. 5.

Cowell, J . E . , et a l ; Arch. Environ. Contam. Toxicol 1987, 16, 327-332. Davis, J . E . ; Minimizing Occupation Exposure to Pesticides Residue Reviews 1980, 75, 34-50. Lauer, R. and Arras, D.D.; Aerial and Ground Applicator Exposure Studies with Lasso Herbicide 1982, Monsanto Final Report MSL-1889; EPA Accession No. 70591. Libich, S.; To, J.; Frank, R.; Sirons, G. Am. Ind. Hyg. Assoc. J . 1984, 45:56-62. Lavy, T. L. Project Completion Report to National Forest Products Association, August 30 to October 1978.

RECEIVED November 24, 1987

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 20

by

Quantitative Determination of Haloxyfop in Human Urine Gas Chromatography—Mass Spectrometry R. A. Campbell, P. E. Kastl, B. E. Kropscott, and M . J. Bartels

Analytical and Environmental Chemistry, Health and Environmental Sciences, Dow Chemical Company, Midland, MI 48674

An analytical method has been developed for the quantitativ low to sub pp human urine. This method could be used to determine potential worker exposure to VERDICT herbicide during typical product use. Urine samples containing haloxyfop were fortified with isotopically labeled haloxyfop, extracted using benzene, derivatized, cleaned-up via HPLC and analyzed by gas chromatography-mass spectrometry. The mean relative recovery (±S.D.) of haloxyfop from urine (test material vs. internal standard) for the 7 concentrations ranging from 100 ng/mL to 0.2 ng/mL, was 107±7%. The coefficient of variation for relative recovery from urine was 3% at the highest (100 ng/mL) concentration and 58% at the lowest (0.2 ng/mL) concentration. A stability evaluation showed haloxyfop to be stable in urine samples stored at room temperature for at least two weeks. H a l o x y f o p m e t h y l e s t e r ( I ) , [methyl 2-(4-((3-chlorq-5(trifluoromethyl)-2-pyridinyl)oxy)phenoxy)propionate], t h e a c t i v e i n g r e d i e n t i n VERDICT h e r b i c i d e f o r m u l a t i o n s , i s b e i n g e v a l u a t e d f o r r e g i s t r a t i o n as a h e r b i c i d e f o r e r a d i c a t i o n o f a n n u a l and p e r e n n i a l grasses. The method d e s c r i b e d i n t h i s r e p o r t was d e v e l o p e d u s i n g a r a c e m i c m i x t u r e o f h a l o x y f o p and does n o t d i f f e r e n t i a t e between t h e S and R f o r m s . 0097-6156/89/0382-0251$06.00/0 • 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

252

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

U n p u b l i s h e d d a t a show h a l o x y f o p m e t h y l e s t e r i s r a p i d l y h y d r o l y z e d t o t h e a c i d i n man. An a n a l y t i c a l method was r e q u i r e d t o d e t e r m i n e low l e v e l c o n c e n t r a t i o n s o f h a l o x y f o p i n human u r i n e . A method u s i n g an i s o t o p i c a l l y l a b e l e d (D/j-haloxyfop) i n t e r n a l s t a n d a r d and gas chromatography-mass s p e c t r o m e t r y w i t h m u l t i p l e i o n d e t e c t i o n (GC/MS MID) was d e v e l o p e d t o q u a n t i t a t i v e l y d e t e r m i n e h a l o x y f o p i n u r i n e as low as 0 . 2 ng/mL. T h i s p r o c e d u r e i n c l u d e s an a u t o m a t e d h i g h r e s o l u t i o n sample c l e a n u p u s i n g r e v e r s e d - p h a s e HPLC.

(I) MATERIALS AND

HALOXYFOP

METHODS

MATERIALS. H a l o x y f o p was s u p p l i e d by A g r i c u l t u r a l P r o d u c t s Department, The Dow C h e m i c a l Company. The chemical p u r i t y of the t e s t m a t e r i a l , a racemic m i x t u r e , was d e t e r m i n e d t o be ± 9 9 % by l i q u i d chromatography. I s o t o p i c a l l y labeled haloxyfop, (99.5% pure) f o r use as an i n t e r n a l s t a n d a r d was s y n t h e s i z e d u s i n g a deuterated intermediate [prepared by r e f l u x i n g w i t h D 2 S O 4 a t 9 5 ° C ] ( B a r t e l s , M. J . ; G a t l i n g , S. C. J . L a b e l l e d Compounds Radiopharm., i n p r e s s .) T h i s procedure r e s u l t e d i n a n e a r l y complete exchange o f d e u t e r i u m f o r t h e f o u r h y d r o g e n s on t h e phenyl (center) r i n g . D i s t i l l e d - i n - g l a s s benzene, m e t h a n o l , UV s p e c t r o p h o t o m e t r y g r a d e hexane, a c e t o n i t r i l e and c o n c e n t r a t e d h y d r o c h l o r i c a c i d were o b t a i n e d from F i s h e r S c i e n t i f i c ( F a i r Lawn, N J ) . WISP L i m i t e d Volume I n s e r t s f o r HPLC a n a l y s i s were o b t a i n e d from Waters D i v . ( M i l l i p o r e , M i l f o r d , MA). Conical GC/MS a u t o s a m p l e r v i a l s were o b t a i n from Wheaton S c i e n t i f i c ( M i l l v i l l e , NJ). Human u r i n e f o r method v a l i d a t i o n was o b t a i n e d from v o l u n t e e r s w i t h i n t h e T o x i c o l o g y Lab, The Dow C h e m i c a l Company. STANDARDS. S t o c k s o l u t i o n s A and B were p r e p a r e d by d i s s o l v i n g a c c u r a t e l y weighed amounts o f h a l o x y f o p and D 4 ~ h a l o x y f o p , r e s p e c t i v e l y , i n methanol. A l i q u o t s of e a c h o f t h e s e s t o c k s o l u t i o n s were combined and d i l u t e d i n methanol t o prepare a t h i r d s t o c k s o l u t i o n (C). E x t e r n a l s t a n d a r d s o l u t i o n s f o r t h e GC/MS were p r e p a r e d by m e t h y l a t i n g an a l i q u o t o f s t o c k s o l u t i o n C u s i n g diazomethane, e v a p o r a t i n g t o dryness under n i t r o g e n and t h e n d i l u t i n g w i t h hexane t o a c h i e v e t h e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

20. CAMPBELL ET AL.

Haloxyfop in Human Urine

desired concentration [i.e. ( 2 4 9 ng/mL h a l o x y f o p / 5 0 3 ng/mL i n t . s t d . ) t o ( 4 . 9 8 ng/mL h a l o x y f o p / 1 0 . 1 ng/mL int. std.)]. S o l u t i o n s f o r s p i k i n g u r i n e were p r e p a r e d by d i l u t i n g an a l i q u o t o f s t o c k s o l u t i o n C with methanol to y i e l d s o l u t i o n s of [ 2 . 4 9 JLlg/mL h a l o x y f o p / 5 . 0 3 Jlg/mL i n t . std.) to ( 0 . 2 4 9 jlg/mL h a l o x y f o p / 0 . 5 0 3 Jlg/mL i n t . s t d . ) . For recovery d e t e r m i n a t i o n s u r i n e s a m p l e s were s p i k e d w i t h 10-125 mL o f t h e a p p r o p r i a t e s p i k i n g s o l u t i o n p r i o r to extraction. E X T R A C T I O N METHOD AND H P L C C L E A N - U P . The appropriate amount o f h a l o x y f o p a n d i n t e r n a l s t a n d a r d were s p i k e d i n t o 30 mL o f u r i n e . C o n c e n t r a t e d HC1 (1 mL) was a d d e d t o t h e u r i n e a n d t h e s a m p l e was h e a t e d a t 80°C f o r 1 hour. A f t e r a l l o w i n g t o c o o l , 5 mL o f b e n z e n e was a d d e d t o t h e s a m p l s e c o n d s on a v o r t e x m i x e at 1500 rpm f o r 5 m i n u t e s . The b e n z e n e e x t r a c t was r e m o v e d b y p i p e t a n d a n a d d i t i o n a l 5 mL o f b e n z e n e was a d d e d t o t h e s a m p l e w h i c h was a g i t a t e d a n d c e n t r i f u g e d again. T h e s e c o n d b e n z e n e e x t r a c t was c o m b i n e d w i t h the f i r s t extract. An e t h e r e a l s o l u t i o n of d i a z o m e t h a n e was g e n e r a t e d u s i n g D i a z a l d (Aldrich C h e m i c a l Company, Inc., Milwaukee), and approximately 1 mL a d d e d t o e a c h s a m p l e . A f t e r complete methylation (-10 m i n . ) t h e s o l u t i o n s were e v a p o r a t e d u n d e r a gentle stream of nitrogen. T h e r e s i d u e was t h e n r e c o n s t i t u t e d i n d i e t h y l e t h e r and quantitatively t r a n s f e r r e d t o a WISP L i m i t e d V o l u m e I n s e r t . The e t h e r was e v a p o r a t e d u n d e r n i t r o g e n a n d t h e s a m p l e was r e c o n s t i t u t e d i n 125 |1L o f m e t h a n o l . For automated c l e a n - u p of the d e r i v a t i z e d urine extracts, a 100 JUL a l i q u o t was i n j e c t e d o n a r e v e r s e d p h a s e HPLC s y s t e m c o l l e c t i n g t h e f r a c t i o n (typically 3.75 t o 5.2 minutes) c o n t a i n i n g t h e h a l o x y f o p m e t h y l e s t e r and the c o - e l u t i n g i n t e r n a l s t a n d a r d peak. The c l e a n - u p was p e r f o r m e d u s i n g a W a t e r s R a d i a l C o m p r e s s i o n M o d u l e (RCM-100) a n d a NOVA-PAK c o l u m n . A g u a r d c o l u m n ( 2 . 5 4 cm x 3 . 9 mm) c o n t a i n i n g j l B o n d a p a k Cl8 ( 3 7 - 5 0 Jim) was u s e d f o r t h e p r o t e c t i o n o f t h e a n a l y t i c a l column. The HPLC c o n d i t i o n s were a s follows: autosampler: W a t e r s WISP ( M o d e l 5 1 0 B ) w i t h L i m i t e d Volume i n s e r t s ; D e t e c t o r : W a t e r s 4 8 1 (X= 227 nm)with s e n s i t i v i t y a t 0 . 0 1 A U F S ; pump: Waters 590; column: N O V A - P A K CIQ (10 cm x 5 mm i d ) ; f l o w r a t e : 1.0 mL/min; pressure: 1700 p s i ; i n j e c t i o n volume: 100 JUL; mobile phase: 70/30 a c e t o n i t r i l e / d i s t i l l e d water. A f r a c t i o n c o l l e c t o r f i t t e d w i t h an a i r a c t u a t e d v a l v e t h a t a l t e r n a t e d the column e f f l u e n t b e t w e e n w a s t e a n d a new c o l l e c t i o n v i a l was u s e d . The t i m i n g o f t h e v a l v e s w i t c h i n g was c o n t r o l l e d b y t h e W a t e r s 590 p u m p .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

253

254

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

T h e UV d e t e c t o r was u s e d o n l y f o r c h e c k i n g t h e r e t e n t i o n window o f s t a n d a r d s . Samples c o n t a i n e d h i g h l e v e l s o f UV a b s o r b i n g c o m p o n e n t s , t h u s f r a c t i o n s w e r e c o l l e c t e d b a s e d on r e t e n t i o n times. The 3 . 7 5 t o 5.2 m i n u t e f r a c t i o n o f t h e effluent f r o m t h e UV d e t e c t o r was c o l l e c t e d i n a 1 d r a m v i a l . 500 |IL o f h e x a n e was a d d e d t o t h e v i a l w h i c h was a g i t a t e d f o r 15 s e c o n d s o n a v o r t e x m i x e r a n d c e n t r i f u g e d a t 3000 rpm f o r 1 m i n u t e . The hexane (top) l a y e r was r e m o v e d a n d p l a c e d i n a 5 0 0 |1L conical vial. T h e s a m p l e was b l o w n t o d r y n e s s under n i t r o g e n a n d r e c o n s t i t u t e d i n 1 0 0 |1L o f h e x a n e prior t o i n j e c t i o n on t h e GC/MS. T h e 10 a n d 1 0 0 ng/|lL s p i k e d u r i n e e x t r a c t s were r e c o n s t i t u t e d i n 1.0 and 10.0 ml hexane, respectively. GAS CHROMATOGRAPHY-MAS GC/MS e q u i p p e d w i t h MAT, S a n J o s e , C A ) , a H e w l e t t P a c k a r d 7 6 7 3 a u t o s a m p l e r and o p e r a t e d i n t h e MID m o d e , was u s e d f o r quantitative analysis of f i n a l extracts. S e p a r a t i o n o f the h a l o x y f o p as i t s m e t h y l e s t e r f r o m i n t e r f e r i n g s p e c i e s i n t h e u r i n e was a c h i e v e d w i t h a 15 m x 0 . 3 2 mm i . d . D X - 3 c a p i l l a r y c o l u m n , 0 . 2 5 Jim f i l m t h i c k n e s s (J&W S c i e n t i f i c , I n c . F o l s o m , CA) . H e l i u m a t 11 p s i g was u s e d a s t h e c a r r i e r g a s . During t h e a n a l y s i s , t h e c o l u m n was m a i n t a i n e d a t a temperature of 220°C, the i n j e c t i o n port and i n t e r f a c e o v e n t e m p e r a t u r e s were 275 a n d 2 7 0 ° C , respectively. T h e i n j e c t i o n v o l u m e was 3 . 0 j l L . A modified fixedr a t i o s p l i t t i n g s y s t e m (1) was u s e d . A 10 cm x 0 . 1 mm f u s e d s i l i c a r e s t r i c t o r t u b e was s e a l e d i n t h e i n j e c t o r s p l i t v e n t on t h e f r o n t i n s t r u m e n t p a n e l . The s p l i t v a l v e was f u l l y o p e n e d , a l l o w i n g f l o w c o n t r o l b y the r e s t r i c t o r tube. T h e e f f l u e n t was v e n t e d t o a f u m e h o o d t h r o u g h 1/8 i n c h t e f l o n t u b i n g . T h e GC/MS was a l s o e q u i p p e d w i t h a r e d u c e d - p r e s s u r e interface (2). O t h e r MS o p e r a t i n g p a r a m e t e r s a r e a s follows: preamp: l O ^ amps/volt; e l e c t r o n energy: 70 e V ; emission current: 0 . 4 mA; m u l t i p l i e r v o l t a g e : 1.4 kV; source temperature: 150°C.; i n d i c a t e d p r e s s u r e : 5 x 10~6 t o r r . H a l o x y f o p was q u a n t i f i e d u s i n g internal s t a n d a r d t e c h n i q u e s by m o n i t o r i n g t h e m o l e c u l a r i o n s , m/z 3 7 5 f o r t h e m e t h y l e s t e r o f h a l o x y f o p a n d m/z 3 7 9 for the deuterated haloxyfop i n t e r n a l standard methyl ester. -

CALCULATIONS I n t e r n a l s t a n d a r d c a l c u l a t i o n s were u s e d i n t h i s analysis. The r e s p o n s e f a c t o r (Rf) f o r a s t a n d a r d c a l c u l a t e d from the f o l l o w i n g equation:

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

was

Rf

where

C

HAL

x

A

I S

A

HAL

X

C

I S

=

(1)

CJJ^L

compounds standard

and in

Cj5

ng/mL

are of

the

compounds The

GC/MS p e a k in

the

the

from

A

the

A

and

of

the

D4-internal

and A J S

are

the

obtained

for

these

L

of

the

recovery

of

spiked

urine

following

standard

haloxyfop

two

solutions.

vs. D 4 -

samples

was

calculated

equation: Rf

% Recovery

H

areas

analysis

relative

haloxyfop

concentrations

haloxyfop

respectively.

respective

from

255

Haloxyfop in Human Urine

20. CAMPBELL ET AL.

x

(A

-BKG)

x

Ng

=

x IS

?HAL

where N g j s i s t h e amount ( i n ng) o f t h e internal standard added to the sample. Ng^^L ^ amount (in ng) o f h a l o x y f o p s p i k e d i n t o t h e u r i n e s a m p l e . Rf is i s t h e mean o f t h e r e s p o n s e f a c t o r s o b t a i n e d f r o m t h e external standards. A and AJS peak a r e a s o b t a i n e d f o r h a l o x y f o p and D 4 - i n t e r n a l s t a n d a r d i n the a n a l y s i s of the sample. BKG i s t h e a v e r a g e o f the b a c k g r o u n d ( a t m/z 3 7 5 ) o b s e r v e d i n t h e a n a l y s i s of b l a n k u r i n e s a m p l e s , a d j u s t e d f o r any d i f f e r e n c e in d i l u t i o n f a c t o r between the c o n t r o l and s p i k e d u r i n e samples. s

H

A

a

L

r

e

t

n

t

n

e

e

The c o n c e n t r a t i o n o f h a l o x y f o p i n unknown u r i n e s a m p l e s was c a l c u l a t e d f r o m t h e f o l l o w i n g equation: Rf C

HAL

(A

H A L

-

BKG)

x

Ng

I

S

x

100

%

recovery

<) 3

AJS RESULTS

x

=

AND

x

volume

of

urine

(mL)

x

DISCUSSION

To d e t e r m i n e t h e r e l a t i v e r e c o v e r y f r o m u r i n e , s t a n d a r d s were p r e p a r e d b y a d d i n g a known amount of h a l o x y f o p a n d i n t e r n a l s t a n d a r d t o 30 mL o f urine. F u l l - s c a n mass s p e c t r a f o r t h o s e two compounds a r e s h o w n i n F i g u r e 1. U r i n e samples were a c i d i f i e d and h e a t e d f o r 1 hour at 80°C t o h y d r o l y z e any conjugates. The u r i n e s a m p l e s were t h e n e x t r a c t e d w i t h b e n z e n e , m e t h y l a t e d and r e c o n s t i t u t e d i n methanol p r i o r to c l e a n - u p by HPLC. The s a m p l e s were t h e n a n a l y z e d by GC/MS u s i n g m u l t i p l e i o n d e t e c t i o n . Relative recovery o f h a l o x y f o p f r o m u r i n e r a n g e d f r o m 120% t o 99% (mean = 1 0 7 ± 7 % ) f o r t h e c o n c e n t r a t i o n r a n g e o f 100 ng/mL t o 0 . 2 ng/mL (see T a b l e I ) . The c o e f f i c i e n t s o f

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

256

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

A) Mass Spectrum of Haloxyfop Methyl Ester >, 100. 0-i


50.

p3968.

O H

59

180

o DC

m/z

T

60

80

100

120

140

160

180

200

220

ioo. On

50. 0-

260 m/z

240

35C

hi 300

260

280

300

320

340

360

380

400

B) Mass Spectrum of D - Haloxyfop Methyl Ester 4

100. O n
r80i6. 180

59

c -

50.0H

6,6

160

94 78 11

m/z 60

80

199 213

jil i|lltojm i f f i ^ i t t i

100

3

120

140 3

100. On

2

160

180

200

•

220

0

292

379

50. 0264 " | — • — | - -

m/z

240

260

Figure 1.

280

r

300

320

340

360

Representative Mass

380

400

Spectra.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

20. CAMPBELL ETAL.

Haloxyfop in Human Urine

v a r i a t i o n f o r t h e highest and lowest urine concentrations w e r e 3 % a t t h e 1 0 0 ng/mL concentration a n d 5 8 % a t t h e 0.2 ng/mL concentration. HPLC p u r i f i c a t i o n o f t h e u r i n e e x t r a c t s w a s performed f o r two reasons. Without t h i s clean-up step t h e r e w e r e l a r g e i n t e r f e r e n c e s p r e s e n t i n t h e GC/MS p e a k f o r t h e u n l a b e l l e d h a l o x y f o p m e t h y l e s t e r (m/z 375). P u r i f i c a t i o n o f t h e s a m p l e s b y t h i s m e t h o d was a l s o found t o s i g n i f i c a n t l y extend t h e l i f e o f t h e f u s e d s i l i c a c a p i l l a r y GC c o l u m n . Another f e a t u r e o f t h e method d e s c r i b e d i n t h i s r e p o r t i s t h e u s e o f a r e d u c e d - p r e s s u r e GC/MS interface. This i n t e r f a c e , developed by Langvardt, et. a l (2), u t i l i z e s a computer-controlled solenoid v a l v e t o d i v e r t t h e m a j o r i t y o f t h e GC e f f l u e n t a w a y f r o m t h e mass s p e c t r o m e t e r s o u r c e ( F i g u r e 2 ) This modification minimize components e n t e r i n g the source l i f e . The i n c o r p o r a t i o n o f t h e s t a b l e - i s o t o p e - l a b e l l e d i n t e r n a l s t a n d a r d i n t o t h i s a s s a y was h i g h l y u s e f u l . Its use automatically corrected f o rloss o f t h e t e s t m a t e r i a l d u r i n g sample e x t r a c t i o n , d e r i v a t i z a t i o n a n d HPLC p u r i f i c a t i o n . I t also corrected for possible e r r o r s i n d i l u t i o n d u r i n g sample p r e p a r a t i o n , due t o the s m a l l sample volumes used i n t h i s assay. Finally, the d e u t e r a t e d h a l o x y f o p compensated f o r t h e v a r i a b i l i t y i n t h e a b s o l u t e r e s p o n s e o f t h e mass spectrometer over t h e course o f t h e a n a l y s i s . The r e l a t i v e r e c o v e r i e s o f 9 9 - 1 2 0 % f o r t h e D Q v s . D 4 ~ h a l o x y f o p (above) i n d i c a t e t h a t t h e r e i s no appreciable difference i nthe absolute recoveries f o r t h e s e t w o compounds f r o m u r i n e . The a b s o l u t e r e c o v e r i e s were n o t r o u t i n e l y c a l c u l a t e d b u t were e s t i m a t e d t o be l e s s t h a n 5 0 % . GC/MS c h r o m a t o g r a m s o f a b l a n k u r i n e e x t r a c t a n d a urine extract spiked a t t h e lowest concentration s t u d i e d a r e p r e s e n t e d i n F i g u r e 3. The r e t e n t i o n t i m e f o r h a l o x y f o p a n d t h e i n t e r n a l s t a n d a r d was 3 . 5 minutes. No p e a k s t h a t w o u l d i n t e r f e r e w i t h t h e a n a l y s i s were n o t e d i n e i t h e r t h e b l a n k hexane, o r standard solutions. H o w e v e r , t h e r e was a n i n t e r f e r e n c e s e e n a t m/z 3 7 5 i n c o n t r o l u r i n e s a m p l e s . To c o m p e n s a t e f o r t h i s i n t e r f e r e n c e , 9 c o n t r o l s a m p l e s w e r e a n a l y z e d a n d t h e a r e a r e s p o n s e f o r m/z 3 7 5 was averaged. This area average o f t h e background ( e q u i v a l e n t t o 0 . 0 2 ng/mL h a l o x y f o p ) w a s t h e n s u b t r a c t e d f r o m t h e h a l o x y f o p a r e a r e s p o n s e (m/z 3 7 5 ) i n t h e spiked u r i n e samples. However, i f t h e sample d i l u t i o n f a c t o r was g r e a t e r t h a n t h e c o n t r o l d i l u t i o n f a c t o r t h e n t h e b a c k g r o u n d a r e a was d i v i d e d b y t h e ratio of thedilution factors prior t o subtracting i t from t h e sample's area response. The d e t e c t i o n limit was d e f i n e d a s t h e mean b a c k g r o u n d r e s p o n s e p l u s three

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

258

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Table

I.

Recovery

Data

for

Haloxyfop

o. Recovery a I n d i v i d u a l Samples

Level (ng/mL)

104 116 97.9 116 124 40.9 161

100 10 1 0.8 0.6 0.4 0.2

Mean % Recovery

104 94 . 1 123 110 99.7 154 89.9

98.3 121 96.0 101 91.4 164 47.0

from

I n d i v i d u a l data p o i n t s not rounded f i g u r e s ; mean o f two injections.

a

ch c

Mean

data

^Average e

point

± standard

Average

standard

is

the

mean o f

Std. Dev.

b

102 110 106 109 105 120 99 707±7

3

C

to

Human

Urine

Coef. of Var.

3 14 15 8 17 68 58

3 13 14 7 16 57 58

35^

32^

significant

determinations.

deviation. deviation

coefficient

of

variation.

To Effluent-Divert Solenoid Valve

29 cm x 10(V/ Id fused silica tubing

GC/MS INTERFACE OVEN

12 cm — 0.7mm i.d.

30 cm * 200// id fused silica tubing 4-5mm gap to MS Ion Source

0.5 mm id

Interface Tee Custom-Made by S G E Using 1/16 in. G L T and 1/16 in. Swagelok Fittings

GC COLUMN OVEN End of Analytical Column 1/16 In. Swagelok® bulkheld union (drilled through)

1/16 In. o.d. * 0.5 mm I.d. G L T sleeve End of 100/i i.d. Restrictor Tube

All ferrules are 1/6 in. graphitized Vespel® (40%)

Analytical Column

Figure 2.

Reduced-Pressure G C / M S

Interface.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

20. CAMPBELL ETAL.

Haloxyfop in Human Urine

259

A) Blank Urine

500 4:10

520 4:20

540 4:30

560 b 4:40

580 4:50

600 Scan 5:00 T

i

m

e

B) Spiked Urine - 0.2 ng Haloxyfop / ml urine

Figure

3.

Representative

Selected

Ion

Chromatograms.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

260

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

times the standard d e v i a t i o n of the background response. The e x t e r n a l s t a n d a r d s o l u t i o n s and f o r t i f i e d u r i n e samples c o n t a i n e d f i x e d r a t i o s o f t e s t m a t e r i a l / i n t e r n a l standard. The GC/MS r e s p o n s e r a t i o (m/z 3 7 5 / 3 7 9) f o r t h e s e compounds was c o n s t a n t , r a n g i n g from 0 . 5 6 t o 0 . 6 0 (mean = 0 . 5 8 ± 0 . 0 2 ) f o r e x t e r n a l s t a n d a r d s o l u t i o n s i n t h e c o n c e n t r a t i o n range o f 2 4 9 / 5 0 3 ng/ml t o 4 . 9 8 / 1 0 . 1 ng/ml (D /D -haloxyfop), respectively. One a s s u m p t i o n made when q u a n t i t a t i n g h a l o x y f o p by t h i s method i s t h a t t h e r e l a t i v e r e s p o n s e o f t h e mass s p e c t r o m e t e r ( D ( ) / D 4 - h a l o x y f o p ) i s l i n e a r over the c o n c e n t r a t i o n s of the e x t e r n a l standard solutions. T h i s i s s u p p o r t e d by t h e c o n s i s t e n t response r a t i o s o b t a i n e d i n t h i s study. However, f u t u r e work w i t h t h i s method would be f u r t h e r s u p p o r t e d by t h e a d d i t i o c o n t a i n i n g f i x e d amount v a r y i n g l e v e l s of the t e s t m a t e r i a l , i n e i t h e r e x t e r n a l standard s o l u t i o n s or f o r t i f i e d c o n t r o l u r i n e samples. 0

4

During the course of a f i e l d study, i t i s l i k e l y t h a t a l l samples c o u l d not be a n a l y z e d immediately. T h e r e f o r e , s t a b i l i t y o f h a l o x y f o p i n u r i n e samples was i n v e s t i g a t e d t o d e t e r m i n e i f t h e samples c o u l d be s t o r e d u n t i l a n a l y s i s c o u l d be p e r f o r m e d . A stability e v a l u a t i o n showed h a l o x y f o p t o be s t a b l e i n u r i n e samples s t o r e d a t room t e m p e r a t u r e f o r a t l e a s t two weeks (data not shown). In summary, a method was d e v e l o p e d f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n o f h a l o x y f o p i n human u r i n e o v e r t h e c o n c e n t r a t i o n range o f 2 0 0 pg/ml t o 1 0 0 ng/ml. The method i n v o l v e d f o r t i f i c a t i o n o f u r i n e samples w i t h a d e u t e r i u m - l a b e l l e d h a l o x y f o p i n t e r n a l s t a n d a r d , h y d r o l y s i s o f a c i d - l a b i l e c o n j u g a t e s , HPLC p u r i f i c a t i o n of d e r i v a t i z e d urine e x t r a c t s , followed by GC/MS a n a l y s i s o f t h e m e t h y l e s t e r o f t h e t e s t material. The HPLC p u r i f i c a t i o n was r e q u i r e d t o o b t a i n samples w i t h o u t s i g n i f i c a n t l e v e l s o f interferences. The i n t e r n a l s t a n d a r d was employed t o c o r r e c t f o r 1) l o s s o f t e s t m a t e r i a l d u r i n g sample p r e p a r a t i o n and 2 ) v a r i a b i l i t y o f t h e mass s p e c t r o m e t e r d u r i n g sample a n a l y s i s . F i n a l l y , t h e MID GC/MS method, e m p l o y i n g a r e d u c e d - p r e s s u r e interface, was f o u n d t o be s u f f i c i e n t l y s e l e c t i v e and s e n s i t i v e f o r the d e t e r m i n a t i o n of haloxyfop at these l e v e l s . ACKNOWLEDGMENTS The a u t h o r s would l i k e t o acknowledge P a t r i c k L a n g v a r d t f o r h i s t e c h n i c a l g u i d a n c e and r e v i e w o f t h i s work, and t h a n k K a r e n F o s t e r and F r a n S t a f f o r d for t h e i r e f f o r t s i n preparing t h i s manuscript.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

20. CAMPBELL ET AL. Haloxyfop in Human Urine 261 LITERATURE CITED 1.

Nestrick, T. J., Lamparski, L. L. and Peters, T. L. (1983). Low-Splitting-Ratio Injector for Capillary Gas Chromatography. Anal. Chem. 55, 2009-2011.

2.

Langvardt, P. W., Shadoff, L. A. and Kastl, P. E. (1985). Proc. 33rd Conf.Amer. Soc. Mass Spectrom. 1985, pp 837-838. RECEIVED February 25, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 21

Enzyme Immunoassay for Aldicarb 1

2

3

James F. Brady , James R. Fleeker , Richard A. Wilson , and Ralph O. Mumma 1

1

Pesticide Research Laboratory, Department of Entomology, Pennsylvania State University, University Park, PA 16802 Biochemistry Department, North Dakota State University, Fargo, ND 58105 Department of Veterinary Science, Pennsylvania State University, 2

3

An enzyme immunoassay (EIA) has been developed. The assay is simple to perform, requires minimal sample prepara­ tion and time of analysis is less than 2.5 hr. The assay has been applied to a variety of sample types including citrus juice products, body fluids and water. The analyses demonstrated a linear response from 15.6 to 2000 ng of aldicarb with coefficients of determination typically exceeding 0.98. The least detectable dose was 0.3 parts per million (ppm). The enzyme immunoassay was characterized by high precision of measurement as coeffi­ cients of variation were less than 5%. The assay is highly specific to aldicarb and has no cross-reactivity with the sulfoxide or sulfone. The a p p l i c a t i o n o f i m m u n o c h e m i c a l t e c h n i q u e s t o t h e analys i s of a g r i c h e m i c a l s has expanded d r a m a t i c a l l y i n r e c e n t years (JL) . I m m u n o c h e m i c a l m e t h o d s o f f e r many a d v a n t a g e s i n c l u d i n g ease of a n a l y s i s , speed, s e n s i t i v i t y , specificity a n d c o s t e f f e c t i v e n e s s (2.) . More i m p o r t a n t l y , they offer an a l t e r n a t i v e means o f q u a n t i t a t i n g c h e m i c a l s whose w a t e r solubility, i n s t a b i l i t y at high temperature, or lack of s t r o n g u l t r a v i o l e t a b s o r b a n c e may r e n d e r c l a s s i c a l analytic a l approaches i n s e n s i t i v e or u n s u i t a b l e . Aldicarb (2-methyl-2-(methylthio)propionaldehyde 0(methyl-carbamoyl)-oxime) i s s u c h a compound. It i s modera t e l y s o l u b l e i n water (0.6% by w e i g h t ) , forms a n i t r i l e d e g r a d a t i o n p r o d u c t at h i g h temperatures and e x h i b i t s a w e a k u l t r a v i o l e t a b s o r b a n c e m a x i m a a t 247 nm. Classical

0097-6156/89/0382-0262$06.75/0 © 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21. BRADY ET AL.

Immunoassay for Aldicarb

263

a n a l y t i c a l techniques developed f o r a l d i c a r b are conse­ q u e n t l y cumbersome o r r e q u i r e s p e c i a l i z e d i n s t r u m e n t a t i o n . A sample i s u s u a l l y e x t r a c t e d w i t h an a c e t o n e - w a t e r s o l u ­ t i o n , o x i d i z e d with p e r a c e t i c a c i d t o the sulfone, cleaned up by p a s s a g e t h r o u g h a F l o r i s i l column, and a n a l y z e d by p y r o l y t i c g a s - l i q u i d chromatography (GLC) (3-5) . A l d i c a r b i s t h u s q u a n t i f i e d i n d i r e c t l y v i a measurement o f an o x i d a ­ t i o n p r o d u c t which, c o i n c i d e n t a l l y , i s a l s o a m e t a b o l i c d e g r a d a t i o n p r o d u c t . D i r e c t h i g h - p r e s s u r e l i q u i d chromato­ g r a p h i c (HPLC) t e c h n i q u e s f o r a l d i c a r b m o n i t o r t h e weak a b s o r p t i o n a t 247 o r 254 nm ( 6 - 8 ) . P o s t - c o l u m n d e r i v a t i z a ­ t i o n HPLC p r o c e d u r e s i n v o l v e h y d r o l y s i s o f a l d i c a r b w i t h base, d e r i v a t i z a t i o n o f t h e l i b e r a t e d methylamine w i t h o r t h o - p h t h a l a l d e h y d e and 2 - m e r c a p t o e t h a n o l , and subsequent a n a l y s i s o f t h e f l u o r e s c e n t p r o d u c t (9,10) . T h i s r e p o r t d e s c r i b e s t h e development o f an enzyme immunoassa f o r aldicarb. The a s s a y form, and r e q u i r e s m i n i m a M a t e r i a l s and Methods Apparatus. K i n e t i c s t u d i e s were p e r f o r m e d on a P e r k i n Elmer Lambda 3B UV/VIS s p e c t r o p h o t o m e t e r . The c u v e t t e was t h e r m o s t a t e d by a Haake A80 t e m p e r a t u r e c o n t r o l l e r . Assay p l a t e s were scanned on an A r t e k V e r t i c a l Beam s p e c t r o p h o ­ tometer. NMR s p e c t r a were o b t a i n e d on a V a r i a n 3 60 spectrophotometer. Reagents. A l l c h e m i c a l s u s e d were o f r e a g e n t grade quality. 4-Aminobutyric a c i d , i s o - b u t y l c h l o r o f o r m a t e , trans-4-(aminomethyl)cyclohexanecarboxylic acid, polyo x y e t h y l e n e s o r b i t a n m o n o l a u r a t e (Tween 20), and a l k a l i n e p h o s p h a t a s e , t y p e V I I - T , from b o v i n e i n t e s t i n a l mucosa, were o b t a i n e d from Sigma C h e m i c a l Co. R a b b i t gama g l o b u ­ l i n s , f r a c t i o n two, were p u r c h a s e d from M i l e s L a b o r a t o r i e s , Inc. HPLC g r a d e a c e t o n i t r i l e was p u r c h a s e d from J.T. Baker C h e m i c a l Co. 4 - N i t r o p h e n y l p h o s p h a t e and 1 - ( 3 - d i m e t h y l a m i n o p r o p y l ) - 3 - e t h y l c a r b o d i i m i d e h y d r o c h l o r i d e were o b t a i n e d from A l d r i c h C h e m i c a l Co., I n c . C a r n i t i n e was p u r c h a s e d from C a l b i o c h e m and c h o l i n e c h l o r i d e was o b t a i n e d from Merck & Co. A l d i c a r b , a l d i c a r b oxime, a l d i c a r b oxime s u l f o x i d e , a l d i c a r b s u l f o x i d e , a l d i c a r b s u l f o n e , methomyl, m e s u r o l and c y a n a z i n e r e f e r e n c e s t a n d a r d s were o b t a i n e d from t h e E n v i r o n m e n t a l P r o t e c t i o n Agency, Reno, NV. Water f o r HPLC a n a l y s i s was d i s t i l l e d and f u r t h e r p u r i f i e d on a M i l l i - Q r e a g e n t grade water system, M i l l i p o r e , I n c . S y n t h e s i s o f L i g a n d s . The p r e p a r a t i o n o f a l d i c a r b oxime and t h e c h l o r o f o r m a t e d e r i v a t i v e have been d e s c r i b e d by Payne e t a l . (11). A c o l d s o l u t i o n o f a l d i c a r b o x i d e c h l o r o f o r m a t e (40 mmole) i n 40 mL o f d i e t h y l e t h e r was added a l t e r n a t e l y w i t h 40 mmole NaOH i n 15 mL water t o a s t i r r e d and c o o l e d s o l u t i o n o f 42 mmole o f trans-4-(aminom e t h y l ) c y c l o h e x a n e c a r b o x y l i c a c i d and 43 mmole NaOH i n 20

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

264

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

mL w a t e r . T h e a d d i t i o n was c a r r i e d o u t o v e r a 20 m i n period. A f t e r t h e a d d i t i o n , s t i r r i n g was c o n t i n u e d a n o t h e r 45 m i n . T h e m i x t u r e was d i l u t e d w i t h 20 mL o f 2 M H C 1 a n d 25 mL d i e t h y l e t h e r and the l a y e r s s e p a r a t e d . The aqueous s o l u t i o n was e x t r a c t e d t w i c e a g a i n w i t h 25 mL p o r t i o n s of e t h e r and t h e combined e t h e r e x t r a c t s washed t w i c e with water. T h e e t h e r s o l u t i o n was d r i e d o v e r a n h y d r o u s N a 2 S 0 4 and t h e s o l v e n t removed w i t h a r o t a r y e v a p o r a t o r . The r e s i d u e was r e c r y s t a l l i z e d f r o m b e n z e n e - h e x a n e , m.p. 102105°C with decomposition. ^-H NMR: (90 M H z , C D C I 3 ) 8 0 . 6 - 2 . 8 [10H, m, C H i ] ; 1 . 5 [6H, s , C ( C H ) ] ; 2 . 0 [3H, s , CH3-S]; 3.1-3.4 [ 2 H , s ( b r o a d ) , C H - N ] ; 6 . 2 [1 H, s ( b r o a d ) , NH] ; 7 . 6 [ 1 H , s , C H = N ] ; 1 2 . 4 [ 1 H , s , C O O E ] . T h i s c o m p o u n d was labeled a l d i c a r b Ligand 1 (Figure 1). L i g a n d 2 ( F i g u r e 2) was p r e p a r e d f r o m 4-aminobutyric a c i d (GABA) a n d a l d i c a r manner as L i g a n d 1 an m.p. 116-117°C. -J-NMR: (90 M H z , a c e t o n e - d ) 5 1 . 5 [6H, s , C ( C H ) ] ; 2 . 0 [3H, s , C H 3 - S ] ; 1.8-2.1 [ 2 H , m, C H C H , C H ] ; 2.3-2.5 [2H, t , C H C O H ] ; 3 . 2 - 3 . 4 [2H, q , N H - C H ] ; 6 . 8 [1H, s ( b r o a d ) , N H ] ; 7 . 6 [1H, s , CH=N]; 1 2 . 0 [1H, s , COOH]. 6

0

3

2

2

6

3

2

2

2

2

2

2

P r e p a r a t i o n o f Immunogen. A l d i c a r b L i g a n d 1 was conjugated t o b o v i n e s e r u m a l b u m i n (BSA) b y a d a p t i n g t h e p r o c e d u r e d e s c r i b e d by E r l a n g e r et a l . (12) ( F i g u r e 1 ) . The ligand (2.25 m m o l e ) was d i s s o l v e d i n 15 mL d i o x a n e a n d 0 . 3 1 mL triethylamine. T h e s o l u t i o n was c o o l e d , d i l u t e d w i t h 0 . 3 mL o f i s o b u t y l c h l o r o f o r m a t e a n d s t i r r e d 20 m i n . This s u s p e n s i o n was a d d e d i n o n e p o r t i o n t o a s o l u t i o n o f 2 . 5 g BSA i n 65 mL w a t e r , 2 . 5 mL 1M NaOH a n d 65 mL d i o x a n e which was c o o l e d a n d s t i r r e d i n a n i c e w a t e r b a t h . A f t e r 2 min, 1 . 2 5 mL o f 1 M NaOH was a d d e d a n d s t i r r i n g c o n t i n u e d 4 h r . T h e s o l u t i o n was d i a l y z e d o v e r n i g h t i n r u n n i n g w a t e r a n d t h e pH a d j u s t e d t o 4 . 5 . A f t e r c o o l i n g 2 hr in i c e water, t h e p r e c i p i t a t e was c o l l e c t e d b y c e n t r i f u g a t i o n a n d t h e p e l l e t d i s s o l v e d i n 150 mL 1% N a H C 0 3 . T h e s o l u t i o n was d i l u t e d w i t h 1 L c o l d acetone and the p r e c i p i t a t e c o l l e c t e d by c e n t r i f u g a t i o n . T h e p e l l e t was s u s p e n d e d i n c o l d a c e t o n e , f i l t e r e d and washed w i t h a c e t o n e . T h e p e l l e t was then d r i e d , ground i n a mortar and s t o r e d d e s i c c a t e d at -20°C. Antibody Formation. The p o w d e r e d immunogen (50 mg) was s u s p e n d e d i n 1 . 5 mL o f 0 . 9 % (w/v) s t e r i l e N a C l and homogen i z e d i n 1 . 5 mL o f F r e u n d ' s c o m p l e t e a d j u v a n t . This prepar a t i o n was i n j e c t e d ( 0 . 5 mL) i n t o t h e t h i g h m u s c l e o f f e m a l e New Z e a l a n d w h i t e r a b b i t s . T h e i n j e c t i o n was repeated every 25-30 days, subcutaneously, at several sites on t h e b a c k . S e r u m was t r e a t e d w i t h a n e q u a l v o l u m e o f 70% s a t u r a t e d ammonium s u l f a t e a n d t h e y g l o b u l i n c o l l e c t e d by centrifugation. T h e y - g l o b u l i n p e l l e t was d i s s o l v e d i n o n e volume of water, d i a l y z e d a g a i n s t p h o s p h a t e - b u f f e r e d saline and freeze-dried.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21.

265

Immunoassay for Aldicarb

BRADY ETAL.

CH 0 0 i ii /v II CH -C-CH = N-0-C-CI + NHg-CHg-^v^r-C-OH 3

0

3

S 4

0^ 3

Aldicar chloroformat

I. iso-butyl chloroformate CH - C - CH = N - 0-C - NH - CH 3

2

-^N^-C-OH

S i CH

2. BSA

3

Ligand I

CH

3

0

0

CH -C-CH=N-0-C-NH-CH -^—^r-C-NH-BSA 3

2

S CH

3

Immunogen Figure 1. immunogen.

Synthesis

of

Ligand

1 and a l d i c a r b

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

266

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

CH

0

3

0

CH - C - C H = N - 0 - C - C I £

+ NH -(CHJ,-C-0H " z

QJ^

C

° ° Base

>

4 3

CH

9

Aldicar chloroformate

0

3

0

^

C-CH= N-O-C-NH - ( C H ^ - C - O H + CH3-C

Enzyme

c

: D

M

A

p

£

C

Ligand 2

CH

3

0

Cr^-C-CH»N-0-C-NH-(Cry

0 3

-C-NH-Enzyme

S 1

CH

3

Enzyme tracer

F i g u r e 2. tracer.

Synthesis

o f Ligand 2 and a l d i c a r b

enzyme

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21.

BRADY ET AL.

267

Immunoassay for Aldicarb

S y n t h e s i s o f Enzyme T r a c e r . A l d i c a r b L i g a n d 2 was c o n j u ­ g a t e d t o a l k a l i n e p h o s p h a t a s e by a method s i m i l a r t o t h a t o f W e i l e r (12.) ( F i g u r e 2 ) . A s o l u t i o n ( 2 0 0 JLIL) o f 1 0 \imol l i g a n d i n 1.0 mL DMF: H 2 O ( 1 : 1 , v/v) was added t o 2 0 0 |iL H 2 O , pH 6.4, i n a 5 mL v i a l i n an i c e b a t h w i t h s t i r r i n g . F o u r 2 5 |i.L a l i q u o t s o f 1 5 |imol 1 - ( d i m e t h y l a m i n o p r o p y l ) - 3 e t h y l c a r b o d i i m i d e h y d r o c h l o r i d e d i s s o l v e d i n 1 0 0 \iL H 2 O were added d r o p - w i s e t o t h e v i a l o v e r 2 min. The s o l u t i o n was a l l o w e d t o s t i r f o r 1 5 min a f t e r w h i c h f o u r 2 5 \iL a l i q u o t s o f a l k a l i n e p h o s p h a t a s e s t o c k s o l u t i o n ( 1 0 mg p r o t e i n p e r mL) were added d r o p - w i s e o v e r 2 min. The r e s u l t i n g c l o u d y s o l u t i o n was l e f t t o s t i r f o r 4 5 min. The s o l u t i o n and a 5 0 0 \iL H 2 O r i n s e were d i a l y z e d a g a i n s t 2 X 1 L o f T r i s - b u f f e r e d s a l i n e (TBS) ( 5 0 mM T r i s , 1 mM magnesium c h l o r i d e and 1 0 mM sodium c h l o r i d e ; pH a d j u s t e d t o 7 . 5 w i t h concentrated hydrochlori centrifuged at 1100 X transferred to a 5 m pelle further evaluation. C h a r a c t e r i z a t i o n o f Enzyme T r a c e r . The a c t i v i t y o f t h e enzyme t r a c e r was compared t o t h a t o f t h e s t o c k a l k a l i n e phosphatase s o l u t i o n . Enzyme a c t i v i t y was measured b y m o n i t o r i n g h y d r o l y s i s o f 4 - n i t r o p h e n o l p h o s p h a t e a t 4 0 5 nm ( A 4 0 5 ) , a n a b s o r b a n c e maxima o f 4 - n i t r o p h e n o x i d e anion. Enzyme was d i l u t e d i n b u f f e r ( 0 . 0 5 M c a r b o n a t e , 2 2 mM d i s o d i u m c a r b o n a t e , 2 8 mM sodium h y d r o g e n c a r b o n a t e and 1 mM magnesium c h l o r i d e , pH 9.8) t o y i e l d a change i n a b s o r b a n c e ( A A 4 0 5 ) o f a p p r o x i m a t e l y 1 . 0 p e r m i n u t e when i n c u b a t e d w i t h 5 mM s u b s t r a t e a t 3 0 ° C . To i n c u b a t i o n s c o n t a i n i n g 2 5 0 |iL o f t h e enzyme p r e p a r a ­ t i o n were added 2 5 0 \iL o f s u b s t r a t e i n b u f f e r . Reactions were m o n i t o r e d f o r f o u r m i n u t e s . E a c h enzyme d i l u t i o n and s u b s t r a t e c o n c e n t r a t i o n was measured t w i c e . Reaction v e l o c i t y was d e t e r m i n e d b y m e a s u r i n g t h e s l o p e o f t h e i n i t i a l l i n e a r p o r t i o n o f the absorbance-time graph. P r e v i o u s e x p e r i m e n t s showed t h a t n o n - e n z y m a t i c h y d r o l y s i s o f s u b s t r a t e d i d n o t a f f e c t a s s a y p e r f o r m a n c e under t h e s e conditions. S l o p e measurement, AA4os/min, were c o n v e r t e d t o v e l o c i t y u n i t s , mM/ng p r o t e i n / m i n , b y i n s e r t i o n o f t h e a b s o r b a n c e v a l u e s i n t o an e q u a t i o n r e g r e s s i n g a b s o r b a n c e s o n t o known c o n c e n t r a t i o n s o f 4 - n i t r o p h e n o l . The i n v e r s e s o f r e a c t i o n v e l o c i t i e s and s u b s t r a t e c o n c e n t r a t i o n s were g r a p h e d i n a d o u b l e r e c i p r o c a l p l o t from w h i c h t h e K and Vmax o f e a c h enzyme p r e p a r a t i o n were c a l c u l a t e d . m

Enzyme Immunoassay. D e t e r m i n a t i o n o f Reagent C o n c e n t r a ­ t i o n . A p p r o p r i a t e c o n c e n t r a t i o n s o f a n t i b o d y and enzyme t r a c e r were d e t e r m i n e d b y c h e c k e r b o a r d a s s a y (14.) . A l l s o l u t i o n s e x c l u d i n g enzyme s u b s t r a t e were c o o l e d t o 4°C p r i o r t o use. L y o p h i l i z e d a n t i s e r u m was r e c o n s t i t u t e d i n H 2 O and d i l u t e d i n b u f f e r ( 0 . 1 M sodium c a r b o n a t e , 3 2 mM d i s o d i u m c a r b o n a t e , 68 mM sodium h y d r o g e n c a r b o n a t e , a n d

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3 mM s o d i u m a z i d e , p H 9 . 6 ) . Adsorption of antiserum to i n d i v i d u a l w e l l s o f N u n c (A/S N u n c , R o s k i l d e , D e n m a r k ) 96w e l l f l a t b o t t o m p o l y s t y r e n e d i s p o s a b l e a s s a y p l a t e s was a c c o m p l i s h e d b y a d d i t i o n o f 1 . 2 5 , 2 . 5 , 5 , 10 a n d 20 ^ig a n t i b o d y i n 100 ^il 0 . 1 M c a r b o n a t e b u f f e r . T h e p l a t e was p l a c e d i n a c l o s e d c h a m b e r l i n e d w i t h wet p a p e r t o w e l s and incubated overnight (12 t o 15 h r ) a t 4 ° C . T h e following m o r n i n g t h e p l a t e ' s c o n t e n t s were removed by a s p i r a t i o n . All w e l l s were washed 3 X 5 m i n w i t h a Tween 2 0 - N a C l s o l u tion (27 g s o d i u m c h l o r i d e a n d 1 . 5 mL T w e e n 20 d i s s o l v e d in 3 L H2O). 50 \iL o f e n z y m e t r a c e r s e r i a l l y d i l u t e d i n T B S f r o m 1 0 - t o 1 6 0 - f o l d were added t o t h e p l a t e s w h i c h were then p l a c e d i n a r e f r i g e r a t o r and i n c u b a t e d at 4 ° C . After 30 m i n , t h e p l a t e s w e r e r e m o v e d f r o m t h e c o l d , aspirated and washed 2 X 5 m i n w i t h Tween 2 0 - N a C l . Substrate solution ( 1 0 0 M.L, 1 mg 4 - n i t r o p h e n y l p h o s p h a t e p e r m l o f 0 . 0 5 M carbonate buffer) at plate set aside to incubat r e a c t i o n was t e r m i n a t e d b y a d d i t i o n o f 50 [iL 1 M NaOH t o each w e l l . E x t e n t o f r e a c t i o n was m e a s u r e d s p e c t r o p h o t o m e t r i c a l l y a t 4 0 5 nm. Each combination of antibody and e n z y m e t r a c e r c o n c e n t r a t i o n was r e p l i c a t e d f o u r t i m e s . The l o w e s t c o n c e n t r a t i o n s o f a n t i b o d y a n d enzyme t r a c e r t h a t r e s u l t e d i n a mean A 4 0 5 o f a p p r o x i m a t e l y 1.3 were s e l e c t e d for use. P r i n c i p l e o f Immunoassay. Reagent c o n c e n t r a t i o n s determined by t h e above p r o c e d u r e were u s e d i n t h e competitive assay. Each s t a n d a r d and sample were run i n q u a d r u p l i c a t e . A n t i b o d y d i l u t e d i n 0 . 1 M c a r b o n a t e b u f f e r was a d s o r b e d t o the p l a t e as d e s c r i b e d above. O u t e r w e l l s were not u s e d due t o n o n - s p e c i f i c c o l o r d e v e l o p m e n t (15.) . Standard c u r v e s w e r e p r e p a r e d b y a d d i n g t o i n d i v i d u a l w e l l s 40 |iL H 2 O a n d 10 |iL o f a s t o c k s o l u t i o n o f a l d i c a r b d i s s o l v e d in acetonitrile (ACN) (1 mg/ml) a n d s e r i a l l y d i l u t e d i n H 2 O . This r e s u l t e d i n f i n a l concentrations of a l d i c a r b ranging f r o m 1 5 . 6 t o 2000 n g . Four wells r e c e i v e d zero c o n c e n t r a tion of aldicarb. An a n t i b o d y c o n t r o l , t o e v a l u a t e nons p e c i f i c b i n d i n g , was i n c l u d e d o n e a c h p l a t e . This cons i s t e d o f w e l l s c o a t e d w i t h non-immune r a b b i t y - g l o b u l i n to w h i c h n o a l d i c a r b was a d d e d d u r i n g t h e i n h i b i t i o n step. T h e p l a t e was i n c u b a t e d 30 m i n a t 4 ° C . A s o l u t i o n o f enzyme t r a c e r (50 |iL) was t h e n a d d e d t o e a c h w e l l a n d t h e p l a t e r e t u r n e d t o t h e c o l d f o r a n a d d i t i o n a l 30 m i n . After t h a t t i m e , c o n t e n t s o f t h e p l a t e were removed by a s p i r a t i o n a n d t h e p l a t e was w a s h e d . A s s a y o f b o u n d e n z y m e t r a c e r was c a r r i e d out as p r e v i o u s l y d e s c r i b e d . Absorption readings, were c o n v e r t e d t o ' P e r c e n t I n h i b i t i o n by t h e following equation: 1

A

Percent

Inhibition

=

[1 -

(

-

3 AQ

A

^ — ) -

]

x

100

A SB N

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21.

BRADY ETAL.

Immunoassay for Aldicarb

269

i n w h i c h As r e p r e s e n t s t h e A 4 0 5 ° i n d i v i d u a l sample or s t a n d a r d w e l l , A N S B r e p r e s e n t s t h e mean A 4 0 5 o f t h e n o n s p e c i f i c b i n d i n g c o n t r o l w e l l s a n d A Q r e p r e s e n t s t h e mean A 4 0 5 o f w e l l s c o a t e d w i t h a n t i - a l d i c a r b serum i n h i b i t e d by zero concentration of a l d i c a r b . These r e s u l t s were u s e d t o c a l c u l a t e the equation of a l i n e describing the relationship of percent i n h i b i t i o n (ordinate) to the logarithm of the concentration of a l d i c a r b (abscissa). T h i s l i n e was p l o t t e d on s e m i - l o g a r i t h m i c p a p e r w i t h e a c h c o n c e n t r a t i o n o f a l d i c a r b e x p r e s s e d as a s i n g l e p o i n t r e p r e s e n t i n g the mean p e r c e n t i n h i b i t i o n o f e a c h l e v e l p l u s o r m i n u s o n e standard deviation. The c o e f f i c i e n t o f v a r i a t i o n (CV) for each l e v e l and c o e f f i c i e n t of determination (r ) of each l i n e were a l s o d e t e r m i n e d . f

a

n

2

Assay S p e c i f i c i t y . The a b i l i t y o f t h e a n t i - a l d i c a r b a n t i body t o d i s c r i m i n a t e betwee w h i c h s h a r e s t r u c t u r a l s i m i l a r i t i e s was e v a l u a t e d . Each c h e m i c a l was t e s t e d a s a b o v e u s i n g t h e c o n c e n t r a t i o n r a n g e o f a l d i c a r b e x c e p t t h a t f o r some w a t e r r e p l a c e d ACN a s t h e carrier solvent. From t h e p e r c e n t i n h i b i t i o n d a t a o b t a i n e d in these experiments, the concentration of each chemical t h a t r e s u l t e d i n a 50% i n h i b i t i o n (I50) o f enzyme t r a c e r b i n d i n g was d e t e r m i n e d . Chemicals s e l e c t e d f o r s c r e e n i n g as p o t e n t i a l l y crossr e a c t i v e included the a l d i c a r b metabolites a l d i c a r b s u l f o x ide, a l d i c a r b s u l f o n e , a l d i c a r b oxime, and a l d i c a r b oxime sulfoxide. A g r i c h e m i c a l s t e s t e d w h i c h may b e p r e s e n t in samples i n c l u d e d the N-methyl carbamates methomyl and m e s u r o l as w e l l as a r e p r e s e n t a t i v e t r i a z i n e herbicide cyanazine. A c e t y l c h o l i n e and i t s h y d r o l y s i s product, c h o l i n e , w e r e e x a m i n e d s i n c e a l d i c a r b was d e s i g n e d a s a s t r u c t u r a l mimic of the n e u r o t r a n s m i t t e r (XI). A biochemi c a l o f s i m i l a r s t r u c t u r e , c a r n i t i n e , was a l s o s c r e e n e d . Sample P r e p a r a t i o n . Lemonade, l i m e a d e and orange juice f r o z e n c o n c e n t r a t e s were p u r c h a s e d from a l o c a l s u p e r m a r k e t and p r e p a r e d a c c o r d i n g t o l a b e l d i r e c t i o n s . A 2 mL a l i q u o t o f l e m o n a d e a n d l i m e a d e was a d j u s t e d t o p H 6 . 0 - 6 . 2 w i t h 2 M NaOH a n d f i l t e r e d w i t h a 0 . 8 pirn f i l t e r ( T y p e AA 25 mm, Millipore Filter Corp.). Two mL o f o r a n g e j u i c e was a d j u s t e d t o pH 6 . 0 - 6 . 2 b y a d d i t i o n o f a c o n c e n t r a t e d T r i s buffer (0.4 M T r i s , 1 mM m a g n e s i u m c h l o r i d e , a n d 10 mM s o d i u m c h l o r i d e a d j u s t e d t o p H 7 . 5 w i t h c o n c e n t r a t e d HC1) and s i m i l a r l y f i l t e r e d . F r e s h w a t e r m e l o n t i s s u e was c r u s h e d and the l i q u i d p r o c e s s e d s i m i l a r l y . Stream water was c o l l e c t e d f r o m a l o c a l s o u r c e a n d f i l t e r e d i n t h e same fashion. Human u r i n e was f i l t e r e d ( 0 . 8 jum) a n d s a l i v a was p r e p a r e d by p a s s i n g t h r o u g h a c e l l u l o s e a b s o r b e n t pad (Millipore Filter Corp.). R a b b i t s e r u m was c o l l e c t e d f r o m f e m a l e w h i t e New Z e a l a n d r a b b i t s a n d f i l t e r e d w i t h a 0 . 8 \im

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filter. P l a s m a was c o l l e c t e d f r o m S u f f o l k - D o r s e t s h e e p , p a s s e d t h r o u g h a 0 . 8 \im f i l t e r a n d d i l u t e d (1:1, v/v). A l l s a m p l e s were c o o l e d a t 4 ° C p r i o r Results

and

mix-breed w i t h TBS to assay.

Discussion

Assay Design. Design considerations i n c o r p o r a t e d i n t o the a l d i c a r b enzyme immunoassay i n c l u d e d t a k i n g a d v a n t a g e o f the s e n s i t i v i t y o f e n z y m e - l i n k e d immunosorbent assays (ELISA) w h i l e r e d u c i n g t i m e o f a n a l y s i s and sample handling. ELISAs d e v e l o p e d as a l t e r n a t i v e pesticide r e s i d u e methods (1) a r e h e t e r o g e n e o u s a s s a y s t y p i c a l l y r e q u i r i n g 18-22 h r t o c o m p l e t e e x c l u d i n g t h e 12-15 h r needed f o r a d s o r p t i o n of c o a t i n g a n t i g e n to the w e l l s of a microtitre plate. A n t i b o d y i n h i b i t i o n by sample or s t a n d a r d s o l u t i o n s i s c a r r i e d out i n s e p a r a t e v e s s e l s from which a l i q u o t s are subsequentl A s o l i d - p h a s e enzyme o f W e i l e r (12.) was d e v e l o p e d t o o v e r c o m e t h e s e p r o b l e m s . T h i s method i n v o l v e s c o a t i n g the w e l l s o f the m i c r o t i t r e p l a t e with antibody d i r e c t e d against the hapten of choice. The c o a t e d p l a t e s e r v e s as t h e r e a c t i o n v e s s e l f o r a l l s u b ­ sequent steps, e l i m i n a t i n g t r a n s f e r of s o l u t i o n s incubated' in other containers. Antibody i n h i b i t i o n is followed i m m e d i a t e l y b y i n c u b a t i o n o f an enzyme t r a c e r whose b i n d i n g i s m e a s u r e d by s p e c t r o p h o t o m e t r y e v a l u a t i o n o f an a p p r o ­ priate substrate. The n e t e f f e c t o f t h i s m e t h o d o l o g y i s t o reduce a n a l y s i s t i m e from t h a t o f a t y p i c a l E L I S A o f 18-22 h r t o l e s s than 2.5 h r . T h i s m e t h o d p e r m i t s a t e c h n i c i a n t o make two r u n s i n a s i n g l e e i g h t - h o u r w o r k d a y a n d a l l o w s u s e o f automated equipment and i n s t r u m e n t a t i o n a l r e a d y a v a i l a b l e for ELISA. In c o n s i d e r a t i o n o f t h e s e advantages, it is s u r p r i s i n g t h i s a p p r o a c h has not been w i d e l y a d a p t e d t o other immunological p e s t i c i d e r e s i d u e methods. H u b e r (1H) d e v e l o p e d an E I A f o r a t r a z i n e u t i l i z i n g a n t i b o d y - c o a t e d p l a t e s and had a t o t a l a n a l y s i s time of 4.5 h r . Schwalbe et a l . (16.) s y n t h e s i z e d a n e n z y m e t r a c e r f o r t h e analysis o f d i c l o f o p - m e t h y l and r a n homogeneous a s s a y s t h a t r e q u i r e d s e p a r a t i o n o f f r e e and bound hapten b e f o r e t r a n s f e r t o the microtitre plate. The a l d i c a r b enzyme immunoassay r e q u i r e s an a n t i a l d i c a r b a n t i b o d y a n d t h e s y n t h e s i s o f an enzyme t r a c e r . R a b b i t a n t i b o d i e s were d e v e l o p e d i n r e s p o n s e t o i n o c u l a t i o n o f an immunogen c o n s i s t i n g o f a l d i c a r b c o u p l e d t o BSA i n t h e f o r m o f L i g a n d 1, w h i c h m i m i c s t h e s t r u c t u r e o f aldicarb attached to a cyclohexanecarboxylic a c i d bridge. The enzyme t r a c e r , i n c o n t r a s t , c o n s i s t e d o f L i g a n d 2, i n c o r p o r a t i n g a GABA b r i d g e , c o n j u g a t e d t o a l k a l i n e phosphatase. The use o f l i g a n d s d i f f e r i n g i n geometry and l e n g t h p r e v e n t e d a n t i b o d y w h i c h may b e s p e c i f i c t o the

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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b r i d g i n g s t r u c t u r e o f t h e immunogen from b i n d i n g t o a s i m i l a r s t r u c t u r e on t h e enzyme t r a c e r . Antibody binding o f t h e enzyme t r a c e r was t h e r e f o r e r e s t r i c t e d t o t h e a l d i c a r b component o f t h e c o n j u g a t e d l i g a n d , e f f e c t i v e l y preventing non-specific tracer binding. S u c c e s s o f t h e enzyme immunoassay depends on s y n t h e s i s o f an a p p r o p r i a t e enzyme t r a c e r . T y p i c a l ELISAs u t i l i z e c o m m e r c i a l l y a v a i l a b l e s e c o n d a n t i b o d i e s bound t o an enzyme label. These l a b e l s r e q u i r e a two-hour i n c u b a t i o n , a d d i n g to the a n a l y s i s time. The enzyme t r a c e r u s e d i n t h i s a s s a y , i n c o m p a r i s o n , r e q u i r e d o n l y a 30-minute i n c u b a t i o n . S y n t h e s i s o f t h e t r a c e r was a s t r a i g h t - f o r w a r d application o f W e i l e r ' s p r o c e d u r e (12.) and, s u r p r i s i n g l y , d i d n o t s i g n i f i c a n t l y a f f e c t t h e enzyme's c a t a l y t i c p r o p e r t i e s ( F i g u r e 3) . The K and V o f t h e c o n j u g a t e and AP s t o c k s o l u t i o n were c a l c u l a t e d respectively t o be 2.95 and 2.65 mM and 76.8 and 85.4 a l k a l i n e phosphatase the c o n j u g a t i o n r e a c t i o n . The r e a g e n t a l s o p r o v e d t o have a l o n g s h e l f l i f e and showed no l o s s o f a c t i v i t y o v e r a s i x - m o n t h p e r i o d a t 4°C. T h i s o b s e r v a t i o n supports W e i l e r ' s c o n t e n t i o n t h a t a l a r g e b a t c h o f t r a c e r c o u l d be s y n t h e s i z e d a t a s i n g l e t i m e and u s e d w i t h c o n f i d e n c e f o r months a f t e r w a r d s . C o n c e n t r a t i o n s o f a n t i b o d y and enzyme t r a c e r f o r u s e i n t h e immunoassay were d e t e r m i n e d b y c h e c k e r b o a r d a s s a y (14.) . From t h i s t e s t , 2.5 \ig a n t i - a l d i c a r b a n t i b o d y and an 8 0 - f o l d d i l u t i o n o f enzyme t r a c e r s t o c k were s e l e c t e d . These c o n c e n t r a t i o n s r e s u l t e d i n a l d i c a r b - f r e e s o l u t i o n s p r o d u c i n g a maximal A 4 0 5 o f a p p r o x i m a t e l y 1.3 w i t h a l d i c a r b - f o r t i f i e d s o l u t i o n s p r o d u c i n g a b s o r b a n c e s down t o a p p r o x i m a t e l y 0.3 a t 2000 n g . The a l d i c a r b s t a n d a r d c u r v e ( F i g u r e 4) shows a t y p i c a l dose r e s p o n s e r e l a t i o n s h i p between 15.6-2000 ng o f aldicarb. The r e s p o n s e i s l i n e a r ( r = .980) a c r o s s t h r e e o r d e r s o f magnitude and measurement a t e a c h l e v e l i s p r e c i s e as i n d i c a t e d b y t h e c o e f f i c i e n t s o f v a r i a t i o n f o r a l l l e v e l s l e s s t h a n 3%. The l e a s t d e t e c t a b l e dose was 0.3 ppm w i t h a t i m e o f a n a l y s i s l e s s t h a n 2.5 h r . m

m a x

2

A s s a y S p e c i f i c i t y . I n h i b i t i o n s t u d i e s were c a r r i e d o u t t o determine the a b i l i t y o f the antibody p r e p a r a t i o n used i n t h i s s t u d y t o d i s c r i m i n a t e between a l d i c a r b and s i m i l a r l y s t r u c t u r e d compounds. A l d i c a r b , not s u r p r i s i n g l y , y i e l d e d t h e h i g h e s t p e r c e n t i n h i b i t i o n , 84.6%, and was c a l c u l a t e d t o have an I 5 0 o f 125 ng (Table I ) . A l d i c a r b s u l f o x i d e , however, d e m o n s t r a t e d no i n h i b i t i o n o f t h e a n t i b o d y what­ soever. Nor was any r e c o g n i t i o n o f a l d i c a r b s u l f o n e apparent. These f i n d i n g s a r e s i g n i f i c a n t n o t o n l y b e c a u s e of the i m p l i c a t i o n s f o r residue a n a l y s i s but a r e a l s o o f i n t e r e s t from an i m m u n o l o g i c a l s t a n d p o i n t . Aldicarb i s known t o degrade c h i e f l y t o i t s s u l f o x i d e i n c o t t o n (18,19) and s o i l (18,20) and f u r t h e r t o t h e s u l f o n e i n p o t a t o e s (21). F o r e a s e o f a n a l y s i s , s e v e r a l a n a l y t i c a l methods

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

272

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

-»

O

I

l/SUBSTRATE

2

J

4 1

C O N C E N T R A T I O N (mM)"

F i g u r e 3. Double r e c i p r o c a l p l o t s f o r the h y d r o l y s i s o f 4 - n i t r o p h e n y l p h o s p h a t e by a l k a l i n e p h o s p h a t a s e before (AP) a n d a f t e r ( A L D - A P ) c o n j u g a t i o n t o L i g a n d 2.

NANOGRAMS OF INHIBITOR Figure

4.

Enzyme

immunoassay

aldicarb

standard

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

curve.

21. BRADY ETAL.

273

Immunoassay for Aldicarb

Table I . P e r c e n t I n h i b i t i o n o f S e l e c t e d Chemicals i n the A l d i c a r b Enzyme Immunoassay

CHEMICAL NAME

STRUCTURE

PERCENT I N H I B I T I O N AT 2 0 0 0 NG

, 3

CH.-C-CH=N-0-C-NM-CH,

' 4

ALDC I ARB SULFOXIDE

CH-C-CH = N-0-C-NH- CH,

ALDICARB SULFONE

CH-C-CH«N-0-C-NH-CH 0=S = 0

3

3

5

ALDC I ARB OXM IE

CH,-C-CH-N-OH

ALDC I ARB OXM IE SULFOXD IE

CH.-IC-CH = N-OH 0=S = 0 4H, 5

CH-C=N-O-C-NH-CH,

CH,

0

> 2 0 0 0 NG

-

H-S-^ ^-0-C-NH-CH CM, CI

S

/^N^NH-CH-CH NHC-C-NH 2

S

ACETYLCHOLINE

CHS -N —CIH —z CH.-OH , CH-

CARNITINE

OH O I I 0 CH-N-CH -CH-CH-C-0 ®l

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

274

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

(3,4) have clumped t h e t h r e e m e t a b o l i c s p e c i e s t o g e t h e r as 'total toxic residue (22.), a consequence o f sample t r e a t ­ ment by o x i d a t i o n w i t h a p e r a c i d . However, i n f o r m a t i o n about t h e r e l a t i v e amounts o f e a c h s p e c i e s i s l o s t t h r o u g h such an a p p r o a c h . P o s s e s s i n g the i n a b i l i t y t o r e c o g n i z e e i t h e r o x i d a t i o n p r o d u c t , t h e immunoassay can d e r i v e i n f o r ­ m a t i o n about t h e amount o f p a r e n t p e s t i c i d e i n a sample. Work t o d e v e l o p an a n t i b o d y t o d e t e c t t h e s u l f o n e i s c u r r e n t l y underway. From an i m m u n o l o g i c a l v i e w p o i n t , s u c h s p e c i f i c i t y can be t r a c e d t o t h e s t r u c t u r e o f t h e immunogen. The t h i o m e t h y l group was p o i s e d i n an immunodominant p o s i t i o n , d i s t a l t o the s u r f a c e of the c a r r i e r p r o t e i n . Thus, any s t r u c t u r a l change i n a l d i c a r b a t t h i s p o i n t would be e x p e c t e d t o b r i n g about a d e c r e a s e i n a n t i b o d y r e c o g n i t i o n . However, t h e complet eliminatio f bindin followin th i n c l u s i o n of a s i n g l This e f f e c t i s als oxime s u l f o x i d e t o be r e c o g n i z e d , a l t h o u g h a l d i c a r b oxime i n h i b i t e d t h e b i n d i n g o f t h e enzyme t r a c e r 42%. The oxime has t h e i d e n t i c a l s t r u c t u r e o f a l d i c a r b e x c l u d i n g t h e Nm e t h y l group and t h e c a r b o n y l f u n c t i o n a l i t y . E x c e p t i n g an a c c i d e n t a l i n d u s t r i a l r e l e a s e (2_2.) , i t has not been f o u n d i n n a t u r e and would not be e x p e c t e d t o i n t e r f e r e w i t h a s s a y performance. Other a g r i c h e m i c a l s t e s t e d a l s o f a i l e d t o d i s p l a y a n t i b o d y r e c o g n i t i o n ( T a b l e I ) . Methomyl and m e s u r o l a r e b o t h N-methyl carbamates and e a c h c o n t a i n s a t h i o m e t h y l group, but t h e i r i n a b i l i t y t o b i n d s u g g e s t s t h a t n e i t h e r t h e carbamate l i n k a g e n o r t h e t h i o m e t h y l group by themselves are s u f f i c i e n t t o warrant c r o s s - r e a c t i v i t y . Cyanazine i s a t y p i c a l t r i a z i n e h e r b i c i d e with three side chains. However, t h e N - e t h y l group o r t h e c h a i n c o n t a i n i n g a t e t r a h e d r a l c a r b o n bound t o two m e t h y l groups and a cyano group d i d not c o n t r i b u t e t o a n t i b o d y i n h i b i t i o n a t any level tested. I t i s i n t e r e s t i n g t o note t h a t f a i l u r e of t h e l a t t e r c h a i n t o e l i c i t r e c o g n i t i o n i n l i g h t o f Payne e t al.'s observation (UL) t h a t an i d e n t i c a l group s u b s t i t u t e d i n t o a l d i c a r b i n p l a c e o f t h e t h i o m e t h y l a c t u a l l y enhanced some i n s e c t i c i d a l p r o p e r t i e s . Thus, t h e a n t i c h o l i n e s t e r a s e a c t i v i t y and a n t i b o d y r e c o g n i t i o n o f t h e cyano and t h i o m e t h y l f u n c t i o n a l i t i e s do not c o r r e l a t e . A l d i c a r b was o r i g i n a l l y d e s i g n e d as a s t r u c t u r a l mimic o f a c e t y l c h o l i n e i n an attempt t o i n c r e a s e t h e a c e t y l ­ c h o l i n e s t e r a s e i n h i b i t i o n o f a r y l carbamates (1_1) . T h e r e ­ f o r e , t h e a b i l i t y o f a c e t y l c h o l i n e and i t s h y d r o l y s i s p r o d u c t , c h o l i n e , t o b i n d t o a n t i - a l d i c a r b a n t i b o d y were examined. B o t h compounds showed a s m a l l d e g r e e o f i n h i b i ­ t i o n (13.8%) s u g g e s t i n g some c o m p l e m e n t a r i t y o f f i t w i t h the a n t i b o d y combining s i t e . However, b o t h c h e m i c a l s i n h i b i t e d 9% l e s s a t t h e h i g h e s t l e v e l t e s t e d (2000 ng) t h a n d i d t h e l o w e s t l e v e l (15.6 ng) o f a l d i c a r b u s e d so n e i t h e r m a t e r i a l should a f f e c t assay r e l i a b i l i t y . A bio­ c h e m i c a l o f s i m i l a r s t r u c t u r e , c a r n i t i n e , was a l s o t e s t e d . 1

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21. BRADY ET AL.

Immunoassay for Aldicarb

275

No c r o s s - r e a c t i v i t y was o b s e r v e d a t a n y l e v e l , p e r h a p s d u e t o t h e p r e s e n c e o f t h e h y d r o x y g r o u p on t h e ( J - c a r b o n . A p l o t to the percent i n h i b i t i o n of a l d i c a r b r e l a t i v e to the p e r c e n t i n h i b i t i o n of a l d i c a r b oxime, a c e t y l c h o l i n e , and c h o l i n e o v e r t h e same r a n g e o f c o n c e n t r a t i o n s i s s h o w n i n F i g u r e 5. A p p l i c a t i o n s . T h e a l d i c a r b e n z y m e i m m u n o a s s a y was a p p l i e d t o a v a r i e t y of sample t y p e s . S a m p l e s i n c l u d e d human a n d a n i m a l f l u i d s w h i c h may b e a n a l y z e d t o e v a l u a t e a l d i c a r b exposure, j u i c e products d e r i v e d from crops to which a l d i c a r b i s a p p l i e d and stream water r e p r e s e n t a t i v e of water samples examined t o monitor e n v i r o n m e n t a l dispersal o f t h e compound. M i n i m a l s a m p l e p r e p a r a t i o n was r e q u i r e d , typically i n v o l v i n g p a s s a g e t h r o u g h a 0 . 8 pjn f i l t e r . The n e c e s s i t y o f m a i n t a i n i n g s a m p l e p H a t a t h r e s h o l d l e v e l was i n d i c a t e d by e a r l y experiment showed t h e a n t i b o d y p r e p a r a t i o t i o n s h a v i n g a pH l o w e r t h a n 6 . 0 . A d j u s t m e n t t o pH 6 . 0 - 6 . 2 b y t h e a d d i t i o n o f e i t h e r 2 M NaOH o r a s p e c i a l T r i s buffer c o n t a i n i n g e i g h t t i m e s t h e amount o f b a s e e l i m i n a t e d t h i s problem. To a s s e s s t h e l i n e a r i t y o f r e s p o n s e o v e r a r a n g e of f o r t i f i c a t i o n s , samples were s p i k e d w i t h 1 5 . 6 t o 2 0 0 0 ng of a l d i c a r b . R e g r e s s i o n l i n e s and c o e f f i c i e n t s of determ i n a t i o n were c a l c u l a t e d from t h e s e d a t a . Each f o r t i f i c a t i o n l e v e l was r u n i n q u a d r u p l i c a t e s o t h e c o e f f i c i e n t o f v a r i a t i o n c o u l d be d e t e r m i n e d . A l d i c a r b i s a p p l i e d to the s o i l , u s u a l l y at time of planting. Uptake by a p l a n t ' s r o o t system r e n d e r s the entire plant toxic to potential pests. However, irrigation and r a i n f a l l tend t o enhance m i g r a t i o n of the p e s t i c i d e through sandy or a c i d i c s o i l s . As a r e s u l t , t h e l e a c h i n g o f a l d i c a r b i n t o d r i n k i n g w a t e r s u p p l i e s has become a w i d e s p r e a d c o n c e r n , p a r t i c u l a r l y i n F l o r i d a (ZA) , S u f f o l k C o u n t y , New Y o r k ( 2 5 , 2 6 ) , W i s c o n s i n ( 2 7 , 2 8 ) a n d E a s t e r n Canada ( 2 £ ) . The p r i n c i p a l a n a l y t i c a l p r o c e d u r e s f o r t h e a n a l y s i s o f a l d i c a r b i n w a t e r a r e i n d i r e c t HPLC methods ( 9 , 1 0 ) a n d a GLC t e c h n i q u e r e q u i r i n g s a m p l e o x i d a t i o n p r i o r to injection . These techniques are not w i d e l y a p p l i e d , h o w e v e r , s i n c e t h e HPLC m e t h o d r e q u i r e s a s u b s t a n t i a l c a p i t a l investment in dedicated instrumentation and t h e GLC t e c h n i q u e i n v o l v e s e x t e n s i v e m a n i p u l a t i o n o f t h e sample. In c o n t r a s t , enzyme immunoassay o f fortified s t r e a m w a t e r ( F i g u r e 6 ) was s i m p l e t o p e r f o r m a n d , except f o r f i l t e r i n g , d i d not n e c e s s i t a t e sample m o d i f i c a t i o n nor pretreatment. The a s s a y c o u l d d e t e c t a l d i c a r b a t a c o n c e n t r a t i o n o f 0 . 3 ppm a n d d e m o n s t r a t e d h i g h l i n e a r i t y of response (r = . 9 9 5 ) w i t h CVs f o r a l l l e v e l s approximately 5%. Sample c o n c e n t r a t i o n by s o l i d phase e x t r a c t i o n could increase sensitivity even f u r t h e r ( £ ) , m a k i n g enzyme immunoassay an a t t r a c t i v e a n a l y t i c a l alternative. Sampling of body f l u i d s f o r the e s t i m a t i o n of p e s t i c i d e e x p o s u r e can be r e g a r d e d as i n t r u s i v e o r n o n intrusive, d e p e n d i n g upon whether o r not t h e b l o o d s t r e a m o f 2

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

276

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

I

I

I I I 11

§0

O - Aldicorb V - Aldicorb oxime

TO

• - Choline

z o

E

• - Acetylcholine

9 0

CD X

5 eo

o «o

cc u

so so -

1000

100

NANOGRAMS OF INHIBITOR F i g u r e 5. E n z y m e i m m u n o a s s a y i n h i b i t i o n c u r v e s of a l d i c a r b , a l d i c a r b oxime, c h o l i n e and a c e t y l c h o l i n e .

I I I MM

Aldicorb in Streom Woter y » 22.6 LogX-9.87

o

r » 0.995

P to S

2

X

2 >o UJ O 4 0 CC u CL 10

tao

10

1000

100

NANOGRAMS OF INHIBITOR Figure stream

6. Enzyme water.

immunoassay

of

aldicarb

fortified

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21. BRADY ET AL.

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a s u b j e c t must be a c c e s s e d . Contamination of the blood i s determined through a n a l y s i s of plasma or serum. Enzyme immunoassay o f sheep plasma y i e l d e d a l i n e a r response (r = .982) o v e r t h e r a n g e t e s t e d w i t h C V s a t e a c h l e v e l typically 5% o r l e s s ( F i g u r e 7 ) . S i n c e t h e p l a s m a was d i l u t e d (1:1, v/v) with Tris buffer, the l e a s t detectable l e v e l was 0.6 p p m . A s s a y o f r a b b i t serum gave a p p r o x i m a t e l y t h e same r e s p o n s e (r = .969) w i t h C V s l e s s t h a n 6% a n d a d e t e c t i o n l i m i t o f 0.3 ppm ( F i g u r e 8 ) . N o n - i n t r u s i v e s a m p l e s i n c l u d e d human s a l i v a a n d u r i n e Immunoassay o f s a l i v a gave a l i n e a r r e s p o n s e a s i n d i c a t e d by a h i g h c o e f f i c i e n t o f d e t e r m i n a t i o n (r = .990) ( F i g u r e 9). T h e d e t e c t i o n l i m i t was 0.3 ppm a n d C V s w e r e 4% o r less. A n a l y s i s of u r i n e by EIA y i e l d e d a l i n e a r response (r = .983), s m a l l v a r i a t i o n o f m e a s u r e m e n t p e r fortification level (CVs l e s s t h a n and a s e n s i t i v i t y of ppm ( F i g u r e 1 0 ) . Body f l u i d matrices tested. T h i s c o m p l e x i t y was r e f l e c t e d t w o w a y s . First, points representing individual fortification levels a r e somewhat d i s p e r s e d a b o u t t h e f i t t e d r e g r e s s i o n lines. However, t h e s e c o e f f i c i e n t s s t i l l i m p l y s t r o n g dose response r e l a t i o n s h i p s . Second, the length of substrate i n c u b a t i o n f o r the a n a l y s i s o f plasma and serum needed t o b e i n c r e a s e d b y a n a d d i t i o n a l 30 m i n t o e n h a n c e c o l o r development such that absorbance values f e l l w i t h i n the r a n g e o f a p p r o x i m a t e l y 1.3 t o 0.3. This increased overall a n a l y s i s time to s l i g h t l y l e s s than 3 hr, but the a d d i t i o n a l t i m e d i d not i n v o l v e any e x t r a m a n i p u l a t i o n . These r e s u l t s p o i n t to the p o t e n t i a l u t i l i t y of the a l d i c a r b enzyme immunoassay as a t o o l t o m o n i t o r exposure of p e s t i c i d e a p p l i c a t o r s and p r o v i d e a n a l y t i c a l results w i t h i n a meaningful time frame. S a l i v a or urine, for e x a m p l e , may b e c o l l e c t e d a t t h e e n d o f a w o r k d a y a n d e x p o s u r e d a t a c o u l d be a v a i l a b l e t h e f o l l o w i n g morning. EIA a n a l y s i s of j u i c e products n e c e s s i t a t e d adjustment o f s a m p l e p H t o 6.0-6.2 a s p r e v i o u s l y d e s c r i b e d . pH a d j u s t m e n t c o n s i s t e d o f t h e a d d i t i o n o f 10 |iL o f 2 M NaOH or a c o n c e n t r a t e d T r i s b u f f e r and had a s m a l l dilution e f f e c t upon t h e s a m p l e s , r e d u c i n g s e n s i t i v i t y f r o m 0.3 t o 0.38 p p m . A n a l y s i s o f l e m o n a d e s a m p l e s was l i n e a r o v e r t h e e n t i r e range of f o r t i f i c a t i o n (r = .990) a n d C V s f o r e a c h l e v e l w e r e 4% o r l e s s ( F i g u r e 1 1 ) . Immunoassay o f limeade samples y i e l d e d s i m i l a r responses with a high c o e f f i c i e n t of determination (r = .993) o v e r t h e c o n c e n t r a t i o n s exami n e d a n d low CVs w i t h o n l y one l e v e l (125 n g ) e x c e e d i n g 4% (Figure 12). T i t r a t i o n of orange j u i c e samples with 2 M NaOH i n d i c a t e d t o o h i g h a c o n c e n t r a t i o n o f a c i d s t o b e c o u n t e r a c t e d w i t h 10 |iL o f b a s e s o a c o n c e n t r a t e d T r i s buffer (pH 7.5) was u t i l i z e d i n i t s p l a c e . Analysis of t h e s e b u f f e r e d o r a n g e j u i c e s a m p l e s was l i n e a r f r o m 200031.3 n g ( r = .988) a n d e x h i b i t e d h i g h p r e c i s i o n o f measurement ( a l l C V s l e s s t h a n 4%) ( F i g u r e 1 3 ) . The least d e t e c t a b l e c o n c e n t r a t i o n was 0.75 p p m . In a s i m i l a r fashion, linearity of watermelon samples (r = .993) 2

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In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21. BRADY ETAL.

Immunoassay for Aldicarb

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In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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e x t e n d e d a c r o s s t h e same r a n g e w i t h a s e n s i t i v i t y of 0.75 ppm ( F i g u r e 1 4 ) . T h e c o e f f i c i e n t o f v a r i a t i o n f o r all l e v e l s d i d n o t e x c e e d 5%. Although not r e g i s t e r e d f o r use on w a t e r m e l o n s , the i l l e g a l a p p l i c a t i o n of a l d i c a r b to t h i s c r o p i n 1 9 8 5 i n C a l i f o r n i a (21.) was w i d e l y p u b l i c i z e d . L i t e r a l l y t h o u s a n d s o f watermelons were s a m p l e d t o determine the extent of contamination (22). The coste f f e c t i v e n e s s o f enzyme immunoassay w o u l d h a v e b e e n d r a m a t i c a l l y demonstrated i n such a s i t u a t i o n . The expense i n c u r r e d by e x t e n s i v e s a m p l i n g c o u l d have been sharply r e d u c e d by p r i o r s c r e e n i n g o f t h e samples by EIA followed by t h e a p p l i c a t i o n of c l a s s i c a l p r o c e d u r e s t o s u s p e c t samples. Conclusion The enzyme immunoassa applicable to a variet to perform and e x h i b i t r a p i d time of a n a l y s i s . A microt i t r e p l a t e adsorbed w i t h a n t i b o d y serves as the reaction v e s s e l for a l l steps in the assay reducing time of analysis while e l i m i n a t i n g the t r a n s f e r of s o l u t i o n s incubated in other vessels. The l e a s t d e t e c t a b l e a l d i c a r b concentration was 0 . 3 p p m . Lower s e n s i t i v i t i e s c a n be a c h i e v e d by solidphase e x t r a c t i o n of a l d i c a r b i n the water. P r e c i s i o n of m e a s u r e m e n t was h i g h w i t h c o e f f i c i e n t s o f v a r i a t i o n for all fortification levels typically 5% o r l e s s . T h i s work s u g g e s t s t h a t EIA c o u l d p r o v e u s e f u l for human o r a n i m a l e x p o s u r e s t u d i e s a n d f o r s c r e e n i n g o f a g r i c u l t u r a l crops for a l d i c a r b contamination. T h i s enzyme immunoassay i s h i g h l y s e l e c t i v e t o a l d i c a r b and does not d e m o n s t r a t e any c r o s s r e a c t i v i t y to the sulfoxide or sulfone. Immunoassay f o r d e t e c t i o n o f t h e s u l f o x i d e or s u l f o n e would r e q u i r e development of a n t i b o d i e s s p e c i f i c to the oxidation products. Acknowledgments S u p p o r t e d i n p a r t by N o r t h e a s t e r n R e g i o n a l R e s e a r c h P r o j e c t NE-115 and R e g i o n a l R e s e a r c h Funds, and t h e N o r t h Dakota A g r i c u l t u r a l Experiment Station.

Literature Cited 1.

2. 3.

Mumma, R.O.; Brady, J . F . In Pesticide Science and Biotechnology; Greenhalgh, R.; Roberts, T . R . , eds.; Blackwell Scientific Publications: Oxford, Great Britain, 1987; pp. 341-348. Hammock, B.D.; Mumma, R.O. ACS Symposium Series 1980, 136, 321-352. Andrawes, N.R.; Romine, R.R.; Bagley, W.P. J . Agric. Food Chem. 1973, 21, 379-386.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

21. BRADY ET AL. Immunoassay for Aldicarb

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

283

Union Carbide Corporation. "A Method for the Deter­ mination of Total Toxic Aldicarb Residues in Agricul­ tural Crops by Gas Chromatography," (May, 1973). Dorough, H.W.; Thorstenson, J . H . J . Chromatog. Sci. 1975, 13, 212-222. Cochrane, W.P.; Lanouette, M. J . Assoc. Off. Anal. Chem. 1981, 64, 724-728. Cochrane, W.P.; Lanouette, M. J . Chromatog. 1982, 243, 307-314. Spalik, J.; Janauer, G . E . ; Lau, M.; Lemley, A . T . J. Chromatog. 1982, 253, 289-294. H i l l , K.M.; Holowell, R.H.; DalCortivo, L . A . Anal. Chem. 1984, 56, 2465-2468. Union Carbide Agricultural Products Co., Inc. "A Method for Determination of Aldicarb Residues in Water by HPLC," (February Payne, L . K . ; Stansbury Agric. Chem. 1966, 14, 356-365. Erlanger, B . F . ; Borek, F . ; Beiser, S.M.; Lieberman, S. J . B i o l . Chem. 1957, 228, 713-727. Weiler, E.W.; Jourdan, P.S.; Conrad, W. Planta 1981, 153, 561-571. Voller, A . ; Bidwell, D . E . ; Bartlett, W. Bull. W.H.O. 1976, 53, 55-56. Clark, M.F.; Adams, A.N. J . Chem. V i r o l . 1977, 34, 475-483. Huber, S . J . Chemosphere 1985, 14, 1795-1803. Schwalbe, M.; Dorn, E.; Beyermann, K. J . Agric. Food Chem. 1984, 32, 734-741. Coppedge, J . R . ; Lindquist, D.A.; Bull, D . L . ; Dorough, H.W. J . Agric. Food Chem. 1967, 15, 902-910. Bull, D.L. J . Econ. Entomol. 1968, 61, 1598-1602. Andrawes, N.R.; Bagley, W.P.; Herret, R.A. J . Agric. Food Chem. 1971, 19, 727-730. Andrawes, N.R.; Bagley, W.P.; Herret, R.A. J . Agric. Food Chem. 1971, 19, 731-737. Richey, F . A . ; Bartley, W.J.; Sheets, K.P. J . Agric. Food Chem. 1977, 25, 47-51. The New York Times 1985, Aug. 12: 1(6) 12(1-3). Foran, J . A . ; Miller, W.L.; Doyan, S.; Krtausch, M. Environ. Pollut. (Series A: Enol. Biol.) 1986, 40, 369-380. Zaki, M.H.; Moran, D.; Harris, D. Am. J. Public Health 1982, 72, 1391-1395. Zaki, M.H. Northeast. Environ. Sci. 1986, 15-22. Rothschild, E.R.; Manser, R . J . ; Anderson, M.P. Ground Water 1982, 20, 437-445. K r i l l , R.M.; Sonzogni, W.C. J . Am. Water Works Assoc. 1986, 78, 70-75.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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29.

Priddle, M.W.; Jackson, R . E . ; Novakowski, K . S . ; Denhoed, S.; Graham, B.W.; Patterson, R . J . ; Chaput, D.; Jardine, D. Water Pollut. Res. J . Can. 1987, 22, 173-186. Lemley, A . T . ; Zhong, W.Z. J . Agric. Food Chem. 1984, 22, 714-719. Chem. Eng. News 1985, 63 (28), 3-4. Agri-Chemical Issues. Union Carbide Agric. Products Co., Inc., Vol. 1(1) (1985).

30. 31. 32.

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29, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 22 Mixer—Loader—Applicator Exposure and Percutaneous Absorption Studies Involving EPTC Herbicide Safety Related to Exposure 1

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James B. Knaak , M . A. Al-Bayati , O. G. Raabe , J. L . Wiedmann , J . W. Pensyl , J. H. Ross , A. P. Leber , and P. Jones 3

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State of California Department of Health Services, Sacramento, CA 95814 Institute for Environmental Health Resources University of California 2

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PPG Industries, Barberton, OH 44203 Pan-Ag, Fresno, CA 93711 4

The skin penetration potential of EPTC was evaluated by the topical application of [propyl-1- C]-EPTC (specific activity, 1.67 mCi/mmole) in water to the clipped back skin (22.9 ug/cm , 25 cm and 21.5 ug/cm , 2.54 cm ) of adult male Sprague-Dawley rats. All glass metabolism cages were used to collect respiratory CO while special skin mounted XAD-2 resin traps were used to collect C-volatiles. Approximately 14.7% of the applied dose was absorbed and/or excreted during 24 hours post adminis­ tration. The remaining dose evaporated (77.8%) or remained on the surface of the skin (1.3—2.8%). Less than 1% was recovered as respired CO . In a separate field study, a mixer/loader/applicator was monitored while applying a PPG brand of EPTC, GENEP 7EC. Air samples, cloth patches, hand rinses, and urine samples were obtained and analyzed for EPTC or metabolites. Total absorbed dose was determined to be 5.6 mg/day (0.074 mg/kg of b.w.) for a 75 kg man applying EPTC to 120 acres of soil/day. On the basis of a percutaneous absorption rate in the rat of 14.7%/24 hours, a worker exposure of 0.074 mg/kg/day of b.w., and a NOEL of 5.0 mg/kg/day from animal toxicity studies, margins of safety of 68 to 340 were determined for workers applying EPTC herbicide. 14

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G E N E P EPTC 7 EC, containing the a c t i v e ingredient, S-ethyl-N,N,-dipropyl thiocarbamate, is registered by PPG Industries, Inc. for use on vegetable and field crops as a preplant herbicide requiring soil incorporation. Toxicity studies conducted with EPTC in the rat indicated that this material can induce early appearance of degenerative heart disease and hind limb paresis frequently as late-in-life changes in this animal. The mouse and dog were refractory to these effects. The finding of a dose related effect in the rat necessitated the development of mixer/loader/applicator exposure and rat dermal absorption data for determining the margin of safety afforded agricultural workers using this chemical. The field portion of the mixer/loader/applicator study was carried out in Chico, California by Pan-Ag, Fresno. Field samples were analyzed at PPG Industries, Inc. The rat dermal absorption studies were conducted at LEHR, University of California, Davis, C.A. c

0097-6156/89/0382-0288$06.00/0 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

22. KNAAK ET AL.

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Percutaneous Absorption of EPTC Herbicide All-Glass Metabolism Cage Study. Young adult male Sprague-Dawley rats weighing 280 g were purchased from Bantin-Kingman, Fremont,C.A.Three rats were prepared for dosing one day prior to treatment according to the procedure of Knaak et al. (1). The animals were individually weighed, and hair removed from their backs using an electric clipper. An area representing 7% of the body surface (25 cm ) was marked off with a rubber template glued to the skin. A Queen Anne collar made of polyethylene sheeting (1.0 mm in thickness, 11.25 cm in diameter) with a neck opening of 2.5 cm in diameter, was placed around the neck of each animal and fastened with staples to prevent the animals from grooming treated areas. An aqueous dosing suspension (500 uL, water emulsion of EPTC) containing 574 ug of [propyl-l-%:]-EPTC (19,400 dpm/ug) was applied using a digital microliter pipet. Following topical application, each animal was immediately placed in an all-glass metabolism cage (Figure 1; Metabolic Cages Series 600 Vangard International Inc., Neptune, NJ.) for the tory carbon dioxide, and i^C-volatile column (drying tube) containing 1.5 g of washed Amberlite XAD-2 resin (10/50 mesh) was placed between the bottom male joint of the feeder arm (joint normally used for feed residue flask) and the CO2 adsorption tower to collect [propyl-l- C] -EPTC lost from skin by evaporation. Approximately 150 mL of 2.0 N KOH was used in the CO2 absorption tower (Medinsky, 2) to collect ^C02 resulting from the metabolism of radiolabeled EPTC. A house vacuum system supplied air at the rate of 600 mL/ minute. Cage flow was split after the XAD-2 adsorption column using two critical flow orifices. Calibrations were made using spirometer volume techniques to determine the actual flow through each orifice. Upstream pressure was monitored to assure constant flow through the system during the experiment. The split allowed a 2:1 separation of all gases leaving the XAD-2 adsorption column. Two-thirds of the total flow was removed through the by-pass orifice, while one-third of the total air volume passed directly through the KOH C 0 2 adsorption tower. This system allowed for total organic vapor collection and 24 hr CO2 quantification without saturating the adsorption media. The animals were sacrificed 24 hr after the topical application of the dose. Each animal was anesthetized with sodium pentothal (70 mg/kg LP.). Skin was removed at the application site and blood collected by cardiac puncture using a disposable 5.0 mL heparinized syringe, 20 gauge needle and a 10 mL BD Vacutainer (heparin). A midline chest incision was made to expose the heart for proper placement of the needle. Heart, liver, kidney, gastrointestinal tract (GIT), fat and the remaining carcass were taken and frozen at 0°C until they were analyzed for radioactivity. Urine, feces, l^C-volatiles XAD-2 resin and C C b in 2N KOH were also collected. Excised skin was stretched across a four-inch funnel and washed with 100 mL of soapy water. Washed skins were frozen to await further analysis. 2

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Skin-Depot/XAD-2 Absorption Study. Two studies were performed using young adult male Sprague-Dawley rats to assess the fate of 1 C-EPTC applied to the skin, volatilization, retention on or in skin, or passage through the skin to internal organs and/or excretion. The first study was a pilot study using three-431 g rats. The second study used four-280 g rats. One day prior to treatment, the hair on the back was clipped between the shoulder blades, and the circular aluminum base (Figure 2) of a skin-depot (Susten et al., .3) was glued (cyanoacrylate glue) and sutured to back skin while the animal was under anesthesia. 4

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Figure 1. All-Glass Metabolism Cage equipped with (1) silica gel absorption column, (2) ascarite/C02 absorption column, (3) glass cage, (6) KOH trap, and (7) silica gel absorption column. This configuration was modified by replacing the vacuum line between the KOH trap and the urine collection device with a vacuum line connected to the feeder side arm (4) via a hose bib (5). An XAD-2 resin absorption column was placed in the line after the feeder side arm. A T was placed in the line between the XAD-2 trap and the KOH trap. The open end of the T was connected to vacuum.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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KNAAK ET AL.

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Figure 2. Aluminum Skin-Depot consisting of base (threaded, male) and XAD-2 resin absorption column (threaded, female) equipped with two fine mesh screens and a retainer ring.

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Twenty-four hours after the operation, the skin area inside the aluminum base (1.8 cm in diameter, 2.54 cm ) was treated with 50 uL of aqueous dosing suspension (emulsion of EPTC and water). In studies 1 and 2, 91.8 and 54.7 ug, respectively, of [propyl-l- C] -EPTC were applied to the skin of rats inside the aluminum base. A specially designed adsorption column containing 1.0 g of XAD-2 resin (Figure 2) was immediately screwed onto the base and the animals individually placed in stainless steel metabolism cages for the complete and separate collection of urine and feces. The animals were sacrificed 24 hr after dosing, and tissues [skin at the application site, blood, heart, liver, kidney, fat, gastrointestinal tract (GIT), and carcass] were taken using the procedure previously described for the animals placed in the all-glass metabolism cages. The skin-depot (base and absorption column) and skin at the application site were removed prior to collecting blood and other tissues. The XAD-2 resin was removed from the absorption column and slurried in 25 mL of ethyl acetate, while the base and column were washed with an equal volume of the solvent. Excised skin was washed with 25 mL of soapy water and frozen to await further work. 2

14

l^C-Analysis. All collected Knaak et al. (1) in a Packard conversion and quench correction. Sealed quench standards were used for standardiza­ tion. Aliquots of liquid samples (plasma, skin surface washings, skin extracts, urine, skin-depot (base/absorption column/resin washings) washings, and KOH] were counted directly in scintillation fluid. Carcasses, GIT, feces, and skins were frozen in dry ice and individually ground in a Model ED-5 Wiley Mill. After extraction with 50 mL of methanol followed by 50 mL of acetone, the ground skins were air dried and reground in the Wiley Mill. Aliquots of carcass, GIT, heart, liver, fat, feces, and kidney (50 mg) were combusted wet, while the dried ground skins were combusted dry in a Harvey Oxidizer, Model OX-300. The resulting CO2 was trapped in 15 mL of the Harvey CO2 absorbent-scintillation fluid and counted. Calculations. The count data were analyzed on an individual animal basis utilizing a C-Calc Spread Sheet Program (Data General) to convert DPM to ug of EPTC herbicide and to calculate the percentage of the applied dose recovered. Worker Exposure Study with EPTC Herbicide Mixing/Loading/Application. The worker exposure study was conducted at Chico, CA during a commercial application of GENEP EPTC to 281 acres prepared for planting red beans. One man, 27 years of age, weighing 75 kg mixed/loaded and applied EPTC herbicide. The mixing operation consisted of hand pouring GENEP 7EC from a 5 gal container to a 2Vi gal clear plastic measuring device. The measured volumes were then poured into a 150 gal spray tank and diluted with water. The mixer/loader wore rubber gloves during this operation. The application equipment consisted of a Farmall 504 high clearance tractor, a 150 gal rear mounted fiber glass spray tank, a rolling cultivator mounted between the tractor wheels, and a power takeoff (PTO) driven sprayer (6 sets of double flat fan nozzles directed to the front and to the rear) mounted in front of the rolling cultivator. Diluted pesticide (23 lb A.I.) was applied to 7 acres at the rate of 3.3 lbs A.I. per acre in 20 gals of water. Exposure was monitored during mixing/loading (15 replicates) and application (15 replicates) of 23 lb. A.I. Separate data were obtained for each batch of EPTC herbicide applied.

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22. KNAAKETAL.

Percutaneous Absorption Studies

Dermal Exposure. For each monitoring replicate, gauze monitoring patches (23.75 cm ) were applied to 11 regions of the body as per Popendorf (4). Ten of the patches were placed on the outside of the clothing and one was placed inside. The monitoring sites are given in Table I. The feet were covered by boots and were not monitored during the study. Hand washes were used to monitor residues on the hands for the mixer/loader with gloves and the applicator without gloves. The worker wore short sleeve shirts, long pants, baseball cap, and boots. Patches were applied prior to mixing/ loading, removed after this operation was completed and new patches applied prior to application of EPTC. Upon completion of the application of EPTC, the patches were removed. Patches were placed individually in 2 oz wide-mouth bottles and frozen to await analysis. Hands were individually washed in 200 mL of water containing surfactant. The wash water was then stored frozen to await analysis. 2

Table I. Positions of Monitoring Sites Used to Represent

Represented Region of the Body Face Front of neck Back of neck Head Shoulders Forearms Hands Chest Back Hips Thighs Calves Feeti/

V V

1/ 4/

Surface Area (cm ) 650 128 100 430 2210 2190 1310 1520 1520 1730 3420 2570 1220 2

Monitoring Site!/ Chest at throat (outside) Chest at throat (outside) Back (outside) Back (outside)?/ Shoulders (outside)?/'!/ Forearm (outside)!/ Handwash Chest (inside)!/ Back (outside)?/ Thighs (outside)?./'!/ Thighs (outside)^/'!/ Shin (outside)?/ J / Nonei/

Inside represents a patch on the worker's skin and outside represents a patch on the outside of clothing. 20% of outside patch values were used as estimate for inside patch value since no corresponding inside values were available. Average of 2 patches. Negligible exposure: not included in the calculation.

Inhalation Exposure. Air sampling was performed continuously in the breathing zone of the worker during mixing/loading and application using charcoal tubes and a Bendix personal monitoring pump operating at 1 L/min. The respiration volume of the mixer/ loader-applicator was assumed to be 25 L/min (Guyton, _5). The mixing/loading operation required 10 to 15 minutes, while application required a minimum of 200 minutes. Urine Monitoring. Urine samples, twenty-one 68 to 319 mL volumes, were collected during the course of the study from the mixer/loader/applicator, immediately frozen, and stored in a freezer to await analysis for S-(N,N-dipropyl carbamoyl) cysteine and S-(N,N-dipropyl carbamoyl)-N-acetylcysteine (Hubbell and Casida,j6).

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Analysis of Gauze Pads. Pads placed in 2 oz wide-mouth bottles in the field were transferred to the laboratory and extracted in situ by shaking for one minute with 40 mL of methanol. After shaking, 20 mL were removed, evaporated to 1.0 mL using a rotary evaporator after the addition of 2.0 mL of internal standard (3 ug/mL azobenzene in 2-propanol), and analyzed by gas chromatography equipped with a thermionic detector. Chromatography was performed on a 30m x 0.25 mm I.D. OV-1701 column with a film thickness of 0.25 um. Column temperature was pro­ grammed from 100° to 150°C at a rate of 5°C/min. Column temperature was initially held for 2 min at 100°C with a final hold time of 20 min. The injection port temperature was 200°C and the detector temperature 300°C. Helium flow rates were set at 1.29 mL/min for carrier, 5.9 mL/min for splitter, and 30 mL/min for make up. Hydrogen was supplied to the detector at 3.8 mL/min and air at 200 mL/min. The injection volume was 1 uL and the split ratio 4.6:1. EPTC and azobenzene had a retention time of 9.13 and 16.78 minutes, respectively. Preliminary data on the recovery of EPTC from cloth patches were obtained prior to the field study by spiking four patches with 0.995 ug of EPTC. Extraction with methanol recovered 88 slightly larger amount (108% 4 hrs, at 70° F and then extracted with methanol. Smaller amounts, however, were recovered when patches were left in the open air after spiking, 71% recovery was obtained for the patch left in the open for 2V2 hrs, while 54% was recovered from a patch left in the open for 4 hrs. Losses from these patches averaged 12% per hr. Eight gauze patches were spiked with 1.09 ug EPTC during the course of the worker expo­ sure study. The recoveries ranged from 57 to 111% with a mean of 78% and a standard deviation of 20%. The patch data (ug EPTC/23.75 cm ) was corrected for recovery and normalized to regional body surface area on the basis of each batch of GENEP applied. A protection value of 80% for clothing was used to adjust the apparent exposure to 20% of the externally monitored value. Exposed areas such as the neck, face and forearm were not adjusted by a protection factor. The adjusted values were then multiplied by the skin adsorption factor (14.7%) determined in the rat percutaneous absorption study. 2

Analysis of Hand Rinses. Hand water rinses containing surfactant (200 ml) were analyzed by high performance liquid chromatography using a 25 cm x 4.6 mm I.D. Zorbax ODS column, 70% methanol and 30% water, and a flow rate of 2.0 mL/min. A 20 uL sample of the rinse water was injected. Detector wavelength was set at 225 nm. A Hewlett-Packard Laboratory Automation System was used to quantify the samples. Calibration was performed using a standard solution containing 4.372 ug/mL of EPTC in water. Samples were spiked in the field with 219 ug of EPTC. Eight recovery extractions had a mean recovery of 80% with a range of 71—101% and a standard deviation of 10%. The spikes were stored for the same time intervals as the samples. The hand rinse data was corrected for recovery and multiplied by 14.7% to determine the absorbed dose. Analysis of Air Samples. The air samples were collected by drawing air through activated charcoal tubes. The EPTC was desorbed (7) from the charcoal with 2 mL of CS2 and analyzed for EPTC using a gas chromatograph equipped with a Supelco Wax 10 capillary column and a flame ionization detector. Storage stability data were obtained from tubes stored for 25 days at 0°C, 23°C and 33°C. Mean EPTC recoveries for three tubes at each temperature were 92.1, 93.3 and 91.8% respectively. The ability of activated charcoal to retain EPTC was determined by passing 0.35 L/min of air through the tube for a period of 2 hrs after 20 mg was adsorbed on the charcoal. No EPTC was detected in the opposite end of the tube. Recoveries of 82 and 91.3% were obtained at 80 and 45% relative humidity, respectively, using 9.37 ug of EPTC. Additional validation was obtained by spiking charcoal tubes in the field during the

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22. KNAAK ET AL.

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295

study with 54 ug of EPTC. The mean recovery was 91% and the standard deviation 5.3%. The EPTC values obtained from the charcoal tubes were corrected for recovery. Inhalation exposure estimates were calculated using the NACA recommended method (Mull and McCarthy, 8). These estimates were reduced by 50% for inhalation exposure on the basis of lung retention data recently developed (9) for a variety of volatile organics. Analysis of Urine Samples. The S-(N,N-dipropylcarbamoyl) cysteine metabolite and its acetylated derivative in 1.0 mL of urine were acetylated in a 15 ml centrifuge tube using 25 mL of 0.1 N NaOH and 25 uL of acetic anhydride for one hr at room tem­ perature. The reaction mixture was adjusted to pH 10 using 250 uL of 10% NaOH and extracted with methylene chloride (2 x 2.0 mL.). The organic phase was discarded, the water phase acidified to pH 3 with 0.5 mL of IN HC1 and reextracted with methylene chloride (2 x 2.0 mL.). The methylene chloride was evaporated off and the N-acetyl derivative was dissolved in 0.5 mL of acetonitrile and methylated with diazo­ methane. The unreacted methylating reagent was removed by evaporation under a gentle stream of nitrogen an 3 uL volume gas chromatographe Scientific, Rancho Cordova, CA) using a thermionic detector. Column temperature was programmed from 80° to 200° C at a rate of 30°C/min. Column temperature was initially held for 2 min. at 80°C with a final hold time of 20 min. The injection port temperature was 200°C and detector temperature 300°C. Flow rate was set at 0.9 mL/min for helium carrier and 25 mL/min for nitrogen makeup. Hydrogen was supplied to the detector at 3.8 mL/min and air at 200 mL/min. The acetylation/methylation method was validated by adding known amounts of the cysteine and N-acetylcysteine metabolites to control urines. The results were compared to an analytical sample of S-(N,N-dipropylcarbamoyl)-N-acetylmethyl cysteine. Results were confirmed using GC/MS analysis. Two urine samples were spiked with each of the two urinary metabolites (10). Recovery of the S-(N,N-dipropylcarbamoyl)-N-acetylcysteine averaged 89%. The S-(N,N-dipropylcarbamoyl) cysteine, which is acetylated prior to analysis, averaged 74% recovery. The mean for all recoveries was 81%. The quantity of EPTC equiva­ lents (ug) found in each urine collection was determined by multiplying the ratio of the molecular weights of EPTC to S-(N,N-dipropyl carbamoy)-N-acetyl cysteine by the ug of this metabolite found in urine. According to Hubbell and Casida (6), in rats this metabolite comprises 28% of the absorbed dose (40% of the absorbed dose was eliminated in urine over a 24 hr period; 70% of the urinary metabolites were identified as the cysteine or N-acetyl derivative). Safety Margins Margins of safety were calculated using an estimated NOEL (no-observable-effect-level) of 5.0 mg/kg/day for myocardial degeneration in a two-year rat chronic/oncogenicity study (1JL), a daily absorbed dose of 0.074 mg/kg/day and a lifetime average exposure of 0.0017 mg/kg/day determined in this study from patch and inhalation data. A margin of safety was also determined using a daily absorbed dose of 0.0147 mg/kg/day estimated from urinary metabolites. This was accomplished by dividing the NOEL in mg/kg/day by the estimated absorbed dosages in mg/kg/day. Results Table II gives the average recovery for the C02/glass metabolism cage study and the two skin-depot studies. According to the recovery data from the three studies, [propyl-l- C] -EPTC rapidly evaporated from the skin and was trapped by the XAD-2 resin. In the glass metabolism cage study, the volatilized EPTC was partially recovered 14

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

on the XAD-2 resin with the remaining EPTC adsorbed to the sides of the glass cage or was absorbed by urine in the collection funnel. Recovery studies were performed prior to conducting the studies, utilizing [ propyl-1- C] -EPTC applied to glass wool in the center of the cage. The studies indicated that the XAD-2 resin adsorbed only 20% of the EPTC evaporating from the glass wool. The remaining radioactivity was partially recovered on the walls of the glass metabolism cage. The poor recovery necessitated the development and use of the skin-depot containing XAD-2 resin. The use of the skin-depot ultimately resulted in the total recovery of 95.3% of the applied dose with 77.8% being recovered on the XAD-2 resin and skin-depot. Approxi­ mately 85.9% of the dose was recovered in the pilot study with 69.8% recovered on the XAD-2 resin and parts of the skin-depot. Similar quantities, 2.63 and 2.77% of the dose, were found on the surface of the skin after 24 hr in the two skin-depot studies. The tissues (heart, liver, kidney, and fat), skin extracts, extracted skin (bound residues), urine, and feces contained in the three studies an equivalent percentage of the topically applied dose. In the two skin-depot studies, 13.7 and 14.7%, respectively, of the topically applied doses were absorbed by the skin 14

Table II. Dermal Absorption of [Propyl-l- C] -EPTC in Young Male Rats One Day Post-Treatment

Sample Type XAD-2 resin (1) Skin-depot (2) Evaporated (1 + 2) Skin surface (H2O) Skin extract (internal) Skin combustion (Bound) Plasma Kidneys Liver Heart GIT Fat Carcass Feces Urine

co

2

Total Recovered

Percentage of Topical Dose Skin-Depot Skin-Depot Glass Cag(: XAD-2/Pilot XAD-2/Study (n = 4) (n = 3) (n=3)

53.14

63.51 6.33 69.84 2.36 1.08 1.81 0.00 0.09 1.63 0.01 1.35 0.02 1.30 0.41 5.78

—

53.14 1.34 0.75 2.19 0.00 0.05 0.92 0.01 1.12 0.03 0.11 0.54 12.20 1.18 76.58

65.38 12.56 77.84 2.77 1.15 2.78 0.00 0.14 1.47 0.01 1.84 0.12 1.27 0.16 5.76

—

—

85.67

95.32

2

Dosages: Glass metabolism cage, 574 ug/25cm Skin-depot XAD-2/Pilot, 91.8 ug/2.54 cm (stainless steel cages) Skin-depot XAD-2/Study, 54.7 ug/2.54 cm (stainless steel cages) Animal weight: Glass metabolism cage, 280 g Skin-depot XAD-2/Pilot, 431 g Skin-depot XAD-2/Study, 280 g 2

2

Tables III and IV summarize the analytical results for the patches (ug of EPTC/ 23.74 cm ) removed from the mixer/loader and applicator. In the mixing operations, the primary locations where EPTC was detected were thighs, shins, and forearms. This was expected because the pouring operation occurs below the waist. The forearm 2

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22. KNAAK ET AL.

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Percutaneous Absorption Studies

patches were located on the underside of the arms. During the transfer of the formu­ lations to the mix tank, arms were often rested on the spray tank while transferring formulations to the tank. Upper body exposure was low and infrequent.

Table III. Summary of Patch Data, Mixer/Loader!/ Position of Patch Back Chest / Shoulder Forearms Thighs Shins 2

-1/ 2J

Total EPTC Detected (ug) 0.460 0.840 0.125 7.860 20.550 366.440

Number of Samples with EPTC 2 4 1 8 8 10

ug EPTC/Patch Highest Values 0.340 0.415 0.125 4.435 13.150 361.860

Amount of EPTC found Chest values are from insid

Table IV. Summary of Patch Data, Applicatorl/ Position of Patch Back Chest / Shoulders Forearms Thighs Shins 2

1/ 2J

Total EPTC Detected (ug) 23.88 5.86 24.14 9.68 27.96 27.23

Number of Samples with EPTC 9 8 10 12 13 13

ug EPTC/Patch Highest Values 13.81 1.84 10.15 3.41 8.09 7.21

2

Amount of EPTC found on skin patch (ug/23.75 cm ). Chest values from inside patches.

The application process involved an even distribution of EPTC to both the upper and lower body with twice as many patches showing detectable residue. The lower portion of the body received 60% of the exposure. No exposure monitoring data was taken for feet. Exposure to the feet was assumed to be very slight since the worker wore boots throughout the study. The two inside patches used in this study (Table V) were placed on either side of the exterior "chest at throat" patch to estimate the amount of EPTC reaching the skin. For the applicator, about 40% of the EPTC measured on the external patches were found inside the shirt. The external patches for the mixer only amounted to 2.5% of the levels found in external patches of the applicator, because the same person alternately performed both mixing and application, and did not change shirts between operations. The EPTC levels on the inside mixer patches are thought to represent amounts transferred from the shirt (which was not changed during operations) following previous spray applications. The applicator levels were also generally low except for the hands as shown in Table VI. Maintenance exposure, involving the unclogging of nozzles plugged by fiber glass particles, doubled the hand values over normal operations. By contrast, the mixing step averages less than 1/3 the exposure experienced in applicator functions where gloves were used on both hands.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE Table V. Exterior and Inside Patch Data

ug, EPTC Mode of Exposure Exterior Chest Patch Inside Left Chest Inside Right Chest Applicator (14 replicates) 0.960 0.320 0.460 Mixer (14 replicates) 0.024 0.057 0.064

Table VI. Summary of Hand Wash Data, Mixer/Loader/Applicator

Time 6/10 6/11 6/11 6/12 6/12 6/16 6/16 6/17 6/17 6/17 6/18 6/18 6/18 6/19 6/19 6/19 6/19 Mean SD U

Amount of EPTC Found in ugl/ Applicator Mixer/Loader /maintenance — — 266.00 — 60.00 55.50 1518.00 69.50 33.50 36.5 —

18.00 38.50 69.00 22.00 47.00 75.30 429.50 —

10.50 48.00 72.50 167.50 83.13 106.75

7.00 0.00 0.00 8.00 —

822.50 310.50 40.00 71.00 74.50 1790.50 —

238.36 479.69

236.50 581.50 527.50 — — — — — — — — —

519.57 535.20

Values are from hand rinses.

The average daily inhalation exposure observed was 28.97 ug for the mixer and 147.42 ug for the applicator. The applicator was expected to be highest due to the longer exposure times (100—200 minutes). Mixing times ranged from 11—23 minutes. Tables VII and VIII give the exposure values and the absorbed dosages, for the mixer/loader and applicator obtained from clothing patches, hand washings, and inhalation data. The exposure values, dermal and inhalation, were converted to absorbed dosages by multiplying the dermal exposure by the dermal absorption percentage, 14.7%, obtained from the rat study and the inhalation exposure values by 50% based on dog and human inhalation studies of Raabe (6). The total absorbed dosages for Tables VII and VIII are given in Table IX along with the absorbed dose expressed in terms of 1.0 and 23 lbs of applied EPTC. The mean dose absorbed per lb of pesticide applied was 14.23 ug. Table X shows the sum of the EPTC urinary metabolites S-(N,N-dipropylcarbamoyl) cysteine and S-(N,N-dipropylcarbamoyl)-N-acetyl cysteine, found in urine samples taken over the course of the study. The detection limit was set at 0.05 ug/ml based on the 0.023 ug/ml background interference peak found in the prestudy control urine. Four samples had metabolite levels of 0.079 to 0.136 ug/ml, which converts to EPTC equivalents excreted of 9.53 to 24.38 ug. The sample with the highest residue

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Table VII. Exposures and Estimated Absorbed Dosages During Mixing/Loading (ug) Dermal Absorbed Dose (14.7%)

Exposure 8387.80 93.50 80.73 51.27 41.53 69.00 22.00 147.18 126.54 664.97 93.99 76.85 72.50 862.63

1234.69 13.76 11.89 7.55 6.11 10.15 3.23 21.66 18.62 97.88

10.67 126.97

58.97 0.00

29.49 0.00

Mean SD

770.75 2206.76

113.45 324.84

28.97 55.48

14.48 27.74

—

—

Exposure 65.79 0.00 0.00 0.00 0.00 25.00 0.00 0.00 15.79 213.16 27.03

Inhalation Absorbed Dose (50%) 32.89 0.00 0.00 0.00 0.00 12.50 0.00 0.00 7.89 106.58 13.51

Time 6/10 6/11 6/11 6/12 6/16 6/16 6/17 6/17 6/17 6/18 6/18 6/19 6/19 6/19 6/19

Table VIII. Exposures and Estimated Absorbed Dosages During Application (ug)

Time 6/10 6/11 6/11 6/121/ 6/121/ 6/16 6/16 6/17 6/17 6/18 6/18 6/19 6/19 6/19 6/19 Mean SD 1/

Exposure 1710.53 426.20 1591.42 1038.44 495.62 380.94 626.26 532.16 14.74 897.07 1436.50 271.04 71.00 228.00 2505.98 815.06 712.08

Dermal Absorbed Dose (14.7%) 251.80 62.72 234.25 152.85 72.95 56.07 92.18 78.33 2.16 132.00 211.45 39.89 10.45 33.56 368.88

Exposure 315.38 215.38 0.00 133.33 89.47 255.26 0.00 71.05 82.14 0.00 618.42 160.53 0.00 51.35 218.92

119.96 104.82

147.42 165.05

Inhalation Absorbed Dose (50%) 157.69 107.69 0.00 66.67 44.74 127.63 0.00 35.53 41.07 0.00 309.21 80.26 0.00 25.68 109.49 73.71 82.53

Applications were 13.8 lbs A.I. and 9.2 lbs A.I. for 6/11 and 6/12 respectively; otherwise, 23 lbs A.I. applications were made.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE Table IX. Summary of Estimated Absorbed Dosages (Total) During Mixing and Application (ug)

Date 6/10 6/11 6/11 6/121/ 6/121/ 6/16 6/16 6/17 6/17 6/17 6/18 6/18 6/18 6/19 6/19 6/19 6/19

Mixer/Loading —

1234.69 13.76 11.89 7.55 18.61 10.15 3.23 29.55 —

125.00 111.39 13.83 25.78 40.16 126.97

Mean SD 1/

126.61 322.06

Applicator 409.49 170.42 234.25 219.52 117.69 183.70 92.18 113.86 43.23 —

Applied 2 3 lbs.

Applied 1.0 lbs.

—

—

1405.11 413.30

61.09 17.97

129.58 191.25 110.79 124.01 46.46

5.63 8.31 4.82 5.39 2.20

—

—

—

—

—

132.00 520.66

645.90

28.00

59.24 478.37

85.02 518.50

3.69 22.50

—

193.68 156.62

—

327.14 392.14

—

14.23 17.03

Applications were 13.8 lbs. A.I. and 9.2 lbs. A.I. for 6/11 and 6/12 respectively; otherwise, 23 lbs. A.I. applications were made.

was selected for further study. The aliquot was submitted to GC-mass spectral analysis and using this technique, the presence of S-(N,N-dipropyl carbarnoyl)-N-acetylmethyl cysteine was confirmed. The exposures that resulted in detectable levels of urinary metabolites occurred on 6/10, 6/11, and 6/17. No urinary metabolites were found on 6/13, 6/14, and 6/15 when there was not exposure and on 6/16, 6/18, and 6/19 when there was considerable exposure based on patch and inhalation monitoring data. Table XI gives the daily exposure for a worker mixing/loading and applying EPTC to 120 acres of soil in mg/person/day and in mg/kg/day along with their mean and standard deviation. The average lifetime exposure expressed in mg/kg/day is also given in Table XI along with the mean value and standard deviation. A margin of safety of 68 was determined for the mixer/loader-applicator by dividing the rat chronic NOEL of 5.0 mg/kg/day by the exposure level of 0.074 mg/kg/day determined in this study from patch and inhalation data. A much wider margin of safety of 340 was calculated using the NOEL of 5.0 mg/kg/day and the daily exposure value of 0.0147 mg/kg/day determined from urinary values. A margin of safety of 2940.0 was determined using the lifetime average estimate of exposure of 0.0017 mg/kg/day determined from patch and inhalation data. Discussion The adsorptive nature of EPTC made it difficult to determine the fate of a topically applied dose in glass metabolism cages normally used by investigators to collect respira­ tory volatiles. Topically applied l^C-EPTC, which volatilized, was not quantitatively collected on the XAD-2 resin as glass surfaces retained approximately 30% of this material. This necessitated the use of a skin-depot as described by Susten et al. (3.) for

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1/ 4/

-?/

11

Time 16:33 22:15 4:30 19:00 5:10 18:30 22:40 5:00

Urine Volume (ml) 286 146 186 275 258 262 170 244 0.00 12.56 0.00 24.38 13.29 0.00 9.53 0.00

ug of Metabolites

64.344/ 111.05

Corrected Absorbed!^/ Dose in ug/23 lbs of Applied EPTC (n=7) 0.00 87.10 0.00 302.892/ 0.00 0.00 60.42 0.00

Twenty-one urine samples were collected 6/9 to 6/19. Metabolites were found in samples collected on 6/10, 6/11, and 6/17. Urine values corrected to 1200 ml and urinary metabolites to S-(N,N-dipropylcarbamoyl)-N-acetylcysteine. Metabolite assumed to be 28% of absorbed dose (40% of absorbed dose eliminated in urine over a 24 hr period and 70% of the urinary metabolites as the cysteine or N-acetyl derivatives). Value obtained for 6/11 and 6/12. Absorbed dose in ug/kg/d = 0.0147. Calculations: 2.8 ug/lb of applied EPTC; 3.3 lb/A x 120 A/d = 396 lbs EPTC applied/d; 2.8 ug/lb x 396 lbs/d = 1.1 mg/75 Kg worker/d.

Mean SD

Day 6/10 6/10 6/11 6/11 6/12 6/17 6/17 6/18

Time Collected

Table X. Total Metabolite Found in Urine During Mixing/Loading and Application

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Table X I . D a i l y a n d Average L i f e t i m e A b s o r b e d Dose F r o m Mixing/Loading and A p p l i c a t i o n

Time 6/11 6/11 6/12 6/12 6/16 6/16 6/17 6/17 6/17 6/18 6/18 6/18 6/19 6/19 6/19 6/19

D a i l y A b s o r b e d ! / Dose ( 1 2 0 acres/day) Per Person (mg/day) (mg/kg/day) 0.320 24.19 7.12 0.090 —

2.23 3.29 1.78 2.13 0.87

1/ 2/

—

6.67-04 9.89-04 5.52-04 6.44-04 2.76-04

—

—

—

—

—

—

11.09 3.95 0.42 1.46 8.90

-

Mean SD

—

0.029 0.043 0.024 0.028 0.012

Average?/ L i f e t i m e A b s o r b e d Dose (mg/kg/day) Commercial Worker 73.60-04 20.70-04

5.62 6.75

0.006 0.019 0.119

0.074 0.089

1.38-04 4.37-04 27.37-04

17.05-04 20.53-04

D a i l y A b s o r b e d Dose = 120 acres x 3.3 lbs/A x u g absorbed dose/A ( f r o m Table I X ) . Average L i f e t i m e A b s o r b e d Dose = D a i l y A b s o r b e d Dose x 0.023 15d/365d x 4 0 yr/70yr = 0 . 0 2 3 .

the mouse a n d m o d i f i e d i n this study t o f i t the rat. T h e removable X A D - 2 resin adsorpt i o n c o l u m n facilitated the t o p i c a l a p p l i c a t i o n a n d t r a p p i n g o f v o l a t i l i z e d l ^ C - E P T C . E P T C was retained b y c l o t h patches l o n g enough to enable us to collect a n d estimate the a m o u n t o f E P T C deposited o n c l o t h i n g . E P T C d i d n o t appear to be retained b y the skin o f the w o r k e r l o n g enough t o be absorbed i n t o the b o d y as i n d i c a t e d b y the small n u m b e r o f urine samples having detectable levels o f E P T C m e t a b o l i t e . T h e use o f gloves d u r i n g m i x i n g / l o a d i n g and n o z z l e cleaning m a y have a c t u a l l y enhanced the a b s o r p t i o n o f E P T C t h r o u g h the skin o f the hands. Studies b y L a v y et a l . (12) indicate that w o r k e r s exposed t o 2 , 4 - D a n d p i c l o r a m o n a d a i l y basis excrete these pesticides i n urine s h o r t l y after exposure a n d d u r i n g the p e r i o d o f e x p o sure. If the E P T C was being absorbed, d a i l y exposure o f the w o r k e r t o E P T C s h o u l d have resulted i n E P T C metabolites i n a l l urines c o l l e c t e d . Evaporative losses o f E P T C f r o m skin m o s t l i k e l y o c c u r r e d r e d u c i n g the a m o u n t o f E P T C available f o r a b s o r p t i o n . A better estimate o f actual exposure/absorption t o E P T C m a y be o b t a i n e d f r o m u r i n a r y metabolites than f r o m c l o t h patches. T h i s estimate reduces the d a i l y absorbed dose f r o m 0 . 0 7 4 t o 0 . 0 1 4 7 mg/kg/day f o r a 75 k g m a n a n d increases the margin o f safety f r o m 68 t o 340. L i f e t i m e absorbed dosages are 1/5 o f the absorbed dose estimated f r o m c l o t h patches w h e n u r i n a r y values are used. Acknowledgments We w i s h t o t h a n k F i o r e l l a G i e l o w a n d S c o t t F o l w a r k o w f o r their expert technical assistance i n c a r r y i n g o u t the analyses required i n the rat percutaneous a b s o r p t i o n study.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

22. KNAAK ET AL. Percutaneous Absorption Studies

303

Literature Cited 1. Knaak, J. B.; Yee, Karin; Ackermann, C. R.; Zweig, G.; Wilson, B. W. Toxicol. Appl. Pharmacol. 1984, 72, 406-416. 2. Medinsky, M. A. J. Anal. Toxicol. 1986, 10, 24-27. 3. Susten, A. S.; Dames, B. L.; Burge, J. R.; Niemeier, R. W. Am. J. Ind. Med. 1985,7, 323-335. 4. Popendorf, W. J. Ph.D. Thesis, University of California, Berkeley, CA, 1976. 5. Guyton, A. C. W. B. Saunders Co., Philadelphia, PA, 1966, 552. 6. Hubbell, J.P.; Casida, J.C. J. Agric. Food Chem. 1977, 25(2), 404-413. 7. Knowles, D. PPG Industries, Inc. Report, Br-24055, 1986. 8. Mull, R. McCarthy, J. F. Vet. Hum. Toxicol. 1986, 28, 328-336. 9. Raabe, O. University of California, Davis; Contract No. A3-132-33 with California Air Resources Board. 10. Ross, J. H. Leber, A. P.; Lucas, F. D.; Chun, S. A. PPG Industries, Inc. Report, Br-24060. 11. Hazelton Laboratories American Study in Rats, 1987. 12. Lavy, T. L.; Norris, L. A.; Mattice, J. D.; and Marx, O. B. Environ. Toxicol, and Chem. 1987, 6, 209-224. ;

;

RECEIVED January 28, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 23

Assessing Risk for Humans on the Basis of Animal Toxicology Data Use of Dermal Absorption Data for Animal Studies E. J . Hixon Data Evaluation Department, Health, Environment and Safety, Mobay Corporation, Stilwell, KS 66085

A method is presente absorption of a collected in animal studies. The data from dermal absorption studies in animals can be fit to the most appropriate equation based on statistical tests. Once the equation has been fit, the coefficients for the parameters of the equation can be estimated. These parameters can then be used to estimate the amount of applied material that is absorbed. After field exposure studies have determined the amount of material contacting the skin and the time of exposure for the workers, the parameter estimates can be used to estimate the amount of the material that will be absorbed. The absorbed dose can then be compared to available toxicology data for hazard determination. One o f t h e most d i f f i c u l t a s p e c t s o f r i s k assessment i s e s t i m a t i n g the r i s k o f c h e m i c a l exposure f o r humans based on a n i m a l t o x i c i t y data. T h i s e s t i m a t i o n i s even more d i f f i c u l t when the a n i m a l study data a r e c o l l e c t e d u s i n g a d i f f e r e n t r o u t e o f exposure than t h a t expected f o r humans. A case i n p o i n t i s e s t i m a t i n g t h e r i s k f o r p e s t i c i d e a p p l i c a t o r s . The a n i m a l t o x i c i t y database f o r a p e s t i c i d e c o n s i s t s l a r g e l y o f data c o l l e c t e d i n f e e d i n g s t u d i e s . The o n l y s t u d i e s a v a i l a b l e t o address t h e r o u t e s o f exposure a p p r o p r i a t e f o r a p p l i c a t o r s a r e t y p i c a l l y a s i n g l e subacute dermal study and (sometimes) a subacute i n h a l a t i o n s t u d y . These s t u d i e s a r e o f t e n conducted u s i n g treatment f i v e days per week, whereas t h e exposure to t h e a p p l i c a t o r may be d a i l y f o r an extended p e r i o d . Subacute studies rarely last longer than three weeks, whereas t h e applicators exposure may be l o n g e r . These d i s c r e p a n c i e s can make i t d i f f i c u l t t o compare t h e exposure s i t u a t i o n t o t h e a n i m a l d a t a . 1

c

0097-6156/89/0382-0304$06.00/0 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

23. HIXON

Assessing Risk for Humans

305

In the p a s t few y e a r s i t has become common t o p e r f o r m a dermal a b s o r p t i o n s t u d y t o determine the e x t e n t t o which a material in c o n t a c t w i t h the s k i n i s absorbed i n t o the g e n e r a l c i r c u l a t i o n . The i n t e n t has been t o use the dermal a b s o r p t i o n s t u d y as a basis for c a l c u l a t i o n o f the dose t o which an i n d i v i d u a l i s exposed. Once the dose " d e l i v e r e d " has been d e t e r m i n e d , a n i m a l s t u d i e s u s i n g any r o u t e of administration could theoretically be used t o e s t i m a t e the l i k e l i h o o d of t o x i c i t y . S e v e r a l p r o t o c o l s have been used t o determine dermal a b s o r p t i o n of a chemical (pesticide). A l l the p r o t o c o l s have a t l e a s t one major d i f f e r e n c e from the way dermal exposure a c t u a l l y o c c u r s i n the field. In the animal studies a measured amount o f m a t e r i a l i s a p p l i e d t o the t r e a t e d s k i n a r e a and l e f t i n c o n t a c t w i t h the skin for a s p e c i f i e d p e r i o d of time. I n c o n t r a s t , an i n d i v i d u a l w o r k i n g i n the f i e l d has a graded exposure to a s l o w l y but steadily increasing amount of m a t e r i a l The "dose" s t a r t s out r e l a t i v e l y s m a l l and i n c r e a s e s s t e a d i l In s t u d i e s conducte field conditions, i t i s s t a n d a r d t o measure the t o t a l amount o f m a t e r i a l on the s k i n a t the end o f the exposure p e r i o d and d i v i d e by the number o f hours (or m i n u t e s ) of exposure t o e x p r e s s the exposure as mass of m a t e r i a l d e p o s i t e d per u n i t t i m e . T h i s measure has been referred to i n some c a s e s as the " r a t e " of dermal exposure. In c o n t r a s t , animal studies may determine the amount o f material absorbed (usually as the amount a p p e a r i n g i n the u r i n e ) p e r u n i t t i m e , and r e f e r t o t h i s as the " r a t e " o f dermal a b s o r p t i o n . C l e a r l y these two "rates" do n o t r e f e r t o the same phenomenon, and s h o u l d not be c o n s i d e r e d comparable. D e s p i t e t h i s key d i f f e r e n c e i n the way the d a t a a r e collected, it i s p o s s i b l e to use the a n i m a l dermal a b s o r p t i o n d a t a t o e s t i m a t e the amount o f a p p l i e d m a t e r i a l a b s o r b e d by an exposed individual. The purpose of this paper i s to p r e s e n t a method f o r making t h i s determination. Dermal A b s o r p t i o n P r o c e s s e s S t u d i e s conducted i n a n i m a l s ( 1 ) and w i t h human v o l u n t e e r s ( 2 ) have clearly demonstrated t h a t the amount o f m a t e r i a l absorbed tends t o i n c r e a s e as the amount applied increases. Several studies have demonstrated that the p e r c e n t absorbed d e c r e a s e s w i t h i n c r e a s i n g dose; however the a b s o l u t e mass absorbed tends to increase ( 1 ) . This may not be, and i n fact u s u a l l y i s not, a l i n e a r process. S i m i l a r r e s u l t s have been o b s e r v e d w i t h r e s p e c t t o t i m e . The amount of material absorbed a c r o s s the skin generally increases with i n c r e a s i n g time of c o n t a c t w i t h the s k i n ( 3 - 4 ) . I t i s not uncommon f o r the amount o f a b s o r p t i o n t o p l a t e a u a f t e r a t i m e . Clearly i t i s necessary f o r dermal absorption studies i n animals to take into account both dose and time. I t i s not practical i n the laboratory s e t t i n g t o a p p l y i n c r e a s i n g doses to e x p e r i m e n t a l a n i m a l s , p a r t i c u l a r l y when the s t u d y i s b e i n g done w i t h radio-labeled material. I t i s p r a c t i c a l , however, t o use more than one dose, and t o l o o k a t more than one time p o i n t a f t e r a p p l i c a t i o n . The protocol outlined by R. P. Z e n d z i a n a d d r e s s e s b o t h o f t h e s e needs ( 5 ) . B r i e f l y , groups o f a n i m a l s a r e t r e a t e d w i t h one of at

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

306

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

least t h r e e doses o f m a t e r i a l w i t h t h e doses spaced a t l o g i n t e r v a l s . The a p p l i c a t i o n a r e a s a r e c o v e r e d and t h e a n i m a l s a r e placed i n metabolism cages f o r c o l l e c t i o n o f u r i n e and f e c e s . A t i n t e r v a l s o f 0.5, 1, 2, 4, 10 and 24 hours a f t e r application subgroups o f a n i m a l s from each t r e a t e d group a r e s a c r i f i c e d f o r d e t e r m i n a t i o n o f r a d i o a c t i v i t y r e m a i n i n g i n s k i n and c a r c a s s . The amount o f m a t e r i a l absorbed i s determined based on t h e amounts o f r a d i o a c t i v i t y e x c r e t e d and t h a t r e m a i n i n g i n t h e c a r c a s s . M a t h e m a t i c a l Treatment

of Results

A l l a n a l y s e s were done u s i n g s o f t w a r e o f SAS I n s t i t u t e I n c . , Cary, NC. T a b l e I p r e s e n t s d a t a c o l l e c t e d i n a study u s i n g t h e Z e n d z i a n p r o t o c o l w i t h f o u r l o g a r i t h m i c a l l y spaced doses and time p o i n t s up to 24 h o u r s . The d a t a from a dermal a b s o r p t i o n s t u d y conducted according t o the Zendzian p r o t o c o l w i l l c o n t a i n two independent v a r i a b l e s , dose and t i m e amount absorbed. The f i r s determine whether linear or quadratic r e g r e s s i o n i s most a p p r o p r i a t e . S i n c e we know from s e p a r a t e e x p e r i m e n t s that the amount absorbed i n c r e a s e s w i t h b o t h time o f exposure and dose, we might expect t o f i n d an i n t e r a c t i o n o f these two f a c t o r s when e f f e c t s of both a r e i n v e s t i g a t e d simultaneously. Thus i t i s i m p o r t a n t t h a t t h e d a t a from each experiment be t e s t e d f o r q u a d r a t i c response.

T a b l e I . Dermal A b s o r p t i o n T e s t Data

(Time) 0.5 1.0 2.0 4.0 10 24

0.04

Dose (mg/kg) 4.0

0.4

.00348 .00277 .00378 .00428 .00498 .00519

a

40 4.77 4.56 5.05 4.44 5.20 5.06

.312 .303 .308 .348 .402 .449

.0267 .0319 .0319 .0323 .0452 .0440

Amount absorbed i n mg e q u i v a l e n t s

T h i s t e s t c a n be done e a s i l y u s i n g t h e SAS procedure RSREG. T h i s procedure f i t s t h e parameters o f a q u a d r a t i c response s u r f a c e (6). The e q u a t i o n d e s c r i b i n g t h e response s u r f a c e i s shown h e r e : 2 y = BQ+ n x ]

l

+ B x 2

+ B x

2

3

x

2 + B x 4

2

+ B x x 5

x

2

where x i s t h e dose a p p l i e d , x i s t h e time o f exposure, B Q i s t h e i n t e r c e p t and B ^ , B e t c . a r e t h e c o e f f i c i e n t s o f each term. The RSREG procedure w i l l p e r f o r m a t e s t t o determine how much o f t h e r e s i d u a l e r r o r i s due t o l a c k o f f i t . T h i s f i t t e s t can be used t o determine whether a q u a d r a t i c response b e s t d e s c r i b e s t h e r e s u l t s . 2

2

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

23. HIXON

Assessing Risk for Humans

307

These d a t a were t e s t e d u s i n g the RSREG p r o c e d u r e , and the r e s u l t s are shown i n T a b l e I I . The procedure^ a p p l i e s f i r s t a l i n e a r r e g r e s s i o n , then adds the q u a d r a t i c terms ( x ^ and x ^ ) , then adds the c r o s s p r o d u c t ( x ^ ) . I n t e s t i n g f o r l a c k of f i t , the procedure RSREG p a r t i t i o n s the t o t a l e r r o r i n t o l a c k of f i t and pure e r r o r . When l a c k of f i t i s s i g n i f i c a n t l y d i f f e r e n t from pure e r r o r , then t h e r e i s v a r i a t i o n i n the model not accounted f o r by random e r r o r . As can be seen from T a b l e I I , l a c k o f f i t i s s i g n i f i c a n t l y d i f f e r e n t from pure e r r o r f o r the t e s t d a t a s e t . T h i s i n d i c a t e s t h a t a q u a d r a t i c response mgdel does not f i t the d a t a , d e s p i t e the apparently favorable R values. Now we a r e f r e e t o p e r f o r m a s i m p l e l i n e a r r e g r e s s i o n on the d a t a . The b e s t way t o approach the d a t a next i s t o determine whether e i t h e r o r b o t h of the independent v a r i a b l e s (dose and time) have an e f f e c t on the dependent v a r i a b l e (amount absorbed). Examining the d a t a i n T a b l e I shows t h a t w h i l e t h e r e appears t o be a reasonable r e l a t i o n s h i r e l a t i o n s h i p between tim can t e s t t h i s u s i n g the SAS procedure RSQUARE. T h i s procedure f i n d s the subset of independent v a r i a b l e s t h a t tjest p r e d i c t s the amount absorbed based on the optimum v a l u e o f R , the c o e f f i c i e n t of determination (7). The r e s u l t s o b t a i n e d when the d a t a i n Table I were t e s t e d u s i n g the RSQUARE procedure are shown i n T a b l e I I I . C l e a r l y u s i n g time as the s o l e r e g r e s s o r r e s u l t e d i n a poor f i t of the d a t a ; i n o t h e r words, the amount absorbed d i d not c o r r e l a t e v e r y w e l l w i t h time alone. I n c o n t r a s t , a good f i t was a c h i e v e d u s i n g dose as the s o l e r e g r e s s o r . However, the s t a t i s t i c s f o r the r e g r e s s i o n analysis u s i n g b o t h dose and time i s s l i g h t l y b e t t e r t h a n t h a t u s i n g dose a l o n e . T h e r e f o r e , a l t h o u g h the e f f e c t o f time i s s m a l l i t does appear t o improve the f i t o f the c u r v e t o the d a t a , so we are b e t t e r o f f i n c l u d i n g the time f a c t o r i n our a n a l y s i s . Now t h a t we have l e a r n e d t h a t a r e g r e s s i o n u s i n g b o t h dose and time r e s u l t s i n a c u r v e t h a t b e s t f i t s the amount absorbed, we are f r e e t o p e r f o r m a s i m p l e l i n e a r r e g r e s s i o n u s i n g these two independent variables. T h i s was done u s i n g the SAS procedure REG. The REG procedure f i t s l e a s t - s q u a r e s e s t i m a t e s t o l i n e a r regression models (8). For our d a t a w i t h two independent v a r i a b l e s , the e q u a t i o n t h a t i s f i t t e d t o the d a t a i s r e p r e s e n t e d by: y = B

Q

+ B

l X l

+ B x 2

2

The p r o c e d u r e REG w i l l e s t i m a t e the v a l u e s of B^, B^ and B^ and will compare the a c t u a l d a t a t o the responses p r e d i c t e d by the model. By comparing the p r e d i c t e d v a l u e s t o those a c t u a l l y o b t a i n e d we can examine the f i t o f the c u r v e t o the d a t a o b t a i n e d . T h i s comparison i s shown i n Table IV. From examining T a b l e IV i t i s c l e a r t h a t f o r t h i s t e s t d a t a s e t the f i t of the p r e d i c t e d v a l u e s t o the a c t u a l v a l u e s i s not good a t v e r y low d o s e / s h o r t time c o m b i n a t i o n s . At these p o i n t s the p r e d i c t e d v a l u e s are n e g a t i v e numbers, which cannot o c c u r i n the r e a l w o r l d . A c t u a l exposure doses i n mg/kg/day can e a s i l y be i n the range of these low doses, however, so i t i s i m p o r t a n t t o f i n d a way

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988. 0.331 0 0.331

18

0

18

Lack o f F i t

Pure E r r o r

Total Error

SS

DF

100.660

RESIDUAL

T o t a l Regress

0.0184

0

0.0184

MEAN SQUARE

0.9967

0.0007

0.0747

Crossproduct

R-SQUARE 0.9952 0.0008

TYPE I SS 100.506 0.0804

DF

Quadratic

REGRESSION Linear

T a b l e I I . RESULTS OF THE RSREG PROCEDURE

PROB 1.0000

9999.990

0.0001

0.0589

0.1412

PROB 0.0001

F-RATIO

1095.18

4.07

2.19

F-RATIO 2733.72

o

o w w o

NH

o

NH

H

3

O

2

H O

o

r

9

NH

O

5

O

00

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

0.04 Actual Predicted .00348 -.0814 .00277 -.0784 .00378 -.0722 .00428 -.0599 .00498 -.0230 .00519 .0630 Actual .0267 .0319 .0319 .0323 .0452 .0440

A c t u a l o r p r e d i c t e d amount absorbed i n mg

Time o f Exposure (Hours) 0.5 1.0 2 4 10 24

-0.0894

0.9952

2

A

equivalents

Predicted -.0375 -.0344 -.0282 -.0160 .0209 .107

Actual .312 .303 .308 .348 .402 .449

Dosage A p p l i e d (mg/kg) 4 Predicted .402 .405 .411 .424 .461 .547

Values

0.12

0.12

Actual 4.77 4.56 5.04 4.44 5.20 5.06

PARAMETER ESTIMATES XI

T a b l e I V . Comparison o f A c t u a l v s P r e d i c t e d

-0.0469

INTERCEPT 1.2674

0.9946

R-SQUARE 0.0006

1

NUMBER IN MODEL 1

T a b l e I I I . RESULTS OF THE RSQUARE PROCEDURE

40 Predicted 4.80 4.80 4.81 4.82 4.86 4.94

0.0061

X2 0.0061

310

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

t o p r e d i c t the amount absorbed a t these low doses. One possibility i s t o do the s t a t i s t i c s on t h e d a t a from j u s t t h e two lower doses. The r e s u l t s o f the RSREG p r o c e d u r e a g a i n showed t h a t the q u a d r a t i c model does n o t f i t the d a t a w e l l ( d a t a not shown). The RSQUARE procedure i n d i c a t e d a g a i n t h a t time a l o n e does n o t f i t the d a t a w e l l but does improve s l i g h t l y the f i t of the d a t a compared t o u s i n g dose as the s o l e r e g r e s s o r ( d a t a n o t shown). I n T a b l e V t h e REG results are shown u s i n g o n l y t h e two l o w e s t dosages of 0.04 and 0.4 mg/kg. T h i s time the p r e d i c t e d v a l u e s agree v e r y w e l l w i t h the a c t u a l values.

T a b l e V.

Comparison o f A c t u a l v s P r e d i c t e d V a l u e s U s i n g the Two Lowest Dosages Dosag A p p l i e d (mg/kg

Time of Exposure (Hours) 0.5 1.0 2 4 10 24

0.0 Actual .00348 .00277 .00378 .00428 .00498 .00519

Predicted .00162 .00181 .00220 .00296 .00526 .0106

a

Actual .0267 .0319 .0319 .0323 .0452 .0440

Predicted .0329 .0331 .0335 .0342 .0365 .0419

3

A c t u a l o r p r e d i c t e d amount absorbed i n mg e q u i v a l e n t s REG S t a t i s t i c s Intercept: -0.0020 Bit 0.08 B : 0.0004 R: 0.95 2

2

The same p r o c e d u r e s were done u s i n g the d a t a from o n l y the two m i d d l e dosages o f 0.4 and 4 mg/kg. As e x p e c t e d , the RSREG p r o c e d u r e i n d i c a t e d t h a t the q u a d r a t i c model does n o t f i t the d a t a w e l l ( d a t a not shown). The RSQUARE procedure showed a g a i n t h a t r e g r e s s i o n based on time o f exposure alone does n o t f i t the d a t a w e l l , but t h a t time and dose t o g e t h e r p r o v i d e a s l i g h t l y b e t t e r f i t than u s i n g dose alone ( d a t a n o t shown). The r e s u l t s o f t h e REG p r o c e d u r e a r e shown i n T a b l e V I : a g a i n t h e p r e d i c t e d v a l u e s agree r e a s o n a b l y well with the a c t u a l v a l u e s . F i n a l l y , t h e d a t a from the h i g h e s t two dosages were t e s t e d . The q u a d r a t i c model d i d n o t f i t the d a t a , and i n t h i s case time p r o v i d e d o n l y a m i n i s c u l e e f f e c t on the r e g r e s s i o n r e s u l t s ( d a t a n o t shown). Table V I I presents the r e s u l t s of the REG p r o c e d u r e , and a g a i n i t can be seen t h a t the p r e d i c t e d v a l u e s agree w i t h the a c t u a l v a l u e s . Discussion The s t a t i s t i c s from the t h r e e s e t s of d a t a a r e summarized i n V I I I . S e v e r a l i n t e r e s t i n g o b s e r v a t i o n s can be made from these

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Table data.

23. HIXON

Assessing Risk for Humans

311

F i r s t , the i n t e r c e p t (or B ) appears t o be a c o n s t a n t d i s t a n c e from the l o w e s t dosage c o n s i d e r e d i n t h e c a l c u l a t i o n s . Second, the e f f e c t o f time ( B ) appears t o i n c r e a s e w i t h i n c r e a s i n g dosage. ?

Table VI.

Comparison of A c t u a l v s P r e d i c t e d V a l u e s U s i n g the Two M i d d l e Dosages Dosage A p p l i e d (mg/kg)

Time of Exposure (Hours) 0.5 1.0 2 4 10 24

Actual .0267 .0319 .031 .032 .045 .0440

0.4 Predicted .0129 .0146

.0461 .0952

4 Predicted .331 .333

Actual .312 .303

.414

.449

A c t u a l or p r e d i c t e d amount absorbed i n mg e q u i v a l e n t s REG S t a t i s t i c s I n t e r c e p t : -0.02 B: 0.09 B: 0.003 R: 0.97 x

2

2

Table V I I .

Comparison of A c t u a l vs P r e d i c t e d V a l u e s U s i n g the Two H i g h e s t Dosages Dosage A p p l i e d (mg/kg)

Time o f Exposure (Hours) 0.5 1.0 2 4 10 24

40

4 Actual .312 .303 .308 .348 .402 .449

Predicted .277 .283 .295 .319 .390 .557

Actual 4.77 4.56 5.05 4.44 5.20 5.06

Predicted 4.77 4.78 4.79 4.81 4.88 5.05

3

A c t u a l or p r e d i c t e d amount absorbed i n mg e q u i v a l e n t s REG S t a t i s t i c s I n t e r c e p t : -0.23 Bj: 0.12 B: 0.012 R: 0.99 2

2

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

T h i r d , the e f f e c t of dose (B-) appears t o be r e l a t i v e l y c o n s t a n t , a l t h o u g h i t may i n c r e a s e v e r y s l i g h t l y w i t h i n c r e a s i n g t i m e . Thus f o r t h i s p a r t i c u l a r d a t a s e t the e f f e c t of time appears t o be e x t r e m e l y s m a l l u n t i l the dosages get f a i r l y l a r g e . A dosage of 4 mg/kg f o r a 60 kg i n d i v i d u a l r e p r e s e n t s a t o t a l exposure of 240 mg, w h i l e a t 40 mg/kg the t o t a l exposure would be 2400 mg. These a r e p r o b a b l y l a r g e r than most of the a c t u a l exposures under normal c o n d i t i o n s o f use. Thus f o r average exposure conditions this a n a l y s i s of these d a t a i n d i c a t e s t h a t we can v i r t u a l l y i g n o r e the d u r a t i o n of exposure and m e r e l y c o n s i d e r the t o t a l dose of m a t e r i a l a p p l i e d t o the s k i n . Under these c i r c u m s t a n c e s i t might be a p p r o p r i a t e t o use the e s t i m a t e d v a l u e o f B^ as a measure of the f r a c t i o n of the a p p l i e d dose t h a t we would expect t o be absorbed. I n o t h e r words, u s i n g the r e s u l t s from the two l o w e s t dosages as an example (Table V I I I ) , we might expect t h a t 0.08, o r 8%, of the a p p l i e d dose would be absorbed r e g a r d l e s s of time o f exposure This approach can o n l y be use i s the case a t these lo

T a b l e V I I I . Summary of S t a t i s t i c s f o r Three Data S e t s Model: Y = B + B. (Dose) + B (Time) n

Intercept

Lowest Dosages -0.002

9

Middle Dosages -0.02

Highest Dosages -0.23

Bi

0.08

0.09

0.12

B

0.0004

0.003

0.012

0.95

0.97

0.99

R

2

G i v e n t h a t these d a t a can be f i t t o a model b e t t e r when not a l l the dosages a r e used a t once, i t c l e a r l y becomes e x t r e m e l y i m p o r t a n t t h a t the dosages used i n the a n i m a l s t u d y b r a c k e t as c l o s e l y as p o s s i b l e the a n t i c i p a t e d range of exposures i n the f i e l d . The data might be e a s i e r t o f i t t o a model i f the dosages used were spaced more c l o s e l y than one l o g a p a r t . For example, dosages spaced at h a l f l o g i n t e r v a l s might g i v e us a b e t t e r f i t , a l t h o u g h the r e s u l t s i n T a b l e V I I I showed o n l y s m a l l d i f f e r e n c e s i n the B ^ and B ^ v a l u e s between the t h r e e s e t s of r e g r e s s i o n d a t a . I t i s o b v i o u s from the r e s u l t s p r e s e n t e d t h a t the p e s t i c i d e used t o produce these d a t a i s not p a r t i c u l a r l y w e l l absorbed r e g a r d l e s s of a p p l i e d dose o r time of exposure. Another compound t h a t i s b e t t e r absorbed might produce a c o m p l e t e l y d i f f e r e n t s e t of statistics. F o r example, one might f i n d t h a t the q u a d r a t i c model f i t a d a t a s e t q u i t e w e l l . I n e i t h e r c a s e , by s u b s t i t u t i n g the e s t i m a t e s o f B Q , B ^ , B « e t c . i n t o the model e q u a t i o n one can determine the a c t u a l amount absorbed u s i n g the amount of dermal exposure measured i n a f i e l d s t u d y as the dose (x^) and the time over w h i c h the exposure was measured as the time ( x ^ ) . I n t h i s way we can take advantage o f the dose and time e f f e c t s noted i n our

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

23. HIXON

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a n i m a l s t u d i e s and a p p l y these e f f e c t s t o human exposure. An example o f such c a l c u l a t i o n s i s shown i n Table I X . Here the estimates of and B^ from the r e g r e s s i o n u s i n g t h e two l o w e s t dosages are used because the amount of m a t e r i a l a p p l i e d t o the s k i n i s i n the range o f these dosages. By s u b s t i t u t i n g t h e amount a p p l i e d and the time of exposure i n t o the model e q u a t i o n , the amount absorbed i s p r e d i c t e d t o be 0.009 mg e q u i v a l e n t s of m a t e r i a l . Thus it c a n be easy t o p r e d i c t the amount absorbed once we have the c o r r e c t r e g r e s s i o n parameter e s t i m a t e s . The c a l c u l a t i o n s would be done i n a s i m i l a r f a s h i o n i f the q u a d r a t i c model were found to f i t the d a t a b e s t , a l t h o u g h i t would be s l i g h t l y more c o m p l i c a t e d .

Table IX. E s t i m a t i n g Human Exposure U s i n g A n i m a l Study Data Human Data T o t a l Dermal Exposure Body Weight o f S u b j e c t : Dosage A p p l i e d : Time of Exposure:

Study Data Intercept:

60 k g 0.1 mg/kg 8 hours

-0.002 0.08 0.0004

Bi:

B: 2

Amount Absorbed - (0.8)(0.1) + (.0004)(8) - .002 = .009 mg e q u i v a l e n t s

T h i s p r e s e n t a t i o n has c e n t e r e d around a s i n g l e d a t a s e t , and c e r t a i n procedures were f o l l o w e d based on the c h a r a c t e r i s t i c s o f these d a t a . The i m p o r t a n t t h i n g t o remember i s t h a t each d a t a s e t s h o u l d be c o n s i d e r e d u n i q u e , and the t e s t s s h o u l d be a p p l i e d t o each s e q u e n t i a l l y t o determine how b e s t t o proceed. To summarize these steps: 1.

2.

3.

Because t h e r e w i l l always be two independent v a r i a b l e s , the d a t a s h o u l d always be t e s t e d u s i n g q u a d r a t i c and c r o s s - p r o d u c t models. Simple l i n e a r r e g r e s s i o n s h o u l d not be attempted u n t i l you a r e s a t i s f i e d t h a t a more complex e q u a t i o n w i l l not f i t t h e data. Regardless o f t h e type o f r e g r e s s i o n , whether q u a d r a t i c o r l i n e a r , p r e d i c t e d v a l u e s s h o u l d be compared t o a c t u a l v a l u e s t o be sure the e q u a t i o n works i n the r e a l w o r l d . I t may be n e c e s s a r y t o p e r f o r m r e g r e s s i o n s on o n l y p a r t of your d a t a s e t t o g i v e a good f i t t h a t works i n the r e a l w o r l d . You can o n l y j u s t i f y o m i t t i n g p a r t of your d a t a i f the parameters o f the exposure s i t u a t i o n f i t w i t h i n the range o f the d a t a you use.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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In c o n c l u s i o n , t h e a v a i l a b i l i t y o f dermal a b s o r p t i o n study d a t a g r e a t l y enhances our a b i l i t y t o p e r f o r m proper and r e a s o n a b l e h a z a r d assessments f o r f i e l d w o r k e r exposure. The flowscheme f o r d a t a a n a l y s i s proposed here i s i n t e n d e d t o s t a n d a r d i z e o u r approach t o these d a t a , t o m i n i m i z e improper use o f t h e d a t a and t o p r o v i d e a more e f f e c t i v e t o o l f o r human h a z a r d assessment. N o n t e c h n i c a l Summary Dermal a b s o r p t i o n s t u d i e s conducted i n a n i m a l s u s i n g the Z e n d z i a n p r o t o c o l p r o v i d e l a r g e s e t s o f d a t a . These d a t a must be a n a l y z e d c a r e f u l l y f o r p r o p e r use i n p r e d i c t i n g t h e amount o f m a t e r i a l an exposed worker might absorb a c r o s s t h e s k i n . The d a t a must f i r s t be t e s t e d a g a i n s t a q u a d r a t i c model t o determine t h e e f f e c t o f dose, time and dose p l u s t i m e . I f t h e q u a d r a t i c model does n o t f i t , a s i m p l e l i n e a r r e g r e s s i o n can be performed R e g a r d l e s s o f t h e type of r e g r e s s i o n , the mode dose and t i m e . These c o e f f i c i e n t p r e d i c t e d amount o f m a t e r i a l absorbed a f t e r a g i v e n exposure.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8.

Shah, P. V.; Fisher, H.; Sumler, M.; Monroe, R.; Chernoff, N.; Hall, L. J. Toxicol. Env. Health 1987, 21, 353-366. Wester, R. C.; Maibach, H. J . Toxicol. Env Health 1985, 16, 25-37. Burka, L. T.; Sanders, J . ; Kool, C.; Kim, Y.; Matthews, H. Toxicol. Appl. Pharmacol. 1987, 87, 121-126. Feldmann, R. J.; Maibach, H. Toxicol. Appl. Pharmacol., 1974, 28, 126-132. Zendzian, R. P. Procedure for Studying Dermal Absorption; Third edition revised June 14, 1985; California Modifications, October 9, 1985. SAS Institute Inc. SAS User's Guide: Statistics, Version 5 Edition. SAS Institute Inc.: Cary, 1985; Chapter 33. SAS Institute Inc. SAS User's Guide: Statistics, Version 5 Edition. SAS Institute Inc.: Cary, 1985; Chapter 32. SAS Institute Inc. SAS User's Guide: Statistics, Version 5 Edition. SAS Institute Inc.: Cary, 1985; Chapter 31

RECEIVED March

23, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 24

Insecticide Absorption from Indoor Surfaces Hazard Assessment and Regulatory Requirements 1

1

1

Peter E. Berteau , James B. Knaak , Donald C. Mengle , and Jay B. Schreider 2

1

Hazard Evaluation Section, California Department of Health Services, Berkeley, CA 94704 Medical Toxicology Branch, California Department of Food and Agriculture, Sacramento, CA 95814 2

Insecticides are often applied indoors where dermal absorption from surfaces as well as inhalatio have oral exposure centers frequently receive complaints of illness following such applica­ tions. These complaints may be coincidental due to odors or to a systemic toxic action from an ingredient. Using an appropriate algorithm, surface exposure and total absorption was calculated for chlorpyrifos, dichlorvos and propoxur. With each of these insecticides, the dose calculated might provide a toxic dose, particularly to an infant. More data are, therefore, needed for these and other insecticides to ensure that the levels in the air and on treated surfaces will not be injurious to health. Such data will include bioavailability of surface residues, rate of transfer from surfaces and dose-response data. Pending such data, risk assessments based upon health-protective assumptions should be used to decide the safety of various components. Pesticides, particularly those containing cholinesterase inhibitors, are often applied to combat insect infestations in indoor environments. The treated sites may include room air, closets, storage areas, baseboards, window sills, cracks, crevices, inaccessible areas, house plants, pets, pet beds, beds, upholstery, carpets and smooth floor areas. Respirable droplets or vapor may be inhaled during or after pesticide spraying and fogging operations. Human exposure may also occur as a result of dermal contact with treated or exposed surfaces followed by percutaneous absorption of the chemicals. In small children, there is also oral exposure through hand-to-mouth transfer. Thus, the cholinesterase-inhibiting pesticides represent a potential acute human health hazard when used indoors, particularly for small children. An appreciable number of persons in California require poison center and medical attention each year following exposure subsequent to treatment of homes, apartments, and offices by structural pest control operators, landlords, or employers as well as by adult household members. In Table I some of the California Department of Food and Agriculture's (CDFA) priority cases for 1984 which involved structural treatment are indicated. In 1984 the San Francisco Poison Control Center and its Toxic Information Center received 1,180 calls on pesticides. Of these calls, 340 involved insecticides and children under the age of 6. The symptoms and history of exposure were in agreement in 41 percent of the cases involving illnesses. The place where the exposure

0097-6156/89/0382-0315$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

occurred, indoors or outdoors, was not recorded. The most common symptom was nausea. Large numbers of poisoning cases due to cholinesterase inhibitors are handled each year by the Los Angeles Poison Control Center and the San Diego Poison Information Center. The great majority of the exposures to these pesticides occur in the home. Young children clad in little more than diapers are at risk playing on previously sprayed surfaces such as floors, carpets, furniture, and bedding. The higher body surface area to volume ratios of children compared to adults increases the likelihood that children will receive a toxic dose. Mild cholinergic symptoms are difficult to identify from normal childhood functions such as drooling, diarrhea, or excessive urination. In this connection, it should be noted that not all the subjects exposed are sick from symptoms of cholinesterase depression; many reports of illness may be coincidental due to the unpleasant odor of some of the pesticide chemicals and adjuvants used, resulting in a nauseous response. However, as will be shown, these concerns necessitate an assessment of the potential adverse health effects from the indoor use of these types of pesticides. The use of non-volatile pesticide to be hazardous to human health is not a significant route of exposure. Volatile pesticides such as dichlorvos are especially hazardous if the enclosed area is not adequately ventilated prior to reentry.

TABLE I. EXAMPLES OF REPORTED PESTICIDE ILLNESSES FROM STRUCTURAL APPLICATION

Applicator

Number Sick

Landlord

Circumstances

Pesticide

Two residents and three visitors to an apartment house.

Chlorpyrifos

Employer

13

County office building. Building evacuated.

Chlorpyrifos

Structural Pest Control Operator

10

Hospital employees.

Chlorpyrifos, bendiocarb, and diazinon

Four-month-old child.

Chlorpyrifos

All five family members.

Methyl bromide

School district office workers.

Propoxur

Structural Pest Control Operator Structural Pest Control Operator Employer

5

15

Mobay Chemical Company submitted two reports to the California Department of Food and Agriculture concerning the use of propoxur for flea control indoors. According to the reports, infant children are expected to be at risk when they come in contact with propoxur-treated floors and carpets. On the basis of these studies, Mobay made a label amendment on their indoor use product containing propoxur that explicitly limited indoor applications to surfaces along baseboards and inacces-

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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sible areas and recommended against the use of propoxur on large floor areas or floor coverings or as a space spray. In Table II an outline of a worst case situation where a scantily clad infant is playing in a room is given. Many of the parameters stated are measured values which can be located in various textbooks. Others are assumptions or "educated guesses" based upon little or no supporting data.

TABLE II. WORST CASE ASSUMPTIONS FOR AN INFANT PLAYING IN A ROOM

The 50th percentile body weight of a 6—9 month old child is approximately 7.5 kg (I). The body surface area of a 6—9 month old child is approximately 0.45 m (4.8 f t ) (2).

2

2

A n infant's hands account for approximately 4.8 percent of the total body surface area (2). Contact with the carpet will remove the total dislodgeable pesticide (12.9 mg/m ). 2

2

2

A child will play on approximately 18.6 m (200 f t ) of treated carpet. A child is in contact with a maximum of 25 percent of the available floor space (4.6 m ) and dermal contact takes place in four hours or less. 2

Dermal exposure is uniformly surface area.

distributed

over 50 percent of the body

It is assumed that all pesticide on the hands is s u b s e q u e n t l y ingested (approximately 9.6 percent of dermal exposure). It is assumed that airborne exposure is c o n t i n u o u s for 24 hours per day. It is assumed that both the inhalation and oral doses are 100 percent absorbed.

The three pesticide chemicals which were initially considered by us were propoxur (Baygon), dichlorvos (DDVP), and chlorpyvifos (Dursban). The calculations which were used in developing an exposure dose are outlined in the forthcoming tables.

Propoxur P r o p o x u r , a carbamate insecticide the structure of which is s h o w n below, was

II

i

,0-C-N-CH3

introduced in 1971 as a low hazard vector control agent to replace D D T in developing

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

countries. Its low dermal toxicity, > 2400 mg/kg, in the rat and reversible inhibitory action on acetylcholinesterase make it a reasonably safe material to use for mosquito control in and around the home. Its high oral toxicity (LD 50, 83 to 86 mg/kg in the rat), however, should be recognized when handling this material indoors. Propoxur is utilized for its rapid knockdown properties due to its vapor toxicity and its long-term residual properties. Villages in developing countries sprayed with p r o p o x u r were protected from mosquitoes for as long as 30 weeks after spraying (3). Propoxur is often used in combination with dichlorvos in pesticide foggers to provide residual insecticidal properties to the registered product. This residual property is the subject of concern as a possible hazard in dwelling places when propoxur is used alone or in combination with dichlorvos in foggers that deposit residues on furniture and carpets. Acceptable levels for propoxur on surfaces that persons might have dermal contact with have not been established by regulatory agencies. A recent risk assessment made by Hackathorn and Eberhart (4), which is summarized in Table III, involved the use of propoxur on carpets for flea control. It indicated that an adequate safety factor does not exist for infants playing on carpets after treatment If the child spends four hours on the floor, this time would b

TABLE III. CALCULATION OF A WORST CASE EXPOSURE DOSE FOR AN INFANT EXPOSED TO PROPOXUR Total dose

= 100% inh. exp. + 20% dermal exp. + 100% oral exp. body weight = 0.26 mg + 0.2 (53.6 mg) + 5.7 mg 7.5 kg

= 2.2 mg/kg Inhalation exposure

= max. air cone, x resp. min. vol. x exp. period 3

3

0.022 m g / m x 0.5 m / h r x 24 hr = 0.26 mg Dermal exposure

= (carpet area contacted) x (propoxur residue available) — amount of propoxur ingested = 4.6 m

2

2

x 12.9 m g / m - 5.7 mg

59.3 - 5.7 mg = 53.6 mg Oral exposure

= (carpet area contacted) x (propoxur residue available) x (0.096) 59.3 mg x 0.096 5.7 mg

Thus, the worst case exposure value is 2.2 mg/kg.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Based on human exposure, the lowest effect level for a single dose is 0.36 mg/kg which produced mild cholinergic symptoms, stomach discomforts, blurred vision, facial redness, and sweating. In repeated applications 0.15 to 0.20 mg/kg of propoxur was administered orally five times in 30 minutes resulting in 60 percent cholinesterase depression but no symptoms (5). Thus, there is a definite possibility for an adverse reaction in a very young child who plays on a treated carpet. Similar calculations were performed for a 12-year-old child (total dose = 0.04— 0.31 mg/kg) and for an adult (total dose = 0.03-0.11 mg/kg). In these cases the likelihood of a toxic reaction appears to be considerably less. Even less safety would apply to applications to smooth floor surfaces. In the assessment, a number of assumptions were made that may result in estimating more exposure than actually occurs. For instance, studies conducted on the dog by Raabe (6) demonstrated that the lung may absorb only 50 percent of an inhaled air pollutant and not 100 percent. Studies by Knaak and Wilson (7) showed that the rate of absorption of pesticides through skin decreases exponentially as the concentration decreases on the skin. Pesticide concentrations amounting to 1.0 jug/cm of skin are absorbed at rates amounting to 0.01 jug/hr/cm (1.0 percent per hou 1.0 jug of propoxur. Feldma their studies with propoxur. Approximately 20 percent of the topically applied dose was absorbed and eliminated over the study period of five days. Based upon these figures, the average rate of absorption over the five-day period was 0.007 jug/hr/cm . The relationship between the dermal dose of propoxur and its effect (dermal dose cholinesterase response) is not known. Gaines (9) reported a dermal LD50 in the rat of greater than 2400 mg/kg. This work suggests that propoxur is not readily absorbed or the inhibitor-acetylcholinesterase complex is short lived. In any case, data on the relationship of exposure and dermal dose cholinesterase response do not appear to be adequate to assure safe use in households, especially if carpets, open floor areas, furniture, or bedding are treated. 2

2

2

Dichlorvos Dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) the structure of which is shown below was one of the first of the vinyl phosphate insecticides to be discovered.

O CH 0

|| ^ P - 0 - C H = CCl CH 0^ 3

2

3

These compounds are formed by the Perkow rearrangement when a dihaloaldehyde or a dihaloketone is allowed to react with a trialkyl phosphite (10). The high vapor pressure of dichlorvos allows the material to penetrate spaces and kill insects. A concentration of 0.015 mg/m (16 m /day) for several hours is sufficient to control flies and mosquitoes in the home. The rapid disappearance of dichlorvos from air and surfaces in the home makes it ideally suited for treating insect infestations. A n airborne exposure level of 30 mg/m is capable of killing rats within a four-hour period. Industrial hygiene guides limit workplaces concentrations to 1.0 m g / m . A safety factor therefore exists for this chemical between the concentration required to control insects and those we 11-tolerated by adult healthy persons in the workplace. For safe use in the home, it would be desirable not to exceed a level of 0.01 m g / m in the room air for long-term exposure. Maddy et al. (VI) monitored dichlorvos residues in a home after fogging. In one or two hours after fogging, airborne residues were below 1.0 m g / m . Wipe samples taken from smooth surfaces during this study indicated that levels as high as 0.8 mg/100 c m were present on smooth surfaces after fogging. 3

3

3

3

3

3

2

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These surface residues persisted over a period of seven hours. According to the monitoring and efficacy data on hand, the amount of dichlorvos initially released into the rooms, 2.8 to 5.7 m g / m , exceeded the amount, 0.015 m g / m , needed to control insects and might well be hazardous for some persons. A level of 0.015 m g / m was finally reached in one room 24 hours after the aerosol was released into the room. By using the same values as were used for propoxur, a calculation of a worst case exposure for an infant was made and is shown in Table IV. 3

3

3

TABLE IV. CALCULATION OF A WORST CASE EXPOSURE DOSE FOR AN INFANT EXPOSED TO DICHLORVOS Using the same assumptions as were used for propoxur but assuming 100 percent absorption from the skin. 3

Maximum air concentration after 30 minutes aeration = 0.75 m g / m . Maximum surface concentratio Inhalation exposure

For 4.6 m

2

=

cone, x resp. min. vol. x exposure time

=

0.75 m g / m x 0.5 m /hr x 24 hr

=

9 mg

3

3

2

(50 ft ) of carpet contacted by the child

Dermal exposure

=

Surface area contacted x dichlorvos residue available

=

4.6 m

=

368 mg — oral exp.

2

x 0.8 mg/100 c m

2

x 100

Assume all dichlorvos on skin of the hands is licked off. Oral exposure

Net dermal exposure

Total dose

=

Dermal exp. x fractional surface area absorbed

=

368 x 0.096

=

35 mg

=

368 — 35 mg

=

333 mg

=

9 + 333 + 55

„ mg/kg

7.5 =

50 mg/kg

It should be noted, however, that this calculation does not take into consideration the rapid metabolic breakdown of dichlorvos; consequently, a value of 50 mg/kg following a day's play on a treated carpet will be unreasonably high. However, the differences in the concentration attained versus the concentration required for insecticidal purposes suggest that dichlorvos should be reevaluated for efficacy as well as safety.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Chlorpyrifos Chlorpyrifos, an organic phosphorothioate insecticide, the structure of which is shown below,

was introduced into the marketplace by Dow Chemical Company in 1965 for the control of mosquitoes and other household pests. The dermal LD50 of this pesticide was determined to be 202 mg/kg in the rat (9) and the acute oral LD50 in the rat is 135 mg/kg (12). Studies with this cholinesterase inhibitor in several animal species, including man, indicate that plasma cholinesterase activity is depressed to a greater extent than red cell acetylcholinesterase. For example, workers applying chlorpyrifos were exposed to water suspension spraying mosquitoes. Plasma commensurate reduction in red cell cholinesterase (13). A daily dose of 0.08 mg/kg for six months failed to depress red cell cholinesterase in the monkey, while levels of 0.4 and 2.0 mg/kg/day depressed plasma and red cell activity. Dogs wearing flea collars containing 8 percent chlorpyrifos lived without detectable clinical symptoms even though plasma cholinesterase was depressed 100 percent below that of preexposure values (14). A dermal absorption study (15) in human volunteers using 0.5 and 5.0 mg/kg of chlorpyrifos indicated that less than 3 percent of the applied dose was absorbed. A treatment area of less than 100 c m was used. The dose was left on the bare skin of the forearm for a period of 12 to 20 hours and then removed by showering. No changes in red cell acetylcholinesterase or plasma cholinesterase activity were seen. The half-life for the absorption into blood was 14 to 31 hours. The elimination halflife was determined from an oral study. The results of the dog flea collar and human volunteer studies need to be considered together. In the dog study the total body surface area was exposed to several milligrams of chlorpyrifos each day, while in the human study an area of only 100 c m was exposed (100 c m divided by 20,000 c m x 100 = 0.5 percent of total surface) to a single dose of the pesticide. In addition, chlorpyrifos was left on the skin of the dog for an extended period of time, while in the human studies chlorpyrifos was washed off 12 to 20 hours after application. The total surface area involved, the concentration per unit of surface area, and the length of the exposure period are important factors that must be considered. A dermal dose cholinesterase-response study is needed to clarify this relationship. 2

2

2

2

In separate studies involving chlorpyrifos, the safe use of chlorpyrifos was investigated in the home environment. Naffziger et al. (16) monitored the application of chlorpyrifos to floor coverings. Standard air sampling/gas chromatograph analytical procedures were used for air residues. Wiping procedures were used for carpets and vinyl floor covers. Table V gives the dose calculation for exposure of infants to chlorpyrifos using the same assumptions as were used for both propoxur and dichlorvos. In a personal communication from Dow sent on June 17, 1985, a lower dose is reported based upon a number of factors including the statement that chlorpyrifos is only 3 percent absorbed through the skin. However, the possibility of a toxic dose being absorbed by an infant under the conditions of application exists for chlorpyrifos as it does for propoxur and dichlorvos. The acceptable daily intake established by the World Health Organization for chlorpyrifos is 0.0015 mg/kg/day, and the minimal human response level has been reported to be 0.03 mg/kg (17).

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE TABLE V. CALCULATION OF A WORSE CASE EXPOSURE DOSE FOR AN INFANT EXPOSED TO CHLORPYRIFOS Using the same assumptions as were used for propoxur, but assuming 100 percent absorption from the skin. Assume that the 0.5 percent spray is applied undiluted and that one hour is allowed for drying (i.e., prior to contact with the surface). Air concentration

=

0.015 m g / m

3

(kitchen dinette)

Average surface concentration = 0.0433 mg/100 c m Inhalation exposure

For 4.6 m

2

2

=

cone, x resp. min. vol. x exposure time

=

0.015 m g / m x 0.5 m / h r x 24 hour

=

0.18 mg

3

3

2

(50 ft ) of surfac

Dermal exposure

=

Surface area contacted x chlorpyrifos res. available

=

4.6 m

=

4.6 x 0.0433 x 100 mg

=

19.9 mg

2

x 0.0433 mg/100 c m

2

Assuming all chlorpyrifos on the skin of the hands is licked off Oral exposure

Net dermal exposure

Total dose

=

Dermal exp. x fractional surface area absorbed

=

19.9x0.096

=

1.91 mg

=

19.9—1.91

=

18.0 mg

=

0.18 + 18.0+

1.91

7.5 =

2.68 mg/kg

Limitations of the Dose Calculations There are limitations in the validity of the doses calculated, which are mainly: 1. They do not consider metabolic breakdown (e.g., as with dichlorvos). 2. They do not consider cumulative effects. 3. The data are very limited (e.g., surface deposition data were located for only three pesticide chemicals). 4. Dermal absorption data are available for only a few pesticide chemicals. It should be noted that more information on rate of metabolic breakdown and extent of dermal absorption would generally result in lower values for the doses. On the other hand, the calculations do not address the probability that a child will continue to play daily on a treated surface. Although it is recognized that with most pesticides the level of chemical on the surface under consideration will attenuate each day, the repeated exposure, albeit to a successively reduced dose, will contribute to the

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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continued lowering of acetylcholinesterase in blood and other tissues and consequent possible development of toxic symptoms.

Data That Would Be Useful for Setting Safe Levels Should a registrant wish to attempt to develop data which might indicate that a certain level of a cholinesterase inhibitor could be used indoors in room air or on open floor areas, carpets, furniture, or bedding, a considerable amount of new data would be needed. Examples of the kind of data that would be needed are safe indoor levels in the air and on treated surfaces such as smooth floors and carpets as well as furniture and bedding. T o establish these levels for a pesticide, the following information would be required, using standardized methods: 1. Levels of pesticide residues in room air through time after application. 2. Bioavailability of residues on surfaces (smooth floors, carpets, furniture, and bedding) through time after application. 3. Rate of transfer from surfaces (smooth floors, carpets, furniture, and bedding) to the skin and clothing of individuals 4. Animal dose-response dat would be necessary to determin in a product required to inhibit 50 percent red cell cholinesterase activity. These data should be used to evaluate and establish safe levels based on the procedure used for dislodgeable field foliage residues (18).

Methods for Collecting Required Data Methods for collecting required data involve extraction procedures and information on the rate of transfer to the subject, which are as follows: 1. Bioavailability a. Solvent wipes b. Solvent extraction procedures 2. Rate of transfer of dislodgeable pesticide residues a. Use of adults with pesticides b. Use of surrogate tracer materials (e.g., fluorescent clothing brighteners) c. Use of household pets such as small dogs Techniques for measuring bioavailability are currently limited to solvent wipes and solvent extraction procedures. Wiping procedures are preferred when it is impossible to remove samples of carpet or fabric for extraction. Carpet with pile not exceeding 1 cm. in depth should be used. In order to minimize the variability inherent in the wipe procedure, it is recommended that samples of carpet (12 x 12 inches) and fabric be used to collect pesticide residues. These samples may be extracted by a continuous solvent (water-surfactant) extraction procedure similar to the one used for dislodgeable residues on foliage (19). This procedure would make it possible for registrants to develop consistent and reliable information on the amount of dislodgeable pesticide deposited on or remaining on smooth floor surfaces (such as vinyl), carpet, furniture fabrics, and bedding through time subsequent to applications. Studies are needed to determine the rate of transfer (jug/hr) of the dislodgeable pesticide residue to the skin or clothing of individuals coming in contact with the treated surfaces. A study was conducted involving the transfer of the organophosphorus pesticide propetamphos (Safrotin) from a furniture fabric to clothing (20). The study involved a single individual repeatedly sitting and standing in order to simulate a typical home-type exposure. This procedure provided information on the rate of transfer of pesticide residues involving adults, but did not address the transfer of residues to young children playing on treated areas. This is a difficult problem, because studies involving the direct transfer of pesticides to the skin and/or clothing of young children are an unacceptable form of human experimentation. It may be quite possible to address this indirectly by using surrogate tracer materials such as nontoxic

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

fluorescent clothing brighteners to quantify the transfer of residues from surfaces to skin or clothing. If this is possible, information on the relationship (ratio of brightener to pesticides) between the tracer and the pesticide under investigation will have to be independently developed. Dogs might be used as animal models for developing such data in a household setting. Procedures already exist for determining the relationship between a dermal dose and cholinesterase inhibition (7). Those procedures could be extended to cover absorption via lungs and the respiratory tract. The data collected (rate of transfer, dislodgeable residue, and dermal dose response information) could be used to set safe levels in a manner described by Knaak and Iwata (21). The procedures of Knaak et al. (22) require that a safe level be known for some pesticides presently in use indoors. A t the present time, the recommended use of propoxur and chlorpyrifos are being re-examined and therefore cannot be used as standards for setting acceptable levels indoors.

Conclusions and Recommendations This chapter indicates that i set acceptable levels for pesticid to considerable skin contact in households. Information from studies performed by the registrants would be needed on household residues deposited from foggers, compressed air sprayers, and other application procedures to determine the amount of the residues remaining in air and on surfaces after each application. The levels should be compared against safe levels still to be established for residues in air and levels for surface residues for each pesticide. Should a registrant wish to justify the use of a cholinesterase inhibitor in treatment of smooth floor surfaces, rugs, furniture, bedding, or entire rooms where people reside, it is recommended that the following data be submitted on each active ingredient such as propoxur, dichlorvos, and chlorpyrifos or any other cholinesterase inhibitor. 1. Information on room air levels of the pesticide and total dislodgeable residues on smooth floors, carpets, furniture, and bedding after application of the product containing the active ingredient of interest. The dissipation of these residues should be followed through time or until the residues completely dissipate (i.e., 2, 4, 8, and 24 hr; 3, 6, 9, 12, 21, 24, 27, and 30 days). 2. Dissipation data plotted in j u g / c m against time (semi-log paper) should be determined and therefore, if possible, the half-life in the room air and of the dislodgeable residue on the surface from which it was taken. 3. Rate of transfer (jug/hr) of the dislodgeable residue from smooth floors, carpet, furniture-fabric, and to the skin of surrogate animals. Five-month-old Beagle dogs should be used to study contact exposure in treated rooms. The same animals should be exposed sequentially to different levels of dislodgeable residues present in the house, starting with the level deposited at application of twice the recommended amount and then exposing the animals at the recommended amount. These exposures should begin just as soon as the application has been completed and any ventilation period on the label has expired. Separate exposure studies should begin when residues are found to be one-half those at the time of application and also when they reach one-fourth those found at the time of application. Dislodgeable residue in / u g / c m versus dose transferred to the animal in //g/hr should be plotted. The slope of the line in c m / h r should be determined. T o remove residues for measurement, the dogs can be washed with soap and water at sampling times. 2

2

2

4.

Dermal dose-cholinesterase response data in the rat for the technical or active material can be determined using the method of Knaak et al. (22).

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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5.

A table of safe levels should be calculated using acceptable daily exposure levels (use results of dermal dose cholinesterase response studies) as indicated by Iwata etal. (18). In addition, for individual products formulated from these active ingredients, data should be required on air and surface residues resulting from the application of the products. Data is needed on the amount of dislodgeable residues that are deposited on smooth flooring, carpet, furniture, and bedding by applying the pesticide product in accordance with label-use instructions. In presenting the data, the registrant should use a dissipation curve developed with the technical material (active ingredient) to estimate when a safe level would be present from the use of a product in the room; the registrant should determine the level of dislodgeable residues at that time. If air and dislodgeable residues are at a safe level for infants to play in the room immediately at the end of a ventilation reentry period specified on the label, then the product should be considered safe to use as directed. In the State of California, regulatory action has already been taken in the form of advice to the registrant of the data needed on insecticide chemicals in products used indoors if registration is to continue to be permitted (reevaluation). The manufacturers and others who sell chlorpyrifos addition, similar action will carbaryl (Sevin), propetampho

Literature Cited 1. Pomerance, H. H., Growth Standards in Children, Harper and Row, Hagerstown, M D., 1979, 225 pages. 2. Vaughan, V. C., III; McKay, R. J.; Nelson, W. E.; Textbook of Pediatrics, 10th Edition, Saunders, Philadelphia, London, Toronto 1975, 1,876 pages. 3. Wright, J. W.; Fritz, R. F.; Hocking, K. S.; Babione, R.; Gratz, N. G.; Pal, R.; Stiles, A. R.; Vandekar, M. Bull. World Health Org., 1969, 46, 67-90. 4. Hackathorn, D. R.; Eberhart, D. C., 1983, Report No. 1-83-42, Mobay Chemical Corporation. 5. Vandekar, M.; Plestrina, R.; Wilhelm, K. Bull. World Health Org., 1971. 6. Raabe, O. 1985 personal communication. 7. Knaak, J. B.; Wilson, B. W., 1984, American Chemical Society Symposium Series No. 273. 8. Feldman, R. J.; Maibach, H. I. Toxicol. Appl. Pharmacol., 1974, 28, 126-132. 9. Gaines, T. B., Toxicol. Appl. Pharmacol., 1969,14,515-534. 10. Perkow, W., Chem. Ber„ 1954, 87, 755-758. 11. Maddy, K. T.; Edmiston, S.; Fredrickson, A. S., 1981, Calif. Dept. Food Agric. report number HS-897. 12. McCollister, S. B.; Kociba, R. J.; Humiston, C. G.; McCollister, D. D. Food Cosmet. Toxicol., 1974, 12, 45-61. 13. Eliason, D. A.; Cranmer, M. F.; von Windeguth, D. C.; Kilpatrick, J. W.; Suggs, J. E.; Schoof, H. F., Mosquitos News, 1969, 29, 591-595. 14. Boyd, J. Pat, P.A.C.E. International, Dallas, Texas, 1985. 15. Nolan, R. J.; Rick, D. L.; Freshour, N. L.; Sanders, J. H. Toxicol. Appl. Pharmacol., 1984, 73, 8-15. 16. Naffziger, D. H.; Spfenkel, R. J.; Mattler, M. P., Down to Earth. 1985, 41, 7-10. 17. 1972 Evaluations of Some Pesticide Residues in Food. The Monographs. World Health Organization, Geneva, 1973, 161-162. 18. Iwata, Y.; Knaak, J. B.; Carmen, G. E.; Dusch, M. E.; Gunther, F. A., J. Agr. Food Chem., 1982, 30, 215-222. 19. Iwata, Y.; Knaak, J. B.; Spear, R. C.; and Foster, R. J., Bull. Environ Contam. Toxicol., 1977, 18, 649-655. 20. Labriola-Tomkins, T.; Ranion, T., 1982, Safrotin EC Carpet Study, (A) Cholinesterase dose/response; (B) Dislodgeable Residues on Carpets, Sandoz, Inc., Crop Protection, East Hanover, New Jersey, 1977, 18, 649-655.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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21. Knaak, J. B.; Iwata, Y. 1982 American Chemical Society, Symposium Series No. 182, 23. 22. Knaak, J. B.; Schlocker, P.; Ackerman, C. R.; Seiber, J. N., Bulk Environ. Contam. Toxicol., 1980, 24, 796-804. RECEIVED February 10, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 25

The Environmental Protection Agency's Use of Biological Monitoring Data for the Special Review of Alachlor Curt Lunchick, Gary Burin, Joseph C. Reinert, and Karen E. Warkentien Office of Pesticide Programs, Hazard Evaluation Division (TS-769C), Exposure Assessment Branch, Environmental Protection Agency, Washington, DC 20460

In conducting herbicide alachlor Protection Agency (EPA) evaluated biological monitoring studies conducted by the Monsanto Chemical Company, the registrant of alachlor. The biological monitoring studies allowed the Agency to estimate the internal dosage of alachlor received by workers during mixing/ loading and ground boom application of alachlor from enclosed tractor cabs. The Monsanto study contained only four replicates per formulation, used Monsanto employees, and only used enclosed application vehicles. Because internal dosage estimates from bio­ logical monitoring data are chemical-specific and, therefore, not amenable to the use of surrogate data, the Agency evaluated patch exposure studies to ensure representation of a wider range of potential exposure s i t u ­ ations. A Monsanto patch exposure study i n which alachlor was applied similarly to the biological monitoring studies and six expo­ sure studies from the published literature provided approximately 100 exposure r e p l i ­ cates employing a variety of commonly used application equipment. The exposure studies established a two orders of magnitude range of exposure to which the Monsanto exposure estimates were at the low end. This range was transferred to the biological monitoring dosage establishing a dosage range of 0.0054 to 0.54 ug/kg body weight (bwt)/lb active ingredient (ai) when open pour mixing/loading occurs and a range of 0.0034 to 0.34 ug/kg bwt/lb a i when a mechanical transfer loading system is used. This chapter not subject to U.S. copyright Published 1989 American Chemical Society

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328

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE I n t h e U n i t e d S t a t e s , t h e O f f i c e o f P e s t i c i d e Programs (OPP) o f EPA h a s t h e r e s p o n s i b i l i t y o f r e g u l a t i n g t h e use o f p e s t i c i d e s under t h e a e g i s o f t h e F e d e r a l I n s e c t i c i d e , F u n g i c i d e , and R o d e n t i c i d e A c t (FIFRA). Under FIFRA, p e s t i c i d e r e g i s t r a n t s have t h e r e s p o n s i b i l i t y o f p r o v i d i n g t h e h e a l t h and s a f e t y d a t a n e c e s s a r y t o s u p p o r t t h e r e g i s t r a t i o n o f t h e i r p r o d u c t s . The Agency, i n t u r n , i s charged w i t h e v a l u a t i n g t h e s e d a t a and d e t e r m i n i n g t h a t t h e r e g i s t e r e d uses o f t h e p r o d u c t s do not p r o d u c e an u n r e a s o n a b l e a d v e r s e e f f e c t on t h e environment. To a s s i s t t h e r e g i s t r a n t s i n p r o v i d i n g d a t a on t h e exposure t o i n d i v i d u a l s h a n d l i n g p e s t i c i d e s , t h e Agency has p u b l i s h e d t h e P e s t i c i d e Assessment G u i d e l i n e s , S u b d i v i s i o n U: A p p l i c a t o r Exposure M o n i t o r i n g . S u b d i v i s i o n U i s intended t o a i d r e g i s t r a n t s i n conducting f i e l d s t u d i e s t h a t estimate the l e v e l o f exposure t o i n d i v i d u a l s who a r e o c c u p a t i o n a l l y exposed t o p e s t i c i d e s d u r i n g a p p l i c a t i o n an l o a d i n g , and f l a g g i n g H i s t o r i c a l l y , OPP has recommended t h a t a p p l i c a t o r e x p o s u r e s t u d i e s be c a r r i e d o u t u s i n g p a s s i v e d o s i m e t r y . T h i s t e c h n i q u e measures t h e amount o f c h e m i c a l i m p i n g i n g on t h e s u r f a c e o f t h e s k i n o r t h e amount o f c h e m i c a l a v a i l a b l e f o r i n h a l a t i o n as d e t e r m i n e d through t h e use o f a p p r o p r i a t e t r a p p i n g d e v i c e s . P a t c h e s o f v a r i o u s c o n s t r u c t i o n a r e used t o t r a p r e s i d u e s w h i c h c o u l d r e s u l t i n d e r m a l exposure t o v a r i o u s body p a r t s . P o t e n t i a l i n h a l a t i o n e x p o s u r e i s g e n e r a l l y measured u s i n g a p e r s o n a l s a m p l i n g pump w i t h an i n t a k e p l a c e d c l o s e t o t h e w o r k e r ' s b r e a t h i n g zone. A n o t h e r t y p e o f m o n i t o r i n g t h a t may be used t o e s t i m a t e human i n t e r n a l dosage t o p e s t i c i d e s i s b i o l o g i c a l m o n i t o r i n g . T h i s method e s t i m a t e s t h e l e v e l o f i n t e r n a l dosage f r o m e i t h e r a measurement o f body b u r d e n i n s e l e c t e d t i s s u e s o r f l u i d s , o r from t h e amount o f p e s t i c i d e o r m e t a b o l i t e e x c r e t e d from t h e body. Most s t u d i e s u s i n g b i o l o g i c a l m o n i t o r i n g methods have i n v o l v e d t h e c o l l e c t i o n o f b l o o d , urine, or both. P r i o r to designing a b i o l o g i c a l monitoring s t u d y , t h e p h a r m a c o k i n e t i c s ( i n c l u d i n g m e t a b o l i s m and e x c r e t i o n ) o f t h e p e s t i c i d e must be known s o t h a t t h e a p p r o p r i a t e t i s s u e o r f l u i d , as w e l l as t i m e p e r i o d s f o r m o n i t o r i n g , can be chosen. W i t h o u t t h i s i n f o r m a t i o n , e x t r a p o l a t i o n back t o dose i s not p o s s i b l e . E s t i m a t i n g o c c u p a t i o n a l exposure by p a s s i v e dosimetry o r b i o l o g i c a l m o n i t o r i n g o f f e r s d i s t i n c t advantages and d i s advantages (see T a b l e I ) . A major advantage o f b i o l o g i c a l m o n i t o r i n g i s t h a t , when t h e p h a r m a c o k i n e t i c s o f a p e s t i c i d e a r e u n d e r s t o o d s u f f i c i e n t l y , t h e a c t u a l a b s o r b e d dose may be c a l c u l a t e d . W i t h p a s s i v e d o s i m e t r y , o n l y t h e amount o f chemical p o t e n t i a l l y a v a i l a b l e f o r absorption or i n h a l a t i o n i s measured. T h i s amount o f c h e m i c a l i s t h e e x p o s u r e . U n l i k e b i o l o g i c a l monitoring, passive dosimetry requires independent e s t i m a t e s o f d e r m a l and l u n g a b s o r p t i o n t o

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

A c t u a l dose may b e m e a s u r e d . Unnecessary t o a d j u s t f o r value o f garment/protective c l o t h i n g . E f f e c t s o f comp l a c e n c y f a c t o r overcome.

a

a

Advantages Routes and a r e a s o f exposure a r e known. Routine experimental design and e x e c u t i o n . P a r t i c i p a n t under s u p e r v i s i o n of i n v e s t i g a t o r . G e n e r i c d a t a b a s e s may b e created.

C o n s i d e r e d most i m p o r t a n t .

Biological monitoring

Passive dosimetry

P h a r m a c o k i n e t i c s must b e known. Routes o f exposure cannot b e distinguished. D i f f i c u l t t o ensure p a r t i c i p a n t cooperation. P o t e n t i a l p r o b l e m s when u s i n g invasive techniques.

3

a

Disadvantages Dermal and r e s p i r a t o r y a b s o r p t i o n must be e s t i m a t e d . E x t r a p o l a t i o n from p a t c h t o body s u r f a c e a r e a must be made. Not a l l exposure s c e n a r i o s a r e amenable.

T a b l e I . THE PERCEIVED ADVANTAGES AND DISADVANTAGES OF ESTIMATING OCCUPATIONAL EXPOSURE WITH PASSIVE DOSIMETRY AND BIOLOGICAL MONITORING

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estimate dosage. These absorption estimates are generallyvery d i f f i c u l t to derive and interpret using a v a i l a b l e data from i n vivo and i n v i t r o systems. In addition, when using passive dosimetry, some assumptions concerning the value of normal clothes or protective clothing i n intercepting residues must a l s o be used to attempt to estimate actual dose. The major advantage with passive dosimetry i s that the r e l a t i v e contributions of the inhalation and dermal routes and of the d i f f e r e n t body areas to t o t a l dermal exposure can be r e a d i l y established. This i s extremely important f o r evaluating and recommending exposure mitigation measures. For example, i f i t can be demonstrated that a substantial f r a c t i o n of t o t a l exposure for a work day occurs to the hands, forearms, and face during the ten minutes a farmer pours the concentrated solution of a p e s t i c i d e formulation into the mix tank, t o t a chemical-resistant gloves mixing system for t h i s short duration/high exposure period. A b i o l o g i c a l monitoring study, which provides a composite p i c t u r e of dosage, would not d i s t i n g u i s h the various routes of exposure. In addition, since b i o l o g i c a l monitoring i s t y p i c a l l y c a r r i e d out a f t e r a complete work day, the r e l a t i v e contributions of the d i f f e r e n t work a c t i v i t i e s to t o t a l i n t e r n a l dose could not be distinguished, assuming the individual had engaged i n more than one work a c t i v i t y . Registrants have extensive experience with carrying out passive dosimetry studies where the study p a r t i c i p a n t s are t y p i c a l l y under the supervision of the investigator during the e n t i r e monitoring period. This i s generally not the case i n b i o l o g i c a l monitoring studies, f o r example, when 24-hour urine voids are c o l l e c t e d . Many passive dosimetry techniques require extrapolation from the residues on the small surface area of the trapping devices to e n t i r e body surface areas, a process which introduces an error f a c t o r . C r u c i a l areas of exposure may a l s o be missed. I t should be recognized that not a l l exposure scenarios are amenable to passive dosimetry techniques. Examples include measuring exposure t o p e s t i c i d e s i n swimming pools, i n dishwashing detergents, or measuring dermal exposure to v o l a t i l e organic chemicals. On the other hand, there are p o t e n t i a l l e g a l and e t h i c a l problems associated with the use of invasive techniques i n f i e l d studies. B i o l o g i c a l monitoring may hold some promise f o r dealing with what has been c a l l e d the "complacency f a c t o r . " I t i s widely recognized that when workers are handling p e s t i c i d e s that are known to be h i g h l y acutely t o x i c , e.g., parathion, extra care i s exercised. I t i s thought that the r e s u l t s of passive dosimetry studies may not e n t i r e l y capture t h i s extra care, and therefore may overestimate exposure for these p e s t i c i d e s . For example, i f during the course of a routine work cycle a worker's c l o t h i n g becomes contaminated, t h i s i n d i v i d u a l i s l i k e l y to change clothes prompted by h i s

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

25. LUNCHICK ET AL.

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awareness o f t h e h i g h l y t o x i c n a t u r e o f t h e p e s t i c i d e b e i n g h a n d l e d . C o n v e r s e l y , i f d u r i n g a work a c t i v i t y d e s i g n e d t o m o n i t o r e x p o s u r e , some p e s t i c i d e impinges on a t r a p p i n g d e v i c e , w h i c h i n p r i n c i p l e does n o t a l l o w p e n e t r a t i o n t o the c l o t h i n g , work i s l i k e l y t o c o n t i n u e u n i n t e r r u p t e d t o t h e c o n c l u s i o n o f the monitoring p e r i o d . A r e l a t e d advantage o f p a s s i v e d o s i m e t r y i s the a b i l i t y t o create l a r g e generic databases. In t h i s context, " s u r r o g a t e " o r " g e n e r i c " d a t a a r e d e f i n e d as exposure monitoring data c o l l e c t e d f o r other p e s t i c i d e chemicals a p p l i e d u s i n g comparable a p p l i c a t i o n methods and under s i m i l a r c o n d i t i o n s as f o r t h e p e s t i c i d e under assessment. The mechanics o f t h e use o f s u r r o g a t e d a t a have b e e n d i s c u s s e d i n p u b l i c f o r a and i n t h e p u b l i s h e d s c i e n t i f i c l i t e r a t u r e i n recent years [ 1 - 4 ] . A b a s i c assumption i n the c r e a t i o n o f g e n e r i c d a t a b a s e s i s t h a t i n many a p p l i c a t i o n scenarios, the p h y s i c a the chemical p r o p e r t i e i n d e t e r m i n i n g t h e l e v e l o f e x p o s u r e . N o t e t h a t , when u s i n g p a s s i v e d o s i m e t r y methods, what i s measured i s t h e amount o f c h e m i c a l i m p i n g i n g on the s k i n s u r f a c e , o r a v a i l a b l e f o r i n h a l a t i o n , n o t t h e a c t u a l dose r e c e i v e d . F a c t o r s such as d e r m a l and l u n g p e n e t r a t i o n i n f l u e n c e t h e l a t t e r , and a r e , o f c o u r s e , e x p e c t e d t o be h i g h l y chemical-dependent. A r e v i e w o f a l l a v a i l a b l e i n f o r m a t i o n on p e s t i c i d e exposure d u r i n g a p p l i c a t i o n a c t i v i t i e s s u p p o r t s the use o f s u r r o g a t e d a t a f o r exposure a s s e s s m e n t s . Alachlor

Studies

I n November 1984, EPA i s s u e d t h e Guidance f o r t h e R e r e g i s t r a t i o n o f P e s t i c i d e P r o d u c t s C o n t a i n i n g A l a c h l o r as t h e A c t i v e Ingredient"! I n t h a t document, t h e Agency s t a t e d i t s d e t e r m i n a t i o n t h a t a l a c h l o r met one o f t h e r i s k c r i t e r i a ( o n c o g e n i c p o t e n t i a l ) used t o d e t e r m i n e whether i t causes u n r e a s o n a b l e a d v e r s e e f f e c t s [40 CFR 1 6 2 . 1 1 ( a ) ( 3 ) ( i i ) ( A ) ] . I n r e s p o n s e t o t h a t d e t e r m i n a t i o n , a S p e c i a l Review, i n w h i c h t h e r i s k s and b e n e f i t s o f c o n t i n u e d use o f a p e s t i c i d e a r e i n t e n s i v e l y examined, was i n i t i a t e d f o r a l a c h l o r i n December 1984. A l a c h l o r i s an a c e t a n i l i d e h e r b i c i d e t h a t has been p r o d u c e d i n t h e U n i t e d S t a t e s b y Monsanto C h e m i c a l Company s i n c e 1969. Almost a l l o f t h e a l a c h l o r used i n t h e U n i t e d S t a t e s i s a p p l i e d b y t r a c t o r - d r a w n ground boom s p r a y e r s . Monsanto s u b m i t t e d t h r e e b i o l o g i c a l m o n i t o r i n g s t u d i e s t o EPA t o s u p p o r t t h e c o n t i n u e d r e g i s t r a t i o n o f a l a c h l o r . I n a d d i t i o n , s u p p o r t i n g p h a r m a c o k i n e t i c s t u d i e s were e v a l u a t e d . These s t u d i e s i n d i c a t e d t h a t an a v e r a g e o f 87 p e r c e n t o f a l a c h l o r i n l a b o r a t o r y p r i m a t e s i s e x c r e t e d i n t h e u r i n e as m e t a b o l i t e s i n two c h e m i c a l c l a s s e s . U s i n g o n l y t h e s e d a t a , EPA was a b l e t o c a l c u l a t e t h e i n t e r n a l dosage o f a l a c h l o r , based on t h e t o t a l q u a n t i t y o f t h e two m e t a b o l i t e c l a s s e s , e x p r e s s e d as a l a c h l o r , measured i n t h e p a r t i c i p a n t ' s u r i n e . In the f i r s t o f the t h r e e s t u d i e s [ 5 ] , e i g h t a p p l i c a t o r s

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332

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE were s t u d i e d b y Monsanto, f o u r u s i n g t h e L a s s o EC f o r m u l a t i o n and f o u r w i t h t h e L a s s o MT f o r m u l a t i o n . The a l a c h l o r was incorporated i n t o corn f i e l d s a t 4 l b a i per acre ( l b a i / A ) . The s u b j e c t s were Monsanto employees who wore g o g g l e s and e l b o w - l e n g t h r u b b e r g l o v e s d u r i n g m i x i n g and l o a d i n g , and b o o t s , t r o u s e r s , l o n g - s l e e v e d s h i r t , and a cap d u r i n g the e n t i r e o p e r a t i o n . The m i x i n g and l o a d i n g i n v o l v e d open p o u r i n g from t h e p e s t i c i d e c o n t a i n e r s . The a p p l i c a t i o n was conducted from e n c l o s e d cab t r a c t o r s and 20 a c r e s were t r e a t e d b y each s t u d y p a r t i c i p a n t . U r i n e was c o l l e c t e d p r i o r t o use o f a l a c h l o r and f o r f i v e days t h e r e a f t e r . The mean i n t e r n a l dosage f o r L a s s o EC was e s t i m a t e d t o be 0.0066 ug/kg bwt/lb a i . The second s t u d y [ 6 ] m o n i t o r e d t h e i n t e r n a l dosage r e c e i v e d d u r i n g m i x i n g , l o a d i n g , and a p p l y i n g L a s s o M i c r o Tech and L a s s o WDG. Four r e p l i c a t e s were monitored f o r each f o r m u l a t i o n and a l l s t u d ©nployees. As i n t h e p o r a t e d i n t o c o r n f i e l d s a t 4 l b a i / A . Each i n d i v i d u a l t r e a t e d 20 a c r e s and a p p l i e d t h e a l a c h l o r from e n c l o s e d t r a c t o r cabs. The s t u d y p a r t i c i p a n t s wore l o n g - s l e e v e d s h i r t s and l o n g p a n t s a t a l l t i m e s and wore g o g g l e s , r u b b e r g l o v e s , and rubber o v e r s h o e s d u r i n g t h e m i x i n g and l o a d i n g . U r i n e was c o l l e c t e d p r i o r t o use o f a l a c h l o r and f o r f i v e days t h e r e a f t e r . The mean i n t e r n a l dosage o f a l a c h l o r was e s t i m a t e d t o be 0.0038 ug/kg b w t / l b a i f o r L a s s o M i c r o - T e c h and 0.0059 ug/kg b w t / l b a i f o r L a s s o WDG. The t h i r d s t u d y [ 7 ] m o n i t o r e d t h e i n t e r n a l dosage o f a l a c h l o r r e c e i v e d when a c l o s e d l o a d i n g system was employed d u r i n g m i x i n g and l o a d i n g and an e n c l o s e d t r a c t o r cab was u s e d t o i n c o r p o r a t e a l a c h l o r a t 4 l b a i / A . L a s s o EC was t h e f o r m u l a t i o n used and t h e f o u r s t u d y p a r t i c i p a n t s were Mons a n t o employees. The L a s s o EC was t r a n s f e r r e d from a 100g a l l o n b u l k tank t o t h e s p r a y t a n k s u s i n g a S c i e n c o pump. The workers wore l o n g - s l e e v e d s h i r t s , l o n g p a n t s , g o g g l e s , r u b b e r g l o v e s , and rubber overshoes d u r i n g t h e m i x i n g and l o a d i n g r o u t i n e . A l a c h l o r was i n c o r p o r a t e d i n t o c o r n f i e l d s a t 4 l b a i / A . E a c h p a r t i c i p a n t t r e a t e d 20 a c r e s from an e n c l o s e d t r a c t o r cab w h i l e w e a r i n g l o n g - s l e e v e d s h i r t s and l o n g p a n t s . U r i n e was c o l l e c t e d p r i o r t o u s i n g a l a c h l o r and f o r f i v e days a f t e r use. The Agency e s t i m a t e d t h e i n t e r n a l dosage o f a l a c h l o r t o be 0.0034 ug/kg b w t / l b a i . I t should be n o t e d t h a t most o f t h e u r i n e samples c o n t a i n e d n o n d e t e c t a b l e l e v e l s o f a l a c h l o r m e t a b o l i t e s and t h e Agency's dosage e s t i m a t e p r e d o m i n a n t l y r e f l e c t s 50 p e r c e n t o f t h e d e t e c t i o n l i m i t o f t h e a n a l y t i c a l method. The Monsanto dosage e s t i mate, w h i c h was l e s s t h a n t h e Agency's, r e f l e c t e d Monsanto's use o f "0" f o r samples c o n t a i n i n g n o n d e t e c t a b l e l e v e l s o f alachlor metabolites. S e v e r a l f a c t o r s p r e v e n t e d EPA from e s t i m a t i n g t h e r i s k t o u s e r s o f a l a c h l o r based s o l e l y on t h e s e b i o l o g i c a l m o n i t o r i n g s t u d i e s . The s t u d i e s were conducted w i t h o n l y f o u r r e p l i cates per f o r m u l a t i o n . S u b d i v i s i o n U of the P e s t i c i d e

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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Assessment G u i d e l i n e s recommends t h a t 15 r e p l i c a t e s o f e a c h work t u n c t i o n be m o n i t o r e d . The study p a r t i c i p a n t s i n a l l t h r e e Monsanto s t u d i e s were employees o f M o n s a n t o . EPA p r e f e r s t h a t p a r t i c i p a n t s i n exposure s t u d i e s be experienced i n d i v i d u a l s who a r e b e i n g m o n i t o r e d w h i l e p e r f o r m i n g t h e i r n o r m a l work f u n c t i o n s t o more a c c u r a t e l y a s s e s s worker exposure. As p r e v i o u s l y discussed, the l a c k o f c o n t r o l over study p a r t i c i p a n t s i n c o l l e c t i n g urine voids i s a perceived d i s a d v a n t a g e o f b i o l o g i c a l m o n i t o r i n g . Monsanto b e l i e v e d t h e b e n e f i t s o f b e t t e r c o n t r o l o v e r u r i n e c o l l e c t i o n outweighed the b e n e f i t s o f u s i n g a c t u a l farmers and, t h e r e f o r e , e l e c t e d t o use i t s own employees t o m a i n t a i n c o n t r o l o v e r u r i n e collection. F i n a l l y , a l l r e p l i c a t i o n s i n v o l v e d the use o f e n c l o s e d t r a c t o r cabs d u r i n g s p r a y i n g . Enclosed t r a c t o r c a b s , when p r o p e r l y u s e d , p r o v i d e a n e f f i c i e n t p h y s i c a l b a r r i e r between t h e a p p l i c a t o r and t h e p e s t i c i d e s p r a y w h i c h w i l l reduce contact w i t e n c l o s e d t r a c t o r cabs a l a c h l o r , EPA c o n c l u d e d t h a t t h e dosage o f a l a c h l o r r e c e i v e d b y i n d i v i d u a l s on t r a c t o r s w i t h o u t c a b e n c l o s u r e s would b e g r e a t e r than that r e c e i v e d b y the i n d i v i d u a l s i n the three s u b m i t t e d Monsanto s t u d i e s . The c o n c e r n s s t a t e d above l e a d EPA t o c o n c l u d e t h a t t h e dosages e s t i m a t e d from t h e Monsanto b i o l o g i c a l m o n i t o r i n g s t u d i e s w o u l d n o t a c c u r a t e l y r e f l e c t t h e r a n g e o f dosages r e c e i v e d by a l l u s e r s o f a l a c h l o r i n the U n i t e d S t a t e s . A l a r g e range would b e e x p e c t e d b e c a u s e o f d i f f e r e n c e s i n s p r a y equipment, i n d i v i d u a l work h a b i t s , and v a r i a t i o n s i n e n v i ­ ronmental c o n d i t i o n s a t the time o f a l a c h l o r use. In order t o e s t i m a t e the range o f exposure l i k e l y t o o c c u r d u r i n g t h e ground boom a p p l i c a t i o n o f a l a c h l o r , EPA e v a l u a t e d a Monsanto a p p l i c a t o r e x p o s u r e s t u d y and o t h e r e x p o s u r e s t u d i e s a v a i l ­ a b l e i n t h e p u b l i s h e d l i t e r a t u r e w h i c h employed p a s s i v e dosimetry. EPA e v a l u a t e d a t o t a l o f s i x s t u d i e s [ 8 - 1 3 ] a v a i l a b l e i n the p u b l i s h e d l i t e r a t u r e that contained s u f f i c i e n t informa­ t i o n t o c o n d u c t an e x p o s u r e assessment f o r ground boom a p p l i c a t o r exposure. The s i x s t u d i e s c o n t a i n e d 101 r e p l i ­ cates. The e x p o s u r e e s t i m a t e s were c a l c u l a t e d assuming t h a t t h e a p p l i c a t o r wore "normal" work a t t i r e c o n s i s t i n g o f l o n g p a n t s and a s h i r t . Because o f v a r i a t i o n s i n how t h e d a t a were r e p o r t e d and i n placement o f t h e p a t c h e s , t h e u s e o f a l o n g - s l e e v e d o r s h o r t - s l e e v e d s h i r t and t h e d e g r e e o f p r o ­ t e c t i o n afforded by the s h i r t v a r i e d . T a b l e I I p r o v i d e s the mean e x p o s u r e s w i t h s p e c i f i c c l o t h i n g a s s u m p t i o n s u s e d f o r each study. In a d d i t i o n t o the s i x p u b l i s h e d s t u d i e s that were r e v i e w e d , a s e v e n t h s t u d y t h a t was c o n d u c t e d b y Monsanto w i t h a l a c h l o r was a l s o e v a l u a t e d [ 1 4 ] . In a l l c a s e s , the e x p o s u r e e s t i m a t e s were n o r m a l i z e d t o a n a p p l i c a t i o n r a t e o f 1.0 l b a i / a c r e t o p e r m i t c o m p a r i s o n o f e x p o s u r e w i t h o u t t h e a p p l i c a t i o n r a t e as v a r i a b l e . The e x p o s u r e e s t i m a t e s f o r t h e s e v e n g r o u n d boom a p p l i ­ c a t o r s t u d i e s v a r i e d o v e r a range o f g r e a t e r t h a n two o r d e r s

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333

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988. 40.0

72.0

9.4

18

12

21

20

23

7

2

Abbott

Dubelman

Maitlen

Staiff

Wojeck

Wolfe

Monsanto

P o t e n t i a l dermal exposure reduced b y 80%

Short-sleeved s h i r t , long pants, 100% p r o t e c t i o n

Long-sleeved s h i r t , long pants, 100% p r o t e c t i o n

Short-sleeved s h i r t , long pants, 100% p r o t e c t i o n

Short-sleeved s h i r t , long pants, 100% p r o t e c t i o n

L o n g - s l e e v e d s h i r t , 50% p r o t e c t i o n Long p a n t s , 100% p r o t e c t i o n

Long-sleeved s h i r t , long pants, 50% p r o t e c t i o n

Clothing Assumptions

A l l e x p o s u r e e s t i m a t e s a r e b a s e d on a n a p p l i c a t i o n r a t e o f 1.0 l b a i / A .

0.15

0.40

0.70

0.93

Mean Exposure (mg/hr)

Number o f Replicates

Study Author

T a b l e I I . DERMAL EXPOSURE DURING GROUND BOOM APPLICATION

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o f magnitude from 0.15 mg/hr i n t h e Monsanto s t u d y t o 72 mg/hr i n t h e Wojeck s t u d y . T h i s v a r i a b i l i t y was n o t unexp e c t e d and r e s u l t e d from d i f f e r e n c e s i n equipment, weather, i n d i v i d u a l work h a b i t s , and s t u d y m e t h o d o l o g i e s . The Mons a n t o and Dubelman s t u d i e s i n v o l v e d a p p l i c a t i o n from e n c l o s e d t r a c t o r c a b s . The A b b o t t s t u d y u s e ! e n c l o s e d t r a c t o r cabs w i t h t h e r e a r window open. Open t r a c t o r s , e n c l o s e d t r a c t o r c a b s , h i g h c l e a r a n c e t r a c t o r s , and booms w i t h s h i e l d e d n o z z l e s were a l l used i n t h e Wojeck s t u d y . M a i t l e n , S t a i f f , and W o l f e d i d n o t d e s c r i b e t h e t r a c t o r s used; however, S t a i f f d i d s t a t e t h a t low p r e s s u r e s p r a y e r s were p a r t i a l l y r e s p o n s i b l e f o r t h e low exposure p o t e n t i a l o b s e r v e d i n t h a t s t u d y . EPA a l s o e s t i m a t e d t h e exposure r e c e i v e d d u r i n g m i x i n g / l o a d i n g when t h e p e s t i c i d e i s poured from t h e p e s t i c i d e c o n t a i n e r . The A b b o t t [ 8 ] and Monsanto [ 1 4 ] s t u d i e s p r o v i d e ! a t o t a l o f 20 r e p l i c a t e s i n w h i c h m i x e r / l o a d e r exposure c o u l d be e x p r e s s e h a n d l e d . The mean d e r m a c a t e s was 0.93 mg/lb a i . The two Monsanto r e p l i c a t e s had an e s t i m a t e d exposure o f 0.077 mg/lb a i . A l l e s t i m a t e s assumed t h a t t h e m i x e r / l o a d e r s wore p r o t e c t i v e g l o v e s . Conclusions I n e v a l u a t i n g t h e exposure d a t a from t h e p u b l i s h e d l i t e r a t u r e , EPA c o n c l u d e d t h a t d e r m a l exposure c o u l d v a r y o v e r a p p r o x i m a t e l y one o r d e r o f magnitude d u r i n g m i x i n g / l o a d i n g and o v e r two o r d e r s o f magnitude d u r i n g ground boom a p p l i c a t i o n . Based on t h e s e ranges, i t was assumed t h a t t h e combined exposure f o r m i x i n g / l o a d i n g and a p p l i c a t i o n would r e a s o n a b l y be e x p e c t e d t o v a r y o v e r two o r d e r s o f magnitude. The Monsanto p a s s i v e d o s i m e t r y s t u d y [ 1 4 ] p r o v i d e d t h e l o w e s t e s t i m a t e s f o r b o t h m i x e r / l o a d e r and ground boom a p p l i c a t o r exposures. Because t h e Monsanto b i o l o g i c a l m o n i t o r i n g s t u d i e s were conducted i n a s i m i l a r manner t o the Monsanto p a t c h s t u d y , EPA c o n c l u d e d t h a t t h e dosage e s t i m a t e s d e r i v e d from t h e b i o l o g i c a l m o n i t o r i n g s t u d i e s r e p r e s e n t e d t h e l o w e s t p o i n t o f a range o f dosages l i k e l y t o o c c u r d u r i n g t h e use o f a l a c h l o r . I t was f u r t h e r assumed t h a t t h e range o f dosage r e c e i v e d d u r i n g m i x i n g / l o a d i n g and ground boom a p p l i c a t i o n would a l s o v a r y o v e r two o r d e r s o f magnitude d e p e n d i n g on equipment used, c o n d i t i o n s o f t h e p r o t e c t i v e c l o t h i n g , weather c o n d i t i o n s , and i n d i v i d u a l work h a b i t s . As p r e v i o u s l y d i s c u s s e d , EPA c o n c l u d e d t h a t t h e dosages r e c e i v e d i n t h e Monsanto b i o l o g i c a l m o n i t o r i n g s t u d i e s d u r i n g open p o u r m i x i n g / l o a d i n g and e n c l o s e d cab ground boom a p p l i c a t i o n were 0.0066 ug/kg b w t / l b a i f o r L a s s o EC, 0.0038 ug/kg b w t / l b a i f o r L a s s o M i c r o - T e c h , and 0.0059 ug/kg b w t / l b a i f o r L a s s o WDG. EPA f u r t h e r c o n c l u d e d t h a t t h e d i f f e r e n t f o r m u l a t i o n s had no a p p r e c i a b l e e f f e c t on t h e dosage r e c e i v e d . T h e r e f o r e , t h e mean dosage o f t h e L a s s o EC, L a s s o M i c r o - T e c h , and L a s s o WDG f o r m u l a t i o n s was c a l c u l a t e d t o be 0.0054 ug/kg b w t / l b a i f o r t h e Monsanto s t u d y p a r t i c i p a n t s .

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EPA c o n c l u d e d t h a t 0.0054 ug/kg b w t / l b a i r e p r e s e n t e d t h e l o w end o f t h e range o f l i k e l y a l a c h l o r dosages and t h a t t h e range o f l i k e l y i n t e r n a l dosage would be 0.0054 t o 0.54 u g / k g b w t / l b a i f o r open pour m i x i n g / l o a d i n g and g r o u n d boom a p p l i c a t i o n . Based on t h e dosage e s t i m a t e o f 0.0034 ug/kg b w t / l b a i from t h e Monsanto b i o l o g i c a l m o n i t o r i n g s t u d y employing c l o s e d l o a d i n g systems and e n c l o s e d t r a c t o r cabs, EPA c o n c l u d e d t h a t t h e i n t e r n a l dosage o f a l a c h l o r r e c e i v e d u s i n g c l o s e ! l o a d i n g systems and ground boom a p p l i c a t i o n would range from 0.0034 t o 0.34 u g / k g / l b a i . T h i s c l o s e d l o a d i n g dosage may b e a n o v e r e s t i m a t e o f a c t u a l dosage, b e c a u s e i t r e f l e c t s t h e use o f 50 p e r c e n t o f t h e d e t e c t i o n l i m i t f o r t h e n o n d e t e c t a b l e u r i n e samples. The Agency's r e v i e w o f m i x e r / l o a d e r exposure d a t a i n d i c a t e s t h a t c l o s e d l o a d i n g systems p r o d u c e more t h a n a t w o - f o l d r e d u c t i o n i n t o t a l exposure. N o n t e c h n i c a l Summary S u b d i v i s i o n U o f t h e P e s t i c i d e Assessment G u i d e l i n e s p e r m i t s t h e use o f e i t h e r p a s s i v e d o s i m e t r y s t u d i e s t o e s t i m a t e worker exposure t o p e s t i c i d e s o r b i o l o g i c a l m o n i t o r i n g s t u d i e s t o e s t i m a t e i n t e r n a l dosage o f a p e s t i c i d e i n workers. D u r i n g t h e S p e c i a l Review o f A l a c h l o r , Monsanto s u b mitted three b i o l o g i c a l monitoring s t u d i e s which permitted EPA t o e s t i m a t e t h e dosage o f a l a c h l o r r e c e i v e d d u r i n g m i x i n g / l o a d i n g and ground boom a p p l i c a t i o n o f a l a c h l o r . EPA d i d n o t b e l i e v e t h a t t h e e s t i m a t e s from t h e s t u d y would p r e d i c t t h e a c t u a l range o f dosage r e c e i v e d d u r i n g use o f a l a c h l o r b y U.S. farmers b e c a u s e o f t h e l i m i t e d number o f r e p l i c a t e s , u s e o f Monsanto employees, and u s e o f o n l y e n c l o s e d t r a c t o r cabs. EPA e v a l u a t e d p a s s i v e d o s i m e t r y s t u d i e s i n w h i c h a l a c h l o r and o t h e r p e s t i c i d e s were a p p l i e d b y ground boom t o e s t a b l i s h a range o f e x p o s u r e s l i k e l y t o o c c u r d u r i n g a l a c h l o r use. T h i s range was t r a n s f e r r e d t o t h e b i o l o g i c a l m o n i t o r i n g s t u d y dosage e s t i m a t e s t o e s t a b l i s h a range o f i n t e r n a l dosage l i k e l y t o o c c u r d u r i n g a l a c h l o r u s e . This range o f i n t e r n a l dosage was u s e ! i n EPA's r i s k assessment f o r o c c u p a t i o n a l r i s k from a l a c h l o r u s e .

Literature Cited 1.

Severn, D . J . In Genetic Toxicology. An Agricultural Perspective; Fleck, R.A.; Hollaender, A . , Eds.; Plenum: New York, 1982; pp 235-242.

2.

Hackathorn, D.R.; Eberhart, D.C. In Dermal Exposure Related to Pesticide Use - Discussion of Risk Assess­ ment; Honeycutt, R.C.; Zweig, G . ; Ragsdale, N . N . , Eds.; American Chemical Society: Washington, DC, 1985; pp 341-355.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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3.

Honeycutt, R.C. In Dermal Exposure Related to Pesticide Use - Discussion of Risk Assessment; Honeycutt, R.C.; Zweig, G.; Ragsdale, N.N., Eds.; American Chemical Society: Washington, DC, 1985; pp 369-375.

4.

Reinert, J.C.; Severn, D.J. In Dermal Exposure Related to Pesticide Use - Discussion of Risk Assessment; Honeycutt, R.C.; Zweig, G.; Ragsdale, N.N., Eds.; American Chemical Society: Washington, DC, 1985; pp 357-368.

5.

Klein, J.J., et a l . Urinary Excretion of Alachlor Residue Following Normal Application of Lasso or Lasso Micro-Tech Herbicide Under Commercial Conditions in Indiana; Report No. MSL-4207; Monsanto Chemical Company, 1984.

6.

Danhous, R.G., e Transfer and Application of Lasso EC, Lasso MT and Lasso WDG; Report No. MSL-5398; Monsanto Chemical Company; 1984.

7.

Danhous, R.G., et a l . Operator Exposure from Closed System Transfer and Application of Lasso EC and Lasso MT Herbicides; Report No. MSL-5320; Monsanto Chemical Company; 1986.

8.

Abbott, I.M., et a l . Am. Ind. Hyg. Assoc. J. 1987, 48(2), 167-175.

9.

Dubelman, S., et a l . J . Ag. Food Chem. 1982, 30, 528532.

10.

Maitlen, J.C., et a l . In Pesticide Residues and Expo­ sure; Plimmer, J., Ed.; American Chemical Society: Washington, DC, 1982; pp 83-103.

11.

Staiff, D.C., et a l . Bull. Environ. Contam. Toxicol. 1975, 14, 334-340.

12.

Wojeck, G.A., et a l . Arch. Environ. Contam. Toxicol. 1983, 12, 65-70.

13.

Wolfe, H.R.; Durham, W.F.; Armstrong, J.F. Arch. Environ. Hlth. 1967, 14, 622-633.

14. Lauer, R.; Arras, D.D. Aerial and Ground Applicator Exposure Studies with Lasso Herbicide Under Actual Field Conditions; Report No. MSL-1889; Monsanto Chemical Company; 1981. RECEIVED January 28, 1988

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Chapter 26

Use of Biological Monitoring Data from Pesticide Users in Making Pesticide Regulatory Decisions in California Study of Captan Exposure of Strawberry Pickers 1

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Keith T. Maddy , R. I. Krieger , Linda O'Connell , M . Bisbiglia , and S. Margetich 2

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California Department of Food and Agriculture, 1220 N Street, Sacramento, CA 95814 California Department of Food and Agriculture, 3292 Meadowview Road,

Exposure of users ingredients which have the potential of causing adverse effects, especially those of subchronic and chronic types, has to be accurately measured i n order to make meaningful risk mitigation determinations. Acceptable methodology is usually available to measure inhalation exposure, however, most pesticide exposure is dermal. Analysis of residues on cloth pads that had been on various parts of the body during exposure may provide an overestimate of dermal exposure. Availability of dermal absorption-rate data i n rodents is useful, but at least on some chemicals it is more l i k e l y to overestimate exposure than such studies done on humans or other primates. The most useful data results from development of metabolism data i n humans or a suitable test animal with appropriate pharmacokinetics, including excretory data, and then biologically monitoring blood, urine, s a l i v a or feces of persons being exposed to the chemical under normal use conditions. As an example results of recent measurements of mixer/loader/applicator and hand harvest worker exposures to captan i n strawberry fields included dislodgeable residues, dermal dosimetry, and urine monitoring. Calculation of the urinary levels resulted i n substantially reduced estimates of human exposure. Such data are used to reach safety and regulatory decisions. The goal is to avoid an inaccurate estimate of exposure that can result from less direct methods of exposure assessment. The k i n d s o f worker exposure d a t a needed b y C a l i f o r n i a have been d e s c r i b e d i n two p r e v i o u s r e p o r t s : " P e s t i c i d e S a f e t y Program o f The C a l i f o r n i a D e p a r t m e n t o f F o o d a n d A g r i c u l t u r e B a s e d Upon Measurements o f P o t e n t i a l Workplace Exposure and t h e E l i m i n a t i o n o f Excess E x p o s u r e s " ( 1 ) , and " R i s k Assessment o f E x c e s s P e s t i c i d e Exposure t o Workers i n C a l i f o r n i a " (2) . California i n i t s Department o f Food and A g r i c u l t u r e (CDFA) h a s a more r e s t r i c t i v e p e s t i c i d e r e g u l a t o r y program t h a n t h e U.S. Government o r o f any

c

0097-6156/89/0382-0338$06.00/0 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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other s t a t e i n the U n i t e d States. C a l i f o r n i a a l s o r e q u i r e s more b a s i c t o x i c o l o g y d a t a and exposure d a t a t h a n t h e U.S. E.P.A.in o r d e r t o meet r e c e n t l y imposed t o x i c o l o g y t e s t s t a n d a r d s , b o t h on new r e q u e s t s f o r r e g i s t r a t i o n as w e l l as f o r a n e x t e n s i v e r e r e g i s t r a t i o n and r e e v a l u a t i o n program w h i c h i s now underway. I n C a l i f o r n i a more t h a n 13,000 p r o d u c t s a r e c u r r e n t l y r e g i s t e r e d on an a n n u a l b a s i s w h i c h c o n t a i n more t h a n 700 a c t i v e p e s t i c i d e i n g r e d i e n t s a n d a l m o s t 1000 c h e m i c a l s w h i c h a r e " i n e r t " as p e s t i c i d e s b u t w h i c h may be q u i t e t o x i c t o man. S i n c e 1971, new C a l i f o r n i a laws have emphasized r e q u i r e m e n t s f o r assessing workplace hazards f o r p e s t i c i d e users ( i n c l u d i n g l o n g - t e r m exposure h a z a r d s ) and ways o f m i t i g a t i n g t h e s e h a z a r d s . T h i s has r e s u l t e d i n s p e c i f i c C a l i f o r n i a r e q u i r e m e n t s f o r d a t a w h i c h may be used t o e s t i m a t e t h e e x t e n t o f such h a z a r d s . The p e s t i c i d e s a f e t y program o f CDFA r e q u i r e s t h e p r e s e n t a t i o n and c o n s i d e r a t i o n o f d a t a on : 1) p e s t i c i d e v a p o r s m i s t s o r d u s t s i n t h e b r e a t h i n g zon powders o r l i q u i d s on t h a p p l y i n g p e s t i c i d e s ; 3) p e s t i c i d e r e s i d u e s , i n c l u d i n g t h e more t o x i c breakdown p r o d u c t s , on f o l i a g e and i n s o i l o f f i e l d s where w o r k i s t o t a k e p l a c e w h i c h may l a t e r c o n t a c t s k i n ; a n d , 4 ) r e s i d u e s i n t h e a i r , on f l o o r s , c o u n t e r s , f u r n i t u r e e t c . , f o l l o w i n g a p p l i c a t i o n of p e s t i c i d e s indoors. These measurements a r e o f v a l u e i n d e s i g n i n g methods t o reduce exposure t o p e r s o n s a g r i c u l t u r a l l y exposed, such as commercial a g r i c u l t u r a l a p p l i c a t o r s , b u t a l s o t o p e r s o n s who use p e s t i c i d e s i n non-agricultural settings. CDFA e v a l u a t e s b a s i c t o x i c o l o g y d a t a , exposure measurements and t h e manner i n which t h e p e s t i c i d e p r o d u c t i s t o be used. By m o d i f y i n g t h e way t h e p e s t i c i d e i s t o be used, e s t a b l i s h i n g r e e n t r y i n t e r v a l s , o r s u g g e s t i n g changes t o EPA o f p r e c a u t i o n a r y s t a t e m e n t s on p e s t i c i d e l a b e l s (which t h e y t h e n agree t o make), t h e r i s k o f exposure t o a p o t e n t i a l l y hazardous p e s t i c i d e may be g r e a t l y reduced. In the p a s t , a major d i f f i c u l t y i n c o n d u c t i n g h a z a r d assessments f o r any persons who might be exposed b e f o r e , d u r i n g , and a f t e r a p e s t i c i d e a p p l i c a t i o n was t h e l a c k o f i n f o r m a t i o n on the amount o f p e s t i c i d e t h a t might be i n h a l e d o r might r e a c h t h e s k i n , t h e r a t e a n d amount o f d e r m a l a b s o r p t i o n , t h e r a t e a n d pathway o f b i o t r a n s f o r m a t i o n , and t h e r o u t e and r a t e o f e l i m i n a t i o n from t h e body. Some o f t h e s p e c i f i c d a t a t h a t may be r e q u i r e d by CDFA t o a s s i s t i n making exposure e s t i m a t e s o f persons i n v a r i o u s a c t i v i t i e s i n v o l v i n g the use o f p e s t i c i d e s i n c l u d e : i n d o o r exposure; f i e l d r e e n t r y exposure; m i x e r , l o a d e r , and a p p l i c a t o r exposure, m e t a b o l i s m , dermal a b s o r p t i o n r a t e , dermal dose response d a t a and b i o l o g i c a l m o n i t o r i n g d a t a . TYPES OF EXPOSURE DATA

I n d o o r Exposure. P e s t i c i d e p r o d u c t s t o be used i n d o o r s such as i n houses, apartments, o f f i c e s , o t h e r i n s t i t u t i o n s , and greenhouses may have exposure ( i n h a l a t i o n , dermal, and i n g e s t i o n ) h a z a r d s b o t h d u r i n g t h e a p p l i c a t i o n and upon r e e n t r y . A p a r t from p r o t e c t i v e measures f o r a p p l i c a t o r s , an a p p r o p r i a t e v e n t i l a t i o n p e r i o d may be

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needed to protect residents, inhabitants, or workers in the treated area from i n h a l a t i o n of hazardous chemicals i n a d d i t i o n to mitigation measures such as reentry periods directed at reducing dermal contact of excess r e s i d u e s on c a r p e t s , f u r n i t u r e and countertops. F i e l d Reentry. Certain pesticides pose a potential hazard to persons i f they enter a treated area and have significant contact with treated plants or s o i l . A waiting period long enough to mitigate exposure i s called a reentry i n t e r v a l . The following is a guide used by CDFA in deciding i f reentry data i s needed. Such data w i l l be needed i f the product is to be applied to a commercially grown crop, p a r t i c u l a r l y to i t s foliage or the s o i l , and c u l t u r a l practices (such as pruning or harvesting) of that p a r t i c u l a r crop i n v o l v i n g s u b s t a n t i a l body contact with the f o l i a g e , bark, or s o i l from the f o l i a g e or cholinesterase i n h i b i t o r ; or (b) a s i g n i f i c a n t l y toxic p r i n c i p l e that can cause a detrimental acute systemic toxic reaction or is suspected of causing a chronic effect, and may be r e a d i l y absorbed through the s k i n or i n h a l e d f o l l o w i n g exposure to p e s t i c i d e residues contacted while conducting usual c u l t u r a l practices; p_r (c) a chemical which causes a significant primary skin i r r i t a n t reaction i n appropriate test animals or man; or (d) a chemical which is a significant skin sensitizer in appropriate test animals or man. Reentry (safe waiting period) intervals are now established on the basis of: (1) data on dermal absorption rates or dermal dose response rates; (2) inhalation and dermal acute t o x i c i t y studies in animal models; (3) f o l i a r and s o i l residue and d i s s i p a t i o n rate data; and, (4) available human exposure data. In the past, acute t o x i c i t y was the major reentry regulatory concern; more recently subacute and chronic t o x i c i t y has also become a major concern. Mixer, Loader. Applicator Exposure. Unless the acute and chronic toxicology data on the formulated product indicates negligible t o x i c i t y ; mixer, loader and applicator exposure data is needed, at least on the use most reasonably expected to give the most r i s k . In order to make an appropriate hazard assessment, information i s needed on the amount of pesticide that may be inhaled and/or reach the skin, or more importantly absorbed during and subsequent to a "typical" application. Metabolism Data. Complete metabolism data in mammals is needed i n order to understand the d i s t r i b u t i o n and excretion of the pesticide and i t s breakdown products. Usually radiotracer studies in rodents are provided to define metabolic pathways. Greater attention needs to be given to defining pathways in accidentally exposed humans. Such data would e s t a b l i s h a b e t t e r experimental b a s i s f o r b i o l o g i c a l monitoring.

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Dermal Absorption Rate Or Dermal Dose Response Rate. Dermal a b s o r p t i o n d a t a which i s u s u a l l y developed i n r a t s o r monkeys, i s needed i n c o n d u c t i n g t h e r i s k assessment f o r f i e l d w o r k e r s , m i x e r s / l o a d e r s , a p p l i c a t o r s , f l a g g e r s , o t h e r p e s t i c i d e u s e r s as w e l l as f o r b y s t a n d e r s ; t h e s e d a t a may a l s o be u s e d i n t h e development o f r e e n t r y i n t e r v a l s . The d a t a p r o v i d e s i n f o r m a t i o n t o c a l c u l a t e how much o f t h e c h e m i c a l e n t e r s t h e body a f t e r i t comes i n t o c o n t a c t w i t h t h e s k i n . Data on monkeys i s c o n s i d e r e d t h e most l i k e l y t o approximate human exposure; o f t e n t h e a b s o r p t i o n by monkey s k i n appears t o be slower than i t i s i n r o d e n t s . B i o l o g i c a l Monitoring. Data c o l l e c t e d on dermal deposition with c u r r e n t methods, when used a l o n g w i t h t h e animal dermal a b s o r p t i o n r a t e o r dermal-dose-response d a t a i s o f t e n assumed t o o v e r e s t i m a t e actual exposure. Metabolism data i s used t o determine p h a r m a c o k i n e t i c and e x c r e t i o n parameters and can form t h e b a s i s f o r b i o l o g i c a l monitoring u r i n a r y m e t a b o l i t e ca the amount o f t h e c h e m i c a l i n t h e u r i n e o f u s e r s can be m o n i t o r e d as a measure o f i n t e r n a l dose. A t t h e same t i m e , t h e amount o f c h e m i c a l t h a t might be i n h a l e d and t h e amount t h a t f a l l s on t h e s k i n can a l s o be measured. With such d a t a a much more o b j e c t i v e assessment can be made o f t h e a c t u a l exposure. Use o f such d a t a may i n d i c a t e s a f e use even though dermal d e p o s i t i o n d a t a and t h e animal study dermal a b s o r p t i o n r a t e d a t a a l o n e might s i g n a l an unacceptable r i s k . We h a v e p r e v i o u s l y s u m m a r i z e d b i o l o g i c a l m o n i t o r i n g d a t a on about 50 p e s t i c i d e a c t i v e i n g r e d i e n t s ( 3 ) .

HAZARD EVALUATION PROCESS. HAZARD ASSESSMENT RISK CHARACTERIZATION

IDENTIFICATION,

The CDFA c o n d u c t s i t s h a z a r d e v a l u a t i o n p r o c e s s consideration of the f o l l o w i n g f a c t o r s : data

EXPOSURE

based

on t h e

submitted

by t h e

1.

Review o f t h e b a s i c t o x i c o l o g y registrant;

2.

Review o f o t h e r t o x i c o l o g y d a t a a v a i l a b l e t o CDFA ( j o u r n a l a r t i c l e s , CDFA s t u d i e s , computerized n a t i o n a l d a t a banks, texts, etc.);

3.

Human i l l n e s s i n f o r m a t i o n developed by CDFA o r o t h e r s i n v o l v i n g t h e p e s t i c i d e under c o n s i d e r a t i o n o r s i m i l a r pesticides;

4.

A v a i l a b l e exposure d a t a on t h i s p e s t i c i d e o r t h i s c l a s s o f p e s t i c i d e s developed by CDFA o r any o t h e r group; and,

5.

Work p r a c t i c e s known about o r expected the proposed u s e .

i n California for

A h a z a r d e v a l u a t i o n i s much more than a b a s i c t o x i c o l o g y r e v i e w . For example, a s p e c i f i c p e s t i c i d e can be found i n t h e t o x i c o l o g y

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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review to be extremely t o x i c ; however, i n the hazard evaluation process, i t may be determined that the product i s to be used i n such small quantities with such specialized equipment that a person could only be overexposed i n the unusual case of equipment f a i l u r e . On the other hand, a product could be found to be of low t o x i c i t y ; but, the most common use might involve long hours of exposure to many workers in orchards while using hand-held spray wands spraying the pesticide above their heads with no protective clothing, due to lack of specification i n the precautionary label statements. In another example, the basic toxicology data for a product may only indicate a moderate t o x i c i t y ; however, in assessing the proposed use of the product mid-summer in a c i t r u s grove i n the San Joaquin V a l l e y , there could be s u b s t a n t i a l c o n v e r s i o n of the a c t i v e ingredient to a highly toxic degradation product in the duff under trees under actual f i e l d conditions which would be hazardous to f i e l d workers. As well as a toxicolog include: 1.

Determining the use pattern (geographic, climate, equipment-type e t c . ) of the proposed product;

2.

Determining significant possible human exposure hazards;

3.

E v a l u a t i n g the adequacy of use i n s t r u c t i o n s and/or r e g u l a t i o n s that are i n place to inform users of the possible use hazards and how to avoid excess exposure.

4.

E v a l u a t i n g the adequacy of i n f o r m a t i o n provided recognize i l l n e s s due to exposure i f i t occurs;

5.

Determining the adequacy of f i r s t aid information; and,

6.

Examining the management.

availability

of

data

to

support

season,

to

medical

Data from the toxicology base, plus those from the additional health and safety studies, including exposure, that are sometimes required, allow for the estimation and calculation of potential exposure hazards. For some products, experience already gained allows for a quick determination that adherence to the proposed or e x i s t i n g use i n s t r u c t i o n s should r e s u l t i n a low hazard use situation. On the other hand, a number of the p e s t i c i d e s considered for r e g i s t r a t i o n have significant hazards from either a short-term or long-term exposure standpoint. These hazards are e s t i m a t e d a n d / o r c a l c u l a t e d to d e t e r m i n e i f a f a v o r a b l e recommendation on the proposed r e g i s t r a t i o n can be given, and i f n o t , whether a d d i t i o n a l r e s t r i c t i o n s would be expected to acceptably reduce the hazards of use. For example, a p a r t i c u l a r product might be a highly dusty wettable powder with only moderate acute t o x i c i t y but with demonstrated p o t e n t i a l f o r producing c h r o n i c e f f e c t s . The calculations for the hazard evaluation are based upon the t o t a l workday measurement of the skin and inhalation exposure to this

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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p e s t i c i d e when i t i s used i n a c c o r d w i t h the l a b e l i n s t r u c t i o n s . The p o t e n t i a l d a i l y dermal dose i s then a d j u s t e d by the e s t i m a t e d 24-hour dermal a b s o r p t i o n r a t e . T h i s f i g u r e i s t h e n added t o the i n h a l a t i o n exposure v a l u e and the sum i s compared t o a n i m a l t e s t d a t a f o r the dose expected t o produce a s p e c i f i c adverse e f f e c t . The s a f e t y f a c t o r f o r t h i s s p e c i f i c e f f e c t w i l l t h e n be c a l c u l a t e d t o determine i f i t i s adequate t o p r o t e c t the w o r k e r s . I n some c a s e s , the exposure assessment might not l e a d t o an a c c e p t a b l e s a f e t y f a c t o r f o r a m i x e r / l o a d e r ; but, i f t h i s product were repackaged i n w a t e r - s o l u b l e p a c k e t s f o r m u l a t e d as a d r y f l o w a b l e o r i f i t were r e f o r m u l a t e d t o be used as a l i q u i d product and t h e n r e q u i r e d t o be t r a n s f e r r e d t h r o u g h a c l o s e d system, t h e h a z a r d might be a c c e p t a b l y reduced. Of p a r t i c u l a r concern a r e p o t e n t i a l adverse e f f e c t s such as c a r c i n o g e n i c i t y and developmental t o x i c i t y ( p r i m a r i l y those w h i c h occur p r i o r to b i r t h ) Adequacy Of Mitigation e v a l u a t e d , an assessment i s made r e g a r d i n g t h e adequacy of t h e p o s s i b l e m i t i g a t i o n measures t o p r o t e c t workers from hazards of use. The l a b e l w i t h i t s use i n s t r u c t i o n s may be accepted and t h e product may be r e g i s t e r e d w i t h o u t f u r t h e r c o n c e r n . On the o t h e r hand, one o r more of t h e f o l l o w i n g c o n d i t i o n s may be r e q u i r e d b e f o r e the product i s c o n s i d e r e d f o r r e g i s t r a t i o n by the CDFA: (1) the EPA may be a d v i s e d of the d e s i r a b i l i t y of r e q u i r i n g a l a b e l change g i v i n g more s p e c i f i c use i n s t r u c t i o n s , o r the r e g i s t r a n t may r e c o g n i z e t h e n e e d t o a s k EPA f o r s u c h a l a b e l c h a n g e ; ( 2 ) a C a l i f o r n i a r e g u l a t i o n on the use may be enacted (which w i l l have the same e f f e c t as a l a b e l change, but t h i s can t a k e s e v e r a l months t o a c c o m p l i s h ) ; (3) the product may be made a C a l i f o r n i a r e s t r i c t e d o r r e g u l a t e d m a t e r i a l which w i l l a l l o w i m p o s i t i o n of s p e c i f i c p e r m i t r e q u i r e m e n t s o r r e g u l a t i o n s ( t h i s p r o c e s s can a l s o t a k e a number of months); (4) c l o s e d system t r a n s f e r o f l i q u i d p e s t i c i d e s may be r e q u i r e d , ( t h i s i s c u r r e n t l y r e q u i r e d f o r a l l t o x i c i t y Category I l i q u i d s , when s p e c i f i e d on l a b e l s r e g a r d l e s s of t h e t o x i c i t y c a t e g o r y and when s p e c i f i c a l l y r e q u i r e d by r e g u l a t i o n s ) ; (5) change i n t h e p r o d u c t ' s f o r m u l a t i o n may be r e q u i r e d t o reduce excess hazards ( e . g . , reduce d u s t i n e s s ) ; (6) w a t e r - s o l u b l e packaging of the more t o x i c powders may be r e q u i r e d ; (7) minimum f i e l d r e e n t r y i n t e r v a l s may be set by r e g u l a t i o n (a several-month p r o c e s s u n l e s s they are a d e q u a t e l y s p e c i f i e d on the l a b e l ) ; (8) m e d i c a l s u p e r v i s i o n may be r e q u i r e d by r e g u l a t i o n ; and/or (9) d e t a i l e d s a f e t y t r a i n i n g may be r e q u i r e d f o r s p e c i f i c p e s t i c i d e s . EXAMPLE OF VALUE OF BIOLOGIC MONITORING DATA The c u r r e n t common m e t h o d s f o r m e a s u r i n g w o r k e r e x p o s u r e t o p e s t i c i d e s i n v o l v e measurement of d i s l o d g e a b l e f o l i a r r e s i d u e s or r e s i d u e s i n t h e b r e a t h i n g zone and/or on the s k i n . The dermal d e p o s i t i o n d a t a i s then used w i t h dermal a b s o r p t i o n r a t e d a t a c o l l e c t e d i n r o d e n t s o r monkeys t o c a l c u l a t e p r o b a b l e human exposure. U n c e r t a i n t i e s about the b i o a v a i l a b i l i t y and t r a n s f e r of r e s i d u e s i n w o r k e n v i r o n m e n t s may result i n overestimated exposures. Below we have summarized our r e c e n t study o f captan

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exposures o f s t r a w b e r r y h a r v e s t workers t o i l l u s t r a t e some o f these considerations. Introduction. Studies of t h e dermal p e s t i c i d e exposures of s t r a w b e r r y h a r v e s t e r s have been r e p o r t e d by Popendorf e t a l . ( 4 ) , E v e r h a r t and H o l t (5.), Zweig e t a l . (6.), W i n t e r l i n e t a l . (7.) and R i t c e y e t a l . (8.). Some o f t h e d a t a accumulated from s t u d i e s o f s t r a w b e r r y h a r v e s t e r s f i g u r e d p r o m i n e n t l y i n t h e development o f t h e e m p i r i c a l t r a n s f e r c o e f f i c i e n t (commonly c a l l e d t h e Zweig-Popendorf f a c t o r ) o f 5000 cm^/h w h i c h can be used t o r e l a t e d i s l o d g e a b l e f o l i a r p e s t i c i d e r e s i d u e s t o h o u r l y dermal p e s t i c i d e exposures o f f i e l d w o r k e r s (Zweig e t a l . ( 9 ) ) . The t r a n s f e r c o e f f i c i e n t seems t o be a h e l p f u l t o o l t o p r o v i d e a f i r s t e s t i m a t e o f t h e d e r m a l exposure o f f i e l d w o r k e r s engaged i n work t a s k s such as h a r v e s t i n g f r u i t s and v e g e t a b l e s o r p i c k i n g f l o w e r s i n a greenhouse. The t r a n s f e r c o e f f i c i e n t w i l l v a r y d e p e n d i n g on t h e t a s k and u s e scenario (protective clothin factors influencing i We developed t h r e e e s t i m a t e s o f s t r a w b e r r y h a r v e s t e r exposure t o c a p t a n b a s e d upon d i s l o d g e a b l e f o l i a r r e s i d u e s , d e r m a l d o s i m e t r y , and b i o l o g i c a l m o n i t o r i n g . We w e r e p a r t i c u l a r l y i n t e r e s t e d i n t h e q u a n t i t a t i v e r e l a t i o n s h i p between t h e e s t i m a t e s due t o t h e i r c r i t i c a l importance i n t h e r i s k assessment p r o c e s s . A d d i t i o n a l l y , we sought t o measure t h e degree o f m i t i g a t i o n o f t h e captan exposure p r o v i d e d by t h e use o f c h e m i c a l l y - r e s i s t a n t g l o v e s . METHODS; Setting. I n June 1987, we o b t a i n e d t h e c o o p e r a t i o n o f the C a l i f o r n i a S t r a w b e r r y A d v i s o r y Board and Mr. L a r r y G a l p e r , Watsonville, California. The Board p r o v i d e d f i n a n c i a l support t o compensate t h e producer f o r any l o s t time and p r o d u c t i v i t y when we stopped work t o put on measurement d e v i c e s o r t o c o l l e c t samples. We had complete access t o a 72 a c r e s t r a w b e r r y farm w h i c h was c o n s i d e r e d by a l l c o n c e r n e d t o be r e p r e s e n t a t i v e o f t h e a p p r o x i m a t e l y 16,000 a c r e s o f C a l i f o r n i a s t r a w b e r r y p r o d u c t i o n . The s t r a w b e r r y beds were p l a n t e d w i t h 18,500 p l a n t s / a c r e o f e i t h e r t h e P a j a r o and S e l v a v a r i e t i e s . The p l a n t s were grown on e l e v a t e d beds (14") w i t h 52" c e n t e r s . The p l a n t s were w e l l past t h e i r peak p r o d u c t i o n , e.g. 10-12 c r a t e s p e r row a t peak v e r s u s 2-3 c r a t e s p e r row i n J u l y . T h i s p r o v i d e d maximal s e a s o n a l worker c o n t a c t w i t h t r e a t e d f o l i a g e . P r o d u c t i o n d a t a were r e c o r d e d ( c r a t e - 1 0 pounds). Two crews o f workers ( a p p r o x i m a t e l y 35-50 workers/crew) p i c k the f i e l d s t w i c e i n each 6-day week (8 hour days) o f t h e p i c k i n g season which extends from A p r i l t o October. The crews c o n s i s t e d of men and women i n apparent good h e a l t h (85 percent were below age 35) w i t h up t o a maximum of 20 years e x p e r i e n c e as s t r a w b e r r y pickers. They u s u a l l y wear l o n g pants and l o n g - s l e e v e d s h i r t s t o p r o t e c t themselves from t h e g e n e r a l l y c o o l weather o f t h e a r e a where s t r a w b e r r i e s grow w e l l . Some o f t h e men wear s h o r t - s l e e v e d s h i r t s , and o t h e r s r o l l up t h e i r s l e e v e s as t h e mornings warm. Women a d d i t i o n a l l y wear s c a r v e s w h i c h cover most of t h e i r f a c e . A l l o f t h e women and l e s s than 5 percent o f t h e men n o r m a l l y wear c h e m i c a l l y r e s i s t a n t g l o v e s t o p r o t e c t t h e i r hands from d i r t and strawberry j u i c e .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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For t h i s s t u d y , a crew of 40 male v o l u n t e e r s was assembled by the foreman and the r a n c h manager. Males were s e l e c t e d t o p r o v i d e s u f f i c i e n t numbers of workers of one sex t o meet e x p e r i m e n t a l objectives. A d d i t i o n a l l y , because men d i d not u s u a l l y wear g l o v e s they p e r m i t t e d e s t a b l i s h m e n t of glove/no g l o v e groups w i t h o u t r e d u c i n g any worker's normal p r o t e c t i o n . The g l o v e s were 13", c h e m i c a l l y r e s i s t a n t , and made f r o m L o n g S e r v i c e rubber. Exposure d a t a were c o l l e c t e d d u r i n g t h r e e c o n s e c u t i v e days. The a s s i s t a n c e p r o v i d e d by the foreman was c r i t i c a l t o o b t a i n i n g the e n t h u s i a s t i c c o o p e r a t i o n of the p i c k e r s . Throughout the s t u d y , the ranch management served as an e f f e c t i v e l i a i s o n between CDFA s t a f f and the f i e l d w o r k e r s . t m

Captan Application. Four days b e f o r e h a r v e s t , each p a r t of the f i e l d was t r e a t e d w i t h Captan 50 WP (4 l b s . a . i . / a c r e ) tank mixed w i t h B e n l a t e 50 WP (1 l b a . i . / a c r e ) and Vendex (2 l b s a . i . / a c r e ) Captan had not been p r e v i o u s l g a l l o n s of spray mix wer sprayer. S a m p l e s of t a n k m i x were c o l l e c t e d f o r c a p t a n and t e t r a h y d r o p h t h a l i m i d e (THPI), a m e t a b o l i t e of captan, a n a l y s i s . F o l i a r Residue Monitoring. P r i o r t o the captan application and d u r i n g e a c h day o f t h e t h r e e - d a y s t u d y , f o l i a g e s a m p l e s w e r e collected. Each sample c o n s i s t e d of f o r t y 2.54 cm diameter l e a f d i s c s ( B i r k e s t r a n d punch) r e p l i c a t e d t h r e e times b e f o r e and a f t e r harvest. Samples were taken a l o n g a d i a g o n a l l i n e from 10 rows of s t r a w b e r r i e s i n a g i v e n s e c t i o n of the f i e l d . E l e v e n s e c t i o n s of the f i e l d were sampled. The sample j a r s were s e a l e d w i t h aluminum f o i l , capped, and kept on wet i c e u n t i l they were t r a n s p o r t e d t o the l a b o r a t o r y f o r captan r e s i d u e a n a l y s i s . Dermal Dosimetry. Measurements of dermal exposure and e v a l u a t i o n of the p r o t e c t i o n p r o v i d e d by g l o v e s were made on days 2 and 3 of the study. I n o r d e r t o h a v e m i n i m a l e f f e c t s on n o r m a l w o r k p r a c t i c e s and t o a s s u r e t h a t CDFA s t a f f c o u l d a d e q u a t e l y a s s i s t each w o r k e r , the crew was randomly d i v i d e d i n t o two groups of 20 t h a t were m o n i t o r e d f o r one day each. On a g i v e n day each group of 20 was p r o v i d e d w i t h a s e t of 100% c o t t o n , t i g h t f i t t i n g l o n g underwear t o be worn f o r f o u r hours beneath normal w o r k c l o t h e s and w h i c h served as dermal d o s i m e t e r s . Each worker was g i v e n t h e underwear the day b e f o r e t h e i r scheduled m o n i t o r i n g . The group was f u r t h e r d i v i d e d i n t o " g l o v e " and "no g l o v e " subgroups. Dermal exposure was m o n i t o r e d d u r i n g a 4-hour, morning work period. The workers c a r e f u l l y removed t h e d o s i m e t e r s i n temporary change rooms c o n s t r u c t e d i n t h e back of two r e n t a l t r u c k s u s i n g sheets and p l a s t i c p i p e . The garments were p l a c e d i n t o Z i p - l o c k bags and s t o r e d on dry i c e u n t i l p r o c e s s i n g . At t h a t time arm and l e g dosimeter samples were p r e p a r e d , i c e d , and t r a n s p o r t e d t o the laboratory. C o n c u r r e n t l y , handwashes were done on each s u b j e c t u s i n g 400 ml of 1% S u r t e n s o l u t i o n c o n t a i n e d i n a one g a l l o n Z i p - l o c k bag. E a c h p e r s o n w a s h e d t h e i r hands f o r two o n e - m i n u t e p e r i o d s . Handwashes (800 ml) were subsampled (500 ml) and s t o r e d on d r y i c e f o r t r a n s p o r t t o the l a b o r a t o r y .

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D u r i n g the t h r e e day study t h e work p e r i o d was a p p r o x i m a t e l y d i v i d e d i n t o t h r e e 7-hour segments. D u r i n g a 4-hour segment of e i t h e r day two o r t h r e e of the s t u d y , each worker wore g l o v e s and l o n g underwear as a dermal d o s i m e t e r . Thus, exposure p o t e n t i a l s might be reduced by a f a c t o r of 21 - 4/21. No d i r e c t e s t i m a t e s of the amount of exposure o c c u r r i n g d u r i n g the p e r i o d of p a s s i v e d o s i m e t r y a r e a v a i l a b l e and no c o r r e c t i o n s w e r e u s e d t o make exposure e s t i m a t e s . Urine. P i c k e r s were p r o v i d e d t h r e e or f o u r p o l y e t h y l e n e u r i n e c o l l e c t i o n b o t t l e s f o r d a i l y 24-hour c o l l e c t i o n s . The b o t t l e s were h e l d i n i n s u l a t e d boxes i n the f i e l d d u r i n g t h e work day and two unused b o t t l e s were taken home each n i g h t . C r e a t i n i n e l e v e l s were measured i n each 24-hour v o i d at a l o c a l c l i n i c a l l a b o r a t o r y as an i n d i c a t o r of compliance. Extraction. Clean-up an l e a f samples were p r e p a r e Samples were shaken t h r e e times w i t h S u r t e n s o l u t i o n and e x t r a c t e d t h r e e times w i t h e t h y l a c e t a t e a f t e r a d d i t i o n of Na2S04» After volume r e d u c t i o n the samples were a n a l y z e d by gas chromatography. Captan was a n a l y z e d on the dermal d o s i m e t e r s i n a s i m i l a r fashion. The i n i t i a l e x t r a c t was prepared by s e p a r a t e l y t u m b l i n g i n d i v i d u a l s e t s of underwear arms and l e g s w i t h e t h y l a c e t a t e . Urinary THPI ( c i s - 1 , 2 - d i c a r b o x i m i d o - 4 - c y c l o h e x e n e ) was determined as r e p o r t e d by W i n t e r l i n et a l . (7_) • T w e n t y - f i v e ml a l i q u o t s were e x t r a c t e d w i t h methylene c h l o r i d e , f i l t e r e d , d r i e d , and t a k e n up i n benzene. The e x t r a c t was a n a l y z e d u s i n g H e w l e t t Packard 5880A gas chromatograph w i t h a N/P i o n i z a t i o n d e t e c t o r . The minimum d e t e c t a b l e l e v e l of THPI i n u r i n e was 0.03 ug/ml and r e c o v e r i e s ranged from 80 t o 89 p e r c e n t . Subsequently a set of 10 u r i n e samples from h i g h exposure workers were f u r t h e r a n a l y z e d by Morse L a b o r a t o r y , Sacramento, C.A. A pH 11 c l e a n - u p and n i t r o g e n s p e c i f i c e l e c t r o l y t i c c o n d u c t i v i t y d e t e c t o r were used t o a c h i e v e a minimum 0.005 ppm s e n s i t i v i t y . At 0.02 and 0.01 ppm s e n s i t i v i t y t h e r e c o v e r i e s of THPI were 75 and 67 percent r e s p e c t i v e l y . Statistics. The d i f f e r e n c e s between means (3 r e p l i c a t e s ) p r e - and p o s t - h a r v e s t d i s l o d g e a b l e f o l i a g e r e s i d u e s were compared u s i n g paired t-tests. A randomized b l o c k d e s i g n was used t o i n v e s t i g a t e p o t e n t i a l m i t i g a t i n g e f f e c t s of g l o v e s on dermal captan exposure. F a c t o r s i n c l u d e d i n the l i n e a r model f o r a n a l y s i s i n c l u d e d g l o v e assignment, day, and a g l o v e i n t e r a c t i n g w i t h day term. I f no g l o v e assignment by day i n t e r a c t i o n was determined (P<0.10), then we made comparisons f o r the two r e m a i n i n g e f f e c t s . A l l analyses were made u s i n g Type I I I Sums of Squares i n the SAS G e n e r a l L i n e a r Model Procedure. S i n c e the exposure d a t a were skewed, the v a r i a b l e i t s e l f , a n a t u r a l l o g a r i t h m i c t r a n s f o r m a t i o n , and rank t r a n s f o r m e d d a t a were analyzed.

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RESULTS; Production. The 40-man crew p i c k e d 12, 12.2, and 11 a c r e s of s t r a w b e r r i e s on the t h r e e days of the s t u d y ( T a b l e I ) . The number of c r a t e s p i c k e d was 916, 1413, and 1239, r e s p e c t i v e l y . The work p e r i o d was a p p r o x i m a t e l y seven hours each day. The h i g h y i e l d of s t r a w b e r r i e s on day two was made up of 832 c r a t e s of S e l v a s and 581 c r a t e s o f P a j a r o s . I t was the s u b j e c t i v e o b s e r v a t i o n of the foreman and ranch manager t h a t the use of g l o v e s slowed some p i c k e r s on t h e f i r s t day of the study. They r e p o r t e d normal p i c k i n g r a t e s f o r the f o l l o w i n g two days of the study. They a d d i t i o n a l l y n o t e d t h a t a f t e r the study had ended o n l y a few of the men c o n t i n u e d t o wear t h e i r gloves. Dislodgeable Residues. E l e v e n s e p a r a t e l a r g e beds of the ranch w e r e s a m p l e d f o r p r e - and p o s t - h a r v e s t d i s l o d g e a b l e c a p t a n r e s i d u e s . The p r e - h a r v e s t sample mean ± S.D was 2.4 + 0.6 ug/cm^ and the p o s t - h a r v e s t mea p r o v i d e d d i r e c t evidenc t o the areas under study. A d d i t i o n a l l y , the p o s t - h a r v e s t samples c o n t a i n e d 0.3 ug/cm^ l e s s d i s l o d g e a b l e r e s i d u e (P=0.024) as evidence of the s u b s t a n t i a l c o n t a c t by the p i c k i n g crew. Dermal Dosimetry and Handwashes. F i e l d w o r k e r work p r a c t i c e s were c a r e f u l l y observed p r i o r t o our s e l e c t i n g c o t t o n l o n g underwear as a whole body d o s i m e t e r . We m o n i t o r e d o n l y hands, arms, and l e g s s i n c e those were the body p a r t s t h a t had s u b s t a n t i a l c o n t a c t w i t h treated foliage. A s i m i l a r s t r a t e g y was used by R i t c e y et a l . (.8) who a s s e s s e d exposure u s i n g o v e r s l e e v e s and l e g g i n g s . Table I I shows exposure d a t a f o r days two and t h r e e of the s t u d y . T a b l e I I I shows t h e r a n g e o f e x p o s u r e s f o u n d . R e c o v e r i e s from dermal d o s i m e t e r s were about 90-95 p e r c e n t . Gloves v e r y e f f e c t i v e l y reduced hand exposure as i n d i c a t e d by the captan and THPI i n handwashes. A p p r o x i m a t e l y 50 percent of the captan was r e c o v e r e d as THPI i n the handwashes. No o t h e r samples c o n t a i n e d more than t r a c e amounts of THPI. As expected due t o l i m i t e d worker c o n t a c t d u r i n g p i c k i n g , low amounts (<15 p e r c e n t ) of captan exposure were the r e s u l t of l e g c o n t a c t . S i n c e the g l o v e s covered more than h a l f of the f o r e a r m , the apparent m i t i g a t i n g e f f e c t of g l o v e s may be o v e r e s t i m a t e d t o a s m a l l e x t e n t by the s i m p l e c a l c u l a t i o n of p e r c e n t of t o t a l captan r e c o v e r e d on t h e arm dosimeter. Urinalysis. I n animals captan i s r a p i d l y absorbed through the s k i n (2 p e r c e n t of a p p l i e d dose per hour) and u r i n e i s the p r i m a r y r o u t e of e x c r e t i o n . These f a c t o r s c o n t r i b u t e t o the f e a s i b i l i t y of u s i n g b i o l o g i c a l m o n i t o r i n g t o gauge captan exposure. Our study began on Monday (no Sunday p i c k i n g ) and no captan had been used p r e v i o u s l y d u r i n g 1987. No preexposure u r i n e samples were c o l l e c t e d . There are numerous m e t a b o l i c s t u d i e s of the f a t e o f captan i n r a t s , b u t u n f o r t u n a t e l y none a r e a v a i l a b l e f o r humans. C a r b o n y l has b e e n u s e d t o s t u d y t h e THPI m o i e t y o f c a p t a n by Hoffman et a l . ( 1 1 ) . When l ^ C - c a p t a n was o r a l l y a d m i n i s t e r e d , 85 p e r c e n t of the r a d i o a c t i v i t y was r e c o v e r e d i n u r i n e w i t h i n 96 h o u r s . The m e t a b o l i c scheme i n c l u d e s h y d r o l y t i c c l e a v a g e of captan

American Chemical Society Library 1155

15th S t ,

N.W.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; Washington, O.C. Society: 20036Washington, DC, 1988. ACS Symposium Series; American Chemical

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Table I:

WATSONVILLE STRAWBERRY FIELDWORKER

STUDY

DATE

DAY

ACRES

CRATES

STRAWBERRY

7/20 7/21

1 2

12 12.2

916 1413

7/22

3

11

1239

PAJARO SELVA; PAJARO (832) (581) PAJARO

Table I I STRAWBERRY PICKERS (MILLIGRAMS) DAY 2

ARMS

LEGS

NO GLOVES GLOVES

9.2 3.6

1.2 0.7

DAY 3

ARMS

LEGS

NO GLOVES GLOVES

27.4 10.9

0.8 1.3

HANDWASH 11.4 0.3

TOTAL 21.8 4.6

HANDWASH

TOTAL

14.4 0.2

42.6 12.4

Table I I I : RANGE OF DERMAL CAPTAN EXPOSURES OF STRAWBERRY PICKERS (MILLIGRAMS) Estimates DAY 2

MEAN

NO GLOVES GLOVES DAY 3

22 5 MEAN

NO GLOVES GLOVES

+

43 12

+

S.D.

MEDIAN

MINIMUM

MAXIMUM

6 5

22 3

10 1

30 17

S.D.

MEDIAN

MINIMUM

MAXIMUM

31 10

17 1

32 10

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t o y i e l d THPI w h i c h c o n s t i t u t e d 15 p e r c e n t o f t h e u r i n a r y radioactivity. Subsequently four a d d i t i o n a l m e t a b o l i t e s a r e p r o d u c e d by h y d r o x y l a t i o n , e p o x i d a t i o n , h y d r o l y s i s , a n d hydroxylation-rearrangement. Human e x p o s u r e s w e r e e s t i m a t e d assuming t h a t THPI was a captan m e t a b o l i t e , and t h a t i t c o n s t i t u t e d 15 percent o f the u r i n a r y m e t a b o l i t e s w h i c h were 85 percent o f t h e dose. No c o r r e c t i o n s were made f o r the f a c t t h a t each worker wore a f u l l body dosimeter f o r f o u r hours o f the a p p r o x i m a t e l y 21 hours worked d u r i n g the t h r e e days o f the study. The samples a n a l y z e d by e l e c t r o l y t i c c o n d u c t i v i t y were 72-hour composites and they were e s t i m a t e d t o c o n t a i n about 200 ug captan e q u i v a l e n t s (48 percent a b s o r p t i o n ; 15/85 m e t a b o l i s m ) . The a c t u a l v a l u e s a r e shown i n T a b l e I V . The median was 0.005 ppm and t h e range was <0.005 t o 0.014 ppm. Estimates of Exposure Estimate f fieldworke based upon d i s l o d g e a b l e f o l i a m o n i t o r i n g d i f f e r markedly r e s i d u e l e v e l on f o l i a g e , a d a i l y dermal exposure o f 96 mg/day was c a l c u l a t e d (5000 cm /h; 8 h ) . Based upon dermal a b s o r p t i o n s t u d i e s i n t h e r a t , t h e absorbed dose (24 h) was assumed t o be 48 p e r c e n t (2%/h; 24 h) r e s u l t i n g i n an e f f e c t i v e dose o f 46 mg/captan/day. Based upon t h e r a t m e t a b o l i c work, 85 percent o f an o r a l dose o f captan would be e l i m i n a t e d i n 96 hours and 15 p e r c e n t of that would be the m e t a b o l i t e , THPI. I f a n y t h i n g , l a r g e r amounts o f THPI i n u r i n e might be expected f o l l o w i n g dermal exposure s i n c e t h e metabolic ( e s p e c i a l l y h y d r o l y s i s ) c o n t r i b u t i o n o f g a s t r o i n t e s t i n a l t r a c t would be m i n i m i z e d ( o r t o t a l l y e l i m i n a t e d ) f o l l o w i n g dermal exposure. As a r e s u l t , d a i l y u r i n e would be expected t o c o n t a i n a b o u t 3 mg T H P I o r 2.5 ppm T H P I . T h i s l e v e l o f THPI i s a p p r o x i m a t e l y 500-times t h e minimum d e t e c t a b l e l e v e l (0.005 ppm). A p p a r e n t l y , t h e approach of u s i n g the d i s l o d g e a b l e r e s i d u e and t h e r a t dermal a b s o r p t i o n r a t e data f o r e s t i m a t i n g dermal captan exposure i s o f l i m i t e d u s e f u l n e s s s i n c e i t p r e d i c t s much h i g h e r exposure than o c c u r s based upon u r i n a l y s i s o f the workers. Due t o the l a c k o f s p e c i f i c human a b s o r p t i o n and m e t a b o l i c d a t a and o u r poor u n d e r s t a n d i n g o f t h e f o l i a g e - w o r k e r t r a n s f e r p r o c e s s , t h e b a s i s f o r the o v e r e s t i m a t e can not be i d e n t i f i e d . Dermal d o s i m e t r y a l s o a p p a r e n t l y o v e r e s t i m a t e s dermal captan exposure. Following c a r e f u l p r e l i m i n a r y observations of strawberry p i c k e r s , we measured arm, l e g , and hand exposures u s i n g t i g h t f i t t i n g , long underwear t o c a p t u r e captan which c o n t a c t e d t h e extremities. Handwashes were a n a l y z e d t o e s t i m a t e hand exposure. Workers w i t h o u t g l o v e s had t h r e e t o f o u r times g r e a t e r exposure than g l o v e d workers (P<0.05). I n T a b l e I I g l o v e d worker exposure i s assumed t o be 10 mg captan/day. On t h i s b a s i s t h e p o t e n t i a l THPI e x c r e t i o n would be 0.3 mg THPl/day. The captan exposure (10 mg/day) was about o n e - n i n t h o f t h a t e s t i m a t e d u s i n g d i s l o d g e a b l e residues. The h y p o t h e t i c a l l e v e l o f THPI was a p p r o x i m a t e l y 60times the minimum d e t e c t a b l e l i m i t . Both d i s l o d g e a b l e r e s i d u e and dermal dosimeter based exposures a r e h i g h e r than exposure e s t i m a t e s based on u r i n a r y e x c r e t i o n under t h e assumptions g i v e n i n t h e p r e c e d i n g paragraph. 2

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DISCUSSION T h i s study demonstrates t h e magnitude o f f i e l d w o r k e r exposure generated u s i n g d i s l o d g e a b l e f o l i a r r e s i d u e s , dermal d o s i m e t r y , and u r i n e m o n i t o r i n g ( F i g u r e l ) . The q u a n t i t a t i v e d i f f e r e n c e s a r e s t r i k i n g and would u l t i m a t e l y r e s u l t i n s u b s t a n t i a l l y d i f f e r e n t r i s k assessments and m i t i g a t i o n measures. The d i s l o d g e a b l e f o l i a r r e s i d u e approach g i v e s i n v e s t i g a t o r s an e s t i m a t e o f exposure, b u t i t p r o b a b l y s h o u l d be made more work t a s k s p e c i f i c t o r e f l e c t major d i f f e r e n c e s i n f o l i a g e c o n t a c t between s t r a w b e r r y p i c k e r s and l e t t u c e c u t t e r s on one hand and peach t h i n n e r s and grape p i c k e r s on the o t h e r . A t t h i s t i m e we a r e t r y i n g t o c o n s t r u c t a s e t o f t r a n s f e r c o e f f i c i e n t s t o r e p r e s e n t h i g h , medium, and low c o n t a c t work t a s k s . The p o s s i b l e i n f l u e n c e o f the p h y s i c a l and c h e m i c a l n a t u r e o f t h e d e p o s i t o f t h e a p p l i e d p e s t i c i d e may a l s o be considered. Furthermore a d d i t i o n a l d a t a w i l l be gathered i n a l o n g term e v a l u a t i o n o c o e f f i c i e n t t o estimat In the s p e c i f i c example p r e s e n t e d here the apparent t r a n s f e r c o e f f i c i e n t was 250-650 cm /h f o r g l o v e d f i e l d w o r k e r s as compared to the g e n e r i c 5000 cm /h c o e f f i c i e n t (9). Gloves and l o n g s l e e v e d s h i r t s may be n e c e s s a r y t o m a i n t a i n low f i e l d w o r k e r exposures i n crops c o n t a i n i n g h i g h (>0.5 ug/cm ) d i s l o d g e a b l e f o l i a g e r e s i d u e s . T h i s i s e s p e c i a l l y important f o r c h e m i c a l s such as captan t h a t have s i g n i f i c a n t c h r o n i c t o x i c i t y and p o t e n t i a l l y l o n g p e r i o d s o f exposure. In a more g e n e r a l v e i n , i t i s c l e a r t h a t b i o l o g i c a l m o n i t o r i n g y i e l d s more d i r e c t e s t i m a t e s o f p e s t i c i d e exposures than p a s s i v e d o s i m e t r y o r the d i s l o d g e a b l e r e s i d u e approach. However, a n a l y s i s i s c u r r e n t l y s e v e r e l y l i m i t e d by i n a d e q u a t e a b s o r p t i o n and m e t a b o l i s m d a t a . T h i s l i m i t a t i o n w i l l not d i s a p p e a r and exposures w i l l continue. I f improved exposure assessments are t o be o b t a i n e d , perhaps procedures t o i n d e x worker exposures u s i n g key m e t a b o l i t e s can be developed. M e t a b o l i c i n d i c e s seem t o have a p l a c e i n exposure assessments s i n c e i t i s e x t r e m e l y u n l i k e l y t h a t complete human m e t a b o l i c and p h a r m a c o k i n e t i c d a t a bases w i l l become a v a i l a b l e f o r most p e s t i c i d e s . A p e s t i c i d e m e t a b o l i c index would be s p e c i f i c t o a p a r t i c u l a r a c t i v e i n g r e d i e n t and would i n c l u d e f a c t o r s known t o i n f l u e n c e e x c r e t i o n o f the parent chemical o r i t s d e r i v a t i v e s . In the case o f captan a r e l a t i v e l y s i m p l e model has been suggested. I t assumes measurement o f THPI as a p r i n c i p a l , s t a b l e captan m e t a b o l i t e . A t t h i s time o n l y d a t a from s i n g l e dose, o r a l gavage s t u d i e s i n r a t s a r e a v a i l a b l e . U r i n e and f e c e s r e s p e c t i v e l y c o n t a i n e d 85 p e r c e n t and 15 p e r c e n t o f the a d m i n i s t e r e d dose ( o v e r 97 p e r c e n t accounted f o r i n 96 h o u r s ) . The dosage was 77-92 mg/kg. T h i s i s c o n s i d e r a b l y more than t h e 0.1 - 0.2 mg/kg t h a t we p r e d i c t f o r g l o v e d f i e l d workers based upon p a s s i v e d o s i m e t r y . The dosage c o n s i d e r a t i o n would tend t o i n f l a t e our exposure e s t i m a t e . The r a t s t u d i e s a d d i t i o n a l l y r e v e a l e d t h a t THPI accounted f o r 15 p e r c e n t o f the t o t a l u r i n a r y r a d i o a c t i v i t y . F o r the p r e s e n t we propose t o r e l a t e u r i n a r y THPI (ppb x volume) t o exposure u s i n g an index w h i c h i n c l u d e s a s t o i c h i o m e t r i c f a c t o r (0.5), m e t a b o l i s m f a c t o r (0.15/0.85), and a dermal a b s o r p t i o n f a c t o r (0.5). As a r e s u l t i f 2

2

2

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Table IV: TETRAHYDROPHTHALIMIDE IN THREE-DAY COMPOSITE URINE SAMPLES FIELDWORKER

THPI(PPM)

1 2 3 4 5 6 7 8 9 10

0.006 0.005 <0.005 <0.005 0.005 0.010 <0.005 0.01

N 0.005

MLD - 0 . 0 0 5 PPM; i f a response l e s s t h a n MDL b u t g r e a t e r t h a n 0 was o b s e r v e d t h e amount o f THPI was l i s t e d as < 0 . 0 0 5 .

RESIDUE

2.4 ug/cm 2

t

DOSIMETRY

96 mg/day 46 mg/day

10 mg/day

1

5 mg/day

3 mg THPI/day BIOLOGICAL 0.2 mg/day

I 0.3 mg THPI/day

T 1 0.1 mg/day

T 6ug/1200 ml

T

0.005 ug THPI/ml F i g u r e 1. E s t i m a t e s o f THPI u r i n e u s i n g f o l i a r r e s i d u e and p a s s i v e dosimetry data. Same assumptions about a b s o r p t i o n , m e t a b o l i s m , and e x c r e t i o n were used i n each case. Measured v a l u e s a r e i n b o l d type. The e s t i m a t e d c a p t a n exposure ranges from 96 mg/day ( f o l i a r r e s i d u e ) t o 0 . 2 mg/day ( b i o l o g i c a l m o n i t o r i n g ) .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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the amount (ug) o f THPI e x c r e t e d i s m u l t i p l i e d by 30, t h e dermal c a p t a n dose c a n be e s t i m a t e d . We have l i m i t e d d a t a t o v a l i d a t e t h i s model. Ample d a t a a r e a v a i l a b l e t o e s t a b l i s h t h a t THPI i s a human c a p t a n m e t a b o l i t e . Unpublished d a t a ( K r i e g e r and T h o n g s i n t h u s a k ) show t h a t l o w p e r c e n t a g e s o f THPI a r e e x c r e t e d f o l l o w i n g s i n g l e o r a l dosages (0.1 and 1.0 mg/kg). I f t h i s i s g e n e r a l l y t r u e , the metabolic index overestimates t h e a v a i l a b l e c a p t a n dose. This factor requires f u r t h e r e v a l u a t i o n as does t h e p o t e n t i a l r o l e o f r o u t e and r a t e o f exposure. A t t h i s time no a d d i t i o n a l d a t a a r e a v a i l a b l e . Present d a t a make i t p o s s i b l e t o e s t i m a t e exposure w i t h fewer assumptions t h a n r e q u i r e d when e i t h e r d i s l o d g e a b l e r e s i d u e o r p a s s i v e d o s i m e t r y i s u s e d as t h e b a s i s o f an exposure e s t i m a t e . T h e r e i s a need t o be much more a g g r e s s i v e i n e v a l u a t i n g human p e s t i c i d e e x p o s u r e s . Such e s t i m a t e s w i l l meet a growing need f o r b e t t e r human d a t a on t h e f a t e and d i s p o s i t i o n o f p e s t i c i d e s i n humans. T h i s i s n o t a s t u d i e s i n humans. Sensitiv u s e d t o measure v a n i s h i n g l y s m a l l amounts o f c h e m i c a l r e s i d u e on t r e a t e d c r o p s must be adapted t o t h e t r a c e a n a l y s i s o f p e s t i c i d e m e t a b o l i t e s i n u r i n e and o t h e r samples from humans. Fieldworkers, p r o d u c e r s , r e g i s t r a n t s , r e g u l a t o r s , and t h e g e n e r a l p u b l i c will b e n e f i t from t h e s i g n i f i c a n t l y more r e l i a b l e assessments o f r i s k which w i l l r e s u l t .

LITERATURE CITED 1.

2.

3.

4. 5. 6. 7. 8. 9.

Maddy, K.T., Edmiston, S.C. Pesticide Safety Program of the California Department of Food and Agriculture Based Upon Measurements of Potential Workplace Exposure and the Elimination of Excess Exposures: American Chemical Society Symposium Series No. 182, Pesticide Residue and Exposure, 1982, pp. 75-81. Maddy, K.T., Wang, R.G., Knaak, J.B., Liao, C.L., Edmiston, S.C., Winter, C.K. in Risk Assessment of Excess Pesticide Exposure to Workers in California: American Chemical Society Symposium Series, 272, Dermal Exposure Related to Pesticide Use, 1985, pp. 445-465. Coye, M.J., Lowe, J.A. and Maddy, K.T., Biological Monitoring of Agricultural Workers Exposed to Pesticides: II Monitoring Intact Pesticides and Their Metabolites: Journal of Occupational Medicine, 28:628-636, 1986. Popendorf, W.J.; Leffingwell, J.T. Residue Rev. 1978, 82, 125. Everhart, L.P.; Holt, R.F. J. Agric. Food. Chem 1982, 30, 222. Zweig, G.; Gao, R.; Popendorf, W.J. J. Agric. Food Chem. 1983, 31, 1109-1115. Winterlin, W.L.; Kilgore, W.W.; Mourer, C.; Schoen, S.R. J. Agric. Food Chem. 1984, 32, 664-672. Ritcey, G; Frank, R.; McEwen, F.L; Braun, H.E. Bull. Environ. Contam. Toxicol. 1987, 38, 840-846. Zweig, G.; Gao, R.; Witt, J.M.; Popendorf, W.; Bogen, K. J. Agric. Food Chem. 1984, 32, 1232-1236.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

353

26. MADDY ET AL. Monitoring Data in Regulatory Decisions

10.

Gunther, F.A.; Westlake, W.E.; Barkley, J.H.; Winterlin, W.; Langbehn, L. Bull. Environ. Contam. Toxicol. 1973, 9, 243249. 11. Hoffman, L . J . ; DeBaun, J.R.; Knarr, J.; Mann, J . J . Metabolism of N-(Trichloromethylthio)-1,2-dicarboximido- C-fe-4-cyclohexene (Captan) in the Rat and Goat. 1973, Western Research Center, Stauffer Chemical Company. RECEIVED March 29, 1988 14

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 27

Use of Biological Monitoring in the Regulatory Process Leonard Ritter and Claire A. Franklin Environmental and Occupational Toxicology Division, Environmental Health Directorate, Tunney's Pasture, Ottawa, Ontario K1A 0L2, Canada

Occupational risks associated with pesticide use can only be estimated if exposure can be quantified. Dermal exposure ha historicall bee estimated fro patches placed o more recent advance biological monitoring. Biological monitoring of pesticides can be carried out in a variety of body fluids including blood, urine, sweat, saliva, adipose tissue and exhaled breath; indeed, urinary concentration of the chemical or its metabolites has become one of the most frequently reported biological measures of exposure. For pesticides such as 2,4-D, which is excreted largely unmetabolized, urinary concentrations may be quantitatively related to exposure. Similarly, for organophosphorus compounds, data are presently available which establish the relationship between dermal dose and urinary metabolites. It has been postulated that this standard curve approach could then be utilized to estimate exposure in workers based on the metabolite excretion. There are many products, however, for which the metabolite excretion pattern is not known, thus severely limiting the usefulness of the biological monitoring approach as a measure of exposure to such products. O c c u p a t i o n a l exposure t o p e s t i c i d e s may p r e s e n t h e a l t h r i s k s t o b o t h a p p l i c a t o r s and a g r i c u l t u r a l w o r k e r s . I n order t o p r o p e r l y assess t h i s exposure and e s t i m a t e r i s k , a c c u r a t e d a t a on exposure and a b s o r p t i o n a r e n e c e s s a r y . I n a d d i t i o n t o q u a n t i f y i n g exposure, p r e c i s e i n f o r m a t i o n s h o u l d be a v a i l a b l e on the dose l e v e l s f o r b i o l o g i c a l e n d p o i n t s e s t a b l i s h e d through e x p e r i m e n t a l t o x i c i t y studies. This chapter not subject to U.S. copyright Published 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

27.

RITTER AND FRANKLIN

Monitoring in Regulatory Process

355

H i s t o r i c a l l y , r i s k from p e s t i c i d e r e s i d u e s i n food has been e s t i m a t e d by comparing the t h e o r e t i c a l d a i l y i n t a k e of the p e s t i c i d e r e s i d u e t o the a c c e p t a b l e d a i l y i n t a k e . The a c c e p t a b l e e f f e c t l e v e l seen i n e x p e r i m e n t a l a n i m a l s t u d i e s and utilization of an a p p r o p r i a t e s a f e t y f a c t o r f o r the t o x i c o l o g i c end-point b e i n g c o n s i d e r e d . I f the t h e o r e t i c a l d a i l y i n t a k e i s determined to be l e s s than the a c c e p t a b l e d a i l y i n t a k e , the p e s t i c i d e i s g e n e r a l l y c o n s i d e r e d s a f e f o r use on f o o d . S i m i l a r l y , r i s k t o agricultural workers can be e s t i m a t e d by comparing the t h e o r e t i c a l d a i l y exposure to a p e s t i c i d e and the a c c e p t a b l e d a i l y exposure. As i s the case w i t h food r e s i d u e s , i f the t h e o r e t i c a l d a i l y exposure i s e s t i m a t e d to be l e s s than the a c c e p t a b l e d a i l y exposure, the p e s t i c i d e may be c o n s i d e r e d s a f e f o r use by a g r i c u l t u r a l w o r k e r s . These comparisons a r e summarized i n F i g u r e 1. From the f i g u r e i t can be seen t h a t f o r the d e t e r m i n a t i o n of a g r i c u l t u r a l worker s a f e t y t h e r e i s a need to o b t a i n r e l i a b l e and a c c u r a t e e s t i m a t e s of worker exposure The m a j o r i t y of exposur take p l a c e v i a the derma e s t i m a t e d by the use of adsorbent patches l o c a t e d at v a r i o u s s i t e s on the w o r k e r s b o d i e s or c l o t h i n g . C l a s s i c a l p a t c h t e c h n i q u e s may o v e r e s t i m a t e exposure (4) l e a d i n g t o exaggerated r i s k e s t i m a t e s and improper r e s t r i c t i o n on r e g i s t r a t i o n and use of the p e s t i c i d e . More r e c e n t l y , c o n s i d e r a b l e i n t e r e s t has developed i n the t e c h n i q u e of human b i o l o g i c a l m o n i t o r i n g t o supplement or indeed r e p l a c e many of these o l d e r p a t c h t e c h n i q u e s f o r e s t i m a t i n g dermal exposure ( 5 ) . B i o l o g i c a l m o n i t o r i n g , a s p e c i f i c form of m o n i t o r i n g of human exposure, can be used to determine i f an a g r i c u l t u r a l worker has been exposed to a c h e m i c a l and, i n s e l e c t e d c a s e s , the e x t e n t t o which exposure may have taken p l a c e . Measurements can be made i n a v a r i e t y of b i o l o g i c a l f l u i d s i n c l u d i n g u r i n e and b l o o d . The advantages of b i o l o g i c a l m o n i t o r i n g , when compared to c l a s s i c a l p a t c h t e c h n i q u e , l i e i n the a b i l i t y of t h i s approach to more a c c u r a t e l y e s t i m a t e body burden of the c h e m i c a l from a l l r o u t e s of exposure. L i m i t a t i o n s of t h i s t e c h n i q u e i n c l u d e i n d i v i d u a l p h a r m a c o k i n e t i c v a r i a b i l i t y (6) and a requirement f o r e x t e n s i v e knowledge on metabolism and d i s p o s i t i o n of the c h e m i c a l . The r e l i a b i l i t y of t h i s t e c h n i q u e i s t h e r e f o r e l i m i t e d by the g e n e r a l u n a v a i l a b i l i t y of such e x t e n s i v e d a t a f o r the m a j o r i t y of p e s t i c i d e s i n use today. Low c o n c e n t r a t i o n s of a m e t a b o l i t e i n u r i n e c o u l d be the r e s u l t of any one or c o m b i n a t i o n of the f o l l o w i n g : ( i ) the c h e m i c a l i s not w e l l absorbed; ( i i ) the c h e m i c a l i s absorbed but i s s e q u e s t e r e d i n the body; ( i i i ) the c h e m i c a l i s m e t a b o l i z e d i n t o a number of d i f f e r e n t m e t a b o l i t e s a l l of w h i c h are e x c r e t e d at low l e v e l s . I f the k i n e t i c s and metabolism of the c h e m i c a l are not w e l l understood i t would be p o s s i b l e to i n c o r r e c t l y conclude t h a t low l e v e l s of the measured m e t a b o l i t e i n u r i n e i n d i c a t e t h a t o n l y a s m a l l amount of the c h e m i c a l was absorbed. T h i s i n t u r n would r e s u l t i n an u n d e r e s t i m a t e of r i s k . In t h i s paper we w i l l focus on the use of b i o l o g i c a l m o n i t o r i n g f o r q u a n t i f y i n g exposure and w i l l d i s c u s s these cases which show how p r i o r knowledge of the metabolism of the c h e m i c a l can i n f l u e n c e the s u i t a b i l i t y of b i o l o g i c a l m o n i t o r i n g to p r e d i c t exposure. 1

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

1. CHEMICALS WHICH ARE RAPIDLY ABSORBED, NOT SEQUESTERED, AND EXCRETED IN URINE LARGELY UNMETABOLIZED ARE GOOD CANDIDATES FOR ESTIMATING EXPOSURE (2,4-D), An example of a c h e m i c a l t h a t i s a good c a n d i d a t e f o r e s t i m a t i n g exposure from b i o l o g i c a l m o n i t o r i n g i s 2,4-D. I t i s a broad spectrum a g r i c u l t u r a l h e r b i c i d e w i d e l y used throughout the world. I t s a b s o r p t i o n , d i s t r i b u t i o n and metabolism i n animals have been s t u d i e d by s e v e r a l a u t h o r s and r e p o r t e d i n t h e l i t e r a t u r e (7-14). The a b s o r p t i o n and e x c r e t i o n o f 2,4-D i n humans v i a the o r a l r o u t e have been i n v e s t i g a t e d i n a c o n t r o l l e d l a b o r a t o r y s i t u a t i o n . The f i r s t o f these s t u d i e s ( 9 ) i n v o l v e d a s i n g l e o r a l dose of 5 mg 2,4-D a c i d / k g body weight t o each of 5 v o l u n t e e r s . U r i n e was c o l l e c t e d over a p e r i o d o f 144 hours f o l l o w i n g i n g e s t i o n and t h e p e r c e n t a g e o f the o r a l dose of 2,4-D e x c r e t e d was then c a l c u l a t e d . I t was shown t h a t 2,4-D was e x c r e t e d u n m e t a b o l i z e d as f r e e o r g l u c u r o n i d e c o n j u g a t e d 2,4complete r e c o v e r y o f th h o u r s . I n a d d i t i o n , 70 t o 83% o f the i n g e s t e d 2,4-D was r e c o v e r e d i n t h e f r e e form. A l t h o u g h two s u b j e c t s e x c r e t e d up t o 26% as t h e c o n j u g a t e d form, two o t h e r s u b j e c t s e x c r e t e d no more than 10% o f t h e c o n j u g a t e and t h e r e m a i n i n g s u b j e c t e x c r e t e d o n l y f r e e 2,4-D. T h i s study i n man shows t h a t 2,4-D i s absorbed f o l l o w i n g o r a l a d m i n i s t r a t i o n and e l i m i n a t e d l a r g e l y as unchanged parent compound. I t has a l s o been shown i n man t h a t 6% o f a d e r m a l l y a p p l i e d dose o f 2,4-D a c i d i s absorbed ( 1 5 ) . R e s u l t s from our own l a b o r a t o r y ( u n p u b l i s h e d ) have a l s o shown t h a t 15% o f a d e r m a l l y a p p l i e d dose o f 2,4-D a c i d i s absorbed from rhesus monkey forearm and t h a t 29% o f the a p p l i e d dose i s absorbed from the forehand ( F i g u r e 2 ) . I t was a l s o shown t h a t 100% of an i n t r a v e n o u s dose was e x c r e t e d i n d i c a t i n g t h a t the compound was not sequestered ( 1 5 ) . The r e s u l t s r e p o r t e d above show t h a t 2,4-D i s a compound which i s absorbed by t h e o r a l and dermal r o u t e s , i s e x c r e t e d l a r g e l y as unchanged parent compound and i s not s e q u e s t e r e d . Although a d e f i n e d r e l a t i o n s h i p between t h e amount o f 2,4-D a p p l i e d d e r m a l l y and the c o n c e n t r a t i o n o f u r i n a r y m e t a b o l i t e has not been e s t a b l i s h e d , t h e type of i n f o r m a t i o n d e s c r i b e d above g i v e s one some c o n f i d e n c e i n c o n c l u d i n g t h a t low u r i n a r y m e t a b o l i t e l e v e l s i n workers r e f l e c t low absorbed doses. The r e l a t i o n s h i p between dermal exposure and u r i n a r y m e t a b o l i t e e x c r e t i o n under normal use c o n d i t i o n s has been i n v e s t i g a t e d by Grover and h i s co-workers ( 1 6 ) . I n t h i s s t u d y , a t o t a l o f 9 farmers who r e p e a t e d l y sprayed 2,4-D were m o n i t o r e d d u r i n g normal s p r a y a p p l i c a t i o n s . I n a l l cases the t o t a l amount of 2,4-D a v a i l a b l e f o r a b s o r p t i o n was e s t i m a t e d u t i l i z i n g a standard p a t c h t e c h n i q u e , w h i l e a b s o r p t i o n was e s t i m a t e d by measuring 2,4-D i n u r i n e c o l l e c t e d f o r 96 hours f o l l o w i n g the l a s t exposure t o t h e chemical. The r e s u l t s a r e summarized i n T a b l e I I . As can be seen from the t a b l e t h e r e was a v e r y wide range o f c a l c u l a t e d dermal d e p o s i t i o n i n workers a p p l y i n g 2,4-D (75 t o 13,286 ug 2,4-D/kg s p r a y e d ) w h i l e t h e r e was a much narrower range o f u r i n a r y e x c r e t i o n of 2,4-D i n these same w o r k e r s . I t i s noteworthy t h a t d e p o s i t i o n

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

hrs

82

Free

1

2

103

21

Conjugate

* adapted from (9)

Total

0-96

SUBJECT

88

Free

88

-

Conjugate

70

Free

3

96

26

Conjugate

87

Free

4

92

5

Conjugate

83

93

Free

5

10

Conjugate

Table I . Percentages of 2,4-D (Free and Conjugate) Excreted i n Urine Following A Single Oral Dose of 5 mg/kg 2,4-D*

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Food

NOEL Safety Factor

• Acceptable D a i l y Intake

- animal t o x i c i t y data generated using o r a l route of exposure - o r a l route of exposure to humans through food and water - i f T h e o r e t i c a l D a i l y Intake (TDI) i s l e s s than Acceptable D a i l y Intake (ADI), p e s t i c i d e may be safe f o r use on food

Workers

NOEL Safety Factor

• Acceptable D a i l y Exposure

- animal t o x i c i t exposure - dermal route of exposure g e n e r a l l y the greatest f o r workers and bystanders - i f T h e o r e t i c a l D a i l y Exposure (TDE) i s l e s s than Acceptable D a i l y Exposure (ADE), p e s t i c i d e may be safe f o r use by a g r i c u l t u r a l workers.

F i g u r e 1.

Risk Estimation

A B

S 0 R P T 1 0 N 0

1

2

3

4

5

6

7

8

9 10 11 12 13 14

TIME (DAYS) 0

FOREHEAD FOREARM F i g u r e 2.

2,4-D

A c i d - % Recovery i n Monkeys

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988. 75 391 1,529 13,286 1,339 444 1,572 951

CALCULATED DERMAL DEPOSITION OF ug 2,4-Da.e/Kg sprayed

11 6 14 3 17 17 33 17

a

CUMULATIVE URINARY EXCRETION OF ug 2,4-Da.e. /kg sprayed

T o t a l amount excreted during exposure period and f o r 4 days a f t e r l a s t exposure.

24 35 49 127 89 102 186 71

AMOUNT SPRAYED (kg a.e.)

• adapted front (16)

* Acid equivalent

a

B B B F D A G E

SUBJECT

Table I I . Cumulative Estimates of Dermal 2,4-Da.e.* Deposition and Urinary 2,4-Da.e. Excretion f o r Farmers Involved i n Spraying of 2,4-D BY Ground Rig.*

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

l e v e l s were n o t n e c e s s a r i l y c o r r e l a t e d w i t h t h e amount o f 2,4-D sprayed. These d a t a suggest t h a t compounds t h a t a r e r a p i d l y absorbed, not s e q u e s t e r e d and e x c r e t e d i n an unchanged form are w e l l s u i t e d t o the p r i n c i p l e o f b i o l o g i c a l m o n i t o r i n g and t h a t u r i n a r y m e t a b o l i t e l e v e l s may more a c c u r a t e l y r e f l e c t both exposure and body burden than would be e s t i m a t e d by more c l a s s i c a l patch t e c h n i q u e s . 2.

CHEMICALS FOR WHICH URINARY METABOLITES ARE QUANTITATIVELY RELATED TO DERMAL DOSE. (AZINPHOS-METHYL) Azinphos-methyl i s an i n s e c t i c i d e w i d e l y used f o r t h e c o n t r o l o f a v a r i e t y o f o r c h a r d i n s e c t s . E a r l y work w i t h organophosphorus i n s e c t i c i d e s has demonstrated t h a t i n humans (17-20) and i n r a t s (21-22) a r e l a t i o n s h i p e x i s t s between exposure t o organophosphorus p e s t i c i d e s and e x c r e t i o n o f a l k y l phosphate m e t a b o l i t e s . S p e c i f i c a l l y , i t has been shown (23) t h a t a z i n p h o s - m e t h y l i s degraded p r i m a r i l y i n m i c r o s o m a t h i o p h o s p h a t e (DMTP), d i m e t h y a n a l o g . The s t r u c t u r e o f a z i n p h o s - m e t h y l and t h e s i t e o f h y d r o l y s i s a r e shown i n F i g u r e 3. S t u d i e s u n d e r t a k e n i n our l a b o r a t o r y have examined t h e r e l a t i o n s h i p between i n c r e a s i n g doses o f t o p i c a l l y a p p l i e d a z i n p h o s - m e t h y l and t h e u r i n a r y e x c r e t i o n o f t h i s dose as DMTP ( m e t a b o l i t e ) i n r a t s (24) and i n man ( 2 5 ) . As can be seen from T a b l e I I I , d e r m a l l y a p p l i e d doses o f between 100 and 800 microgram per r a t r e s u l t e d i n a p p r o x i m a t e l y 10% o f t h e a p p l i e d dose b e i n g e x c r e t e d as DMTP m e t a b o l i t e . F o l l o w i n g o u r i n i t i a l o b s e r v a t i o n s t h a t a p p r o x i m a t e l y 10% o f a t o p i c a l l y a p p l i e d a z i n p h o s - m e t h y l dose i n r a t s c o u l d be r e c o v e r e d as u r i n a r y DMTP, a s i m i l a r i n v e s t i g a t i o n was u n d e r t a k e n i n man ( T a b l e I V ) . I t was shown t h a t a s i m i l a r 10:1 p r o p o r t i o n o f t h e d e r m a l l y a p p l i e d dose i n humans c o u l d be r e c o v e r e d as u r i n a r y DMTP m e t a b o l i t e . F u r t h e r , as can be seen from Table V, the r e l a t i o n s h i p between d e r m a l l y a p p l i e d dose and t h e amount o f DMTP m e t a b o l i t e e x c r e t e d was r e l a t i v e l y l i n e a r over a dermal dosage range o f 500 t o 6000 micrograms p e r p e r s o n . The r e s u l t s o b t a i n e d from b o t h t h e r a t and human i n v e s t i g a t i o n s have suggested t h a t t h e r e i s a q u a n t i t a t i v e r e l a t i o n s h i p between dermal dose and t h e p r o p o r t i o n o f t h i s dose t h a t c a n be r e c o v e r e d i n u r i n e as a DMTP m e t a b o l i t e . S p e c i f i c a l l y , these r e s u l t s suggest t h a t t h e r e i s a p p r o x i m a t e l y a 10 t o 1 r e l a t i o n s h i p between t h e dose t h a t i s a p p l i e d and t h e c o n c e n t r a t i o n o f u r i n a r y m e t a b o l i t e s ( T a b l e V ) . T h i s r e l a t i o n s h i p c a n be v e r y u s e f u l i n f i e l d m o n i t o r i n g o f a g r i c u l t u r a l workers i n t h a t i t a l l o w s e s t i m a t i o n o f dermal exposure s i m p l y by the d e t e r m i n a t i o n o f l e v e l s o f u r i n a r y m e t a b o l i t e s . I t s h o u l d , however, be noted t h a t w h i l e t h i s method may be u s e f u l f o r e s t i m a t i o n o f dermal exposure l e v e l s , u r i n a r y m e t a b o l i t e s may not be u s e f u l f o r purposes of e s t i m a t i n g r i s k . DMTP r e p r e s e n t s o n l y one o f s e v e r a l m e t a b o l i t e s formed on exposure t o a z i n p h o s - m e t h y l and may not r e f l e c t r e l a t i v e c o n c e n t r a t i o n s o f o t h e r b i o l o g i c a l l y i m p o r t a n t metabolites i n various species. 3. CHEMICALS WHICH UNDERGO COMPLEX METABOLISM AND WHERE DOSE AND ROUTE OF ADMINISTRATION MAY QUALITATIVELY QUANTITATIVELY AFFECT METABOLISM AND BIOLOGICAL OUTCOME (METHYLENE CHLORIDE). Mice exposed t o 3500 and 7000 mg/kg/6 h r day methylene c h l o r i d e (MC) by

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

8

5

5

3

100

200

400

800

Dermal

Dermal

Dermal

Dermal

Route

* Adapted from (24)

N

Dose (ug/rat)

8.3 15.5 34.8 73.6

12.2 23.0 18.9

48 hours

6.9

24 hours

86.1

81.7

11

9

37.3

9

36.4

91.2

120 hours

X Dose Excreted As DMTP

8

96 hours

X Dose

16.0

8.8

72 hours

Cumulative T o t a l (ug DMTP)

Table I I I . Excretion of DMTP Following Dermal Application of Azinphos-methyl i n Male Rats*

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

52 140

36 76

188 120

122 184

1,000

2,000

4,000

6,000

* adapted from (24)

82 25

246 322

264 270

104 76

80 180

110 41

48 hours

278 368

454 354

148 90

92 212

123 41

72 hours

Cumulative Total (ug DMTP)

24 hours

500

Dose (ug/person)

323

404

119

152

85

Average Total

5

10

6

15

17

% dose excreted as DMTP

Table IV. Excretion of DMTP Following Dermal Application of Azinphos-methyl to the Forearm of Human Volunteers*

o

w w

3

W H O

o ^ o

2 3

o

H

S o

P

5

o

ON

27. RITTER AND FRANKLIN

Azinphos-methyl:

Monitoring in Regulatory Process

s-(3,4-dihydro-4-oxobenzo [d]~ [ 123]-triazin-3-yl methyl)-0,O-dimethyl phosphorodithioate

-|—S-CHj

1

hydrolysi

Metabolite:

DMTP

Analysis:

Azinphos-methyl by HPLC

:

Metabolite by a modified shafik method to derlvatize alkyl phosphates followed by G.C. analysis

Figure 3

Table V.

Analysis of Azinphos-methyl by HPLC

Relationship Between Amount of DMTP Excreted and the Amount of Azinphos-methyl (A.M.) Applied*

Dermal Dose Range

Metabolite Excretion/ Amount A.M. Applied

Rat

100 - 800 ug

1 ug DMTP/10 ug A.M.

Human

500 - 6000 ug

1 ug DMTP/10 ug A.M.

* adapted from (25)

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

363

364

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

i n h a l a t i o n f o r 5 days/week demonstrated an excess o f h e p a t o c e l l u l a r adenomas/carcinomas as compared t o unexposed c o n t r o l s ( 2 6 ) . These r e s u l t s c o n t r a s t e d w i t h an e a r l i e r study conducted by t h e N a t i o n a l C o f f e e A s s o c i a t i o n (27) i n w h i c h mice exposed t o MC i n d r i n k i n g water a t c o n c e n t r a t i o n s l e a d i n g t o exposure o f 250 mg/kg/day showed no i n c r e a s e i n t h e i n c i d e n c e o f l i v e r tumours. There a r e two pathways i n v o l v e d i n t h e metabolism o f MC. R e l a t i v e tumour response a s s o c i a t e d w i t h these two pathways i s shown i n F i g u r e 4. The two pathways i n v o l v e d i n c l u d e t h e s a t u r a b l e mixed f u n c t i o n o x i d a s e (MFO) pathway and t h e g l u t a t h i o n e - s - t r a n s f e r a s e (GST) pathway. E s t i m a t e s of t h e c o n c e n t r a t i o n o f MC and t h e r a t e s o f each o f t h e two m e t a b o l i c pathways have been examined ( 2 8 ) . The r e s u l t s o f these s t u d i e s a r e g i v e n i n T a b l e V I and have shown t h a t w h i l e t h e r a t e o f the MFO pathway i s comparable i n the two s t u d i e s , the d e l i v e r e d dose by t h e GST pathway and p a r e n t compound c o n c e n t r a t i o n s d i f f e r s u b s t a n t i a l l y . T h i s would tend t o i m p l y t h a t t h e MFO pathway i s not r e s p o n s i b l e f o r t h e tumou the f a c t t h a t t h e r a t e o d r i n k i n g water and i n h a l a t i o n s t u d i e s , u t i l i z a t i o n o f t h i s m e t a b o l i c pathway a l o n e f o r t h e purpose o f assessment o f p o t e n t i a l r i s k would l e a d t o erroneous c o n c l u s i o n s ; i t c o u l d not be u t i l i z e d t o e x p l a i n the b i o l o g i c a l response seen i n the i n h a l a t i o n s t u d y but absent i n the d r i n k i n g water s t u d y . C l e a r l y , i n t h i s c a s e , t h e e x a m i n a t i o n o f the GST pathway i s o f paramount importance i n a s s e s s i n g t h e o v e r a l l r i s k a s s o c i a t e d w i t h exposure t o MC. The case o f methylene c h l o r i d e , demonstrates r a t h e r e l e g a n t l y t h e need f o r a thorough u n d e r s t a n d i n g o f m e t a b o l i s m p r i o r t o u t i l i z i n g m e t a b o l i t e l e v e l s as an i n d i c a t i o n o f exposure f o r t h e purpose o f e s t i m a t i n g r i s k . 100 •••

80

Multistage Model Prediction

o

Drinking Water Study

+

Inhalation Study

60 -

40

Saturation of MFO Path

20

0

1 I T 0.005 0.010 0.015

/

/

0.85

GST Activity (g/L/day)

Figure 4. Metabolism of methylene chloride. (Adapted from ref. 28.)

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

1.80

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

•adapted from (28)

0 3500 7000

0 60 125 185 250

Drinking water (mg/kg/day)

Inhalation (mg/kg/6 hr day)

Administered Dose

Route of Administration (dose u n i t s )

0.0 3.6 3.7

0.0 1.3 2.8 4.0 5.4

MFO Path (g/L/day)

0.00 0.85 1.80

0 0.003 0.007 0.011 0.016

0 360 770

0 1.3 2.9 4.6 6.4

Delivered Dose GST Path Parent Compound (g/L/day) (mg hr/L)

Table VI. Metabolism of Methylene Chloride i n Female Mice*

366

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

SUMMARY We b e l i e v e t h a t b i o l o g i c a l m o n i t o r i n g , s p e c i f i c a l l y u r i n a r y m e t a b o l i t e l e v e l s , c a n be used t o e s t i m a t e exposure i f t h e a b s o r p t i o n , d i s t r i b u t i o n , m e t a b o l i s m and e x c r e t i o n a r e known f o r the chemical. The r e l a t i o n s h i p between exposure and u r i n a r y m e t a b o l i t e l e v e l s a r e more e a s i l y u n d e r s t o o d f o r c h e m i c a l s t h a t a r e a b s o r b e d , not s e q u e s t e r e d and e x c r e t e d v i r t u a l l y u n m e t a b o l i z e d . Selection of a few such c h e m i c a l s f o r i n depth e v a l u a t i o n c o u l d l e a d t o a b e t t e r e s t i m a t i o n o f dermal exposure than t h a t p r e s e n t l y o b t a i n a b l e from patches. T h i s i n t u r n would a s s i s t i n t h e e v e n t u a l v a l i d a t i o n o f the concept o f a g e n e r i c approach t o e s t i m a t e s o f e x p o s u r e . I t must be emphasized t h a t measurement o f u r i n a r y m e t a b o l i t e l e v e l s f o l l o w i n g e x p o s u r e , i n t h e absence o f adequate i n f o r m a t i o n on a b s o r p t i o n , d i s t r i b u t i o n , m e t a b o l i s m and e x c r e t i o n , i s u s e l e s s . Under such c i r c u m s t a n c e s , i t c o u l d not be d e t e r m i n e d whether a low c o n c e n t r a t i o n o f m e t a b o l i t e i n u r i n e was due t o poor a b s o r p t i o n (and t h e r e f o r e low r i s k ) , whether i t was w e l l absorbed and s e q u e s t e r e d w i t h i n t h e body o r whethe e x c r e t e d as many m e t a b o l i t e s measured. Another p o s s i b i l i t y would be t h a t e x c r e t i o n had taken place v i a other routes. Acknowledgments: The a u t h o r s g r a t e f u l l y m a n u s c r i p t by Wendy M i l k s .

acknowledge t h e t y p i n g o f t h i s

Literature Cited 1.

Wolfe, H.R.; Durham, W.F.; Armstrong, J.F. Arch. Environ. Health, 1967, 14, 622-633. 2. Wolfe, H.R.; Armstrong, J.F.; Staiff D.C.; Comer, S.W. Arch. Environ. Health, 1972, 25, 29-31. 3. Durham, W.F.; Wolfe H.R.; Elliott, J.W. Arch. Environ. Health, 1972, 24, 381-287. 4. Nigg, H.N.; Stamper, J.H. In Dermal Exposure Related to Pesticide Use; Honeycutt, R.C.; Zweig, G.; Ragsdale, N.N., Eds.; ACS Symposium Series No. 273; American Chemical Society: Washington, DC, 1985; pp 95-108. 5. Ashford, N.A.; Spadafor C.J.; Caldart, C.C. Harvard Environmental Law Review, 1984, 8, 263-363. 6. Gompertz Ann. Occup. Hygiene, 19 80, 23, 405-410. 7. Erne, K. Acta Vet. Scand. 1966, 7, 264-271. 8. Kolmodin-Hedman, B.; Hoglund, S.; Akerblom, M. Arch Toxicol 1983, 54, 257-265. 9. Sauerhoff, M.W.; Braun, W.H.; Blau, G.E.; Gehring, P.J. Toxicology, 1977, 8, 3-11. 10. Gutenmann, W.H.; Lisk, D.J. NY Fish Game J., 1965, 12(1), 108-111. 11. Erne, K.; Sperber, I. Acta Pharmacol. Toxicol., 1974, 35, 233-241. 12. Guarino, A.M.; Arnold, S.T. In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M.A.Q., Lech, J . F . , Menn, J.J., Eds.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979; pp 250-258. 13. James, W.H. Lancet, 1977, 1, 603.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

27. RITTER AND FRANKLIN Monitoring in Regulatory Process

367

14. Pritchard, J.B.; Miller, D.S. Fed. Proc., 1980, 39(14), 3207-3212. 15. Feldman, R.J.; Maibach, H.I. Toxicol. Appl. Pharmacol., 1974, 28, 126-132. 16. Grover, R., Franklin, C.A.; Muir, N.I.; Cessna, A.J.; Riedel, D. Toxicology Letters, 1986, 32, 73-83. 17. Kraus, J.F.; Mull, R.; Kurtz, P.; Winterlin, W.; Franti, C.E.; Borhani, N.; Kilgore, W. J . Toxicol. Environm. Hlth. 1981, 8, 169- 184. 18. Wojeck, G.A.; Nigg, H.N.; Stamper, J.H.; Bradway, D.E. Arch. Environm. Contam. Toxicol., 1981, 10, 725-735. 19. Knaak, J.B.; Maddy, K.T.; Khalifa, S. Bull. Environm. Contam. Toxicol. 1979, 21, 375-380. 20. Lores, E.M.; Bradway, D.E.; Moseman, R.F. Arch. Environm. Hlth. 1978, 33, 270-276. 21. Bradway, D.E.; Shafik T.M.; Lores E.M J Agric Food Chem 1977, 25, 1353-1358 22. Bradway, D.E.; Shafik 1342-1344. 23. Motoyama, N.; Dauterman, W.C. Pest. Biochem. Physiol. 1972, 2, 170- 177. 24. Franklin, C.A.; Greenhalgh, R.; Maibach, H.I. In IUPAC Pesticide Chemistry, Human Welfare and the Environment; J. Miyamoto et al Eds.; Perg Press. 1983; pp 221-26. 25. Franklin, C.A.; Muir, N.I.; Moody, R. Toxicology Letters, 1986, 33, 127-136. 26. NTP: National Toxicology Program, CAS No. 75-09-2. National Institute of Health, 1986. 27. EPA/600/8-82/004F. Final Report. United States Environmental Protection Agency, 1985. 28. Krewski, D.; Murdoch, D.J.; Withey, J.R. Drinking Water and Health 1987, 8, 441-448. RECEIVED February 23, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Chapter 28

National Agricultural Chemicals Association Overview on Assessment of Mixer-Loader-Applicator Exposure to Pesticides Biological Monitoring Richard C. Honeycutt National Agricultural Chemicals Association, 1155 15th Street, NW,

Agricultural worker exposure to pesticides is rapidly becoming a major issue in occupational health. With this in mind, the National Agricultural Chemical Association (NACA) along with member companies has over the past few years begun to throughly address this issue. The objective of this paper is to present an overview of how NACA regards mixer-loader-applicator exposure assessment. This paper w i l l briefly consider a historical perspective on the NACA viewpoint on worker exposure and discuss the development of the NACA protocol for worker exposure as well as the development of the NACA - EPA public worker exposure data base to be used for surrogate or generic estimation of mixer-loader-applicator exposure. This paper w i l l also deal with specific field methods of measuring mixer-loader­ -applicator exposure including biological monitoring that are common­ ly used by U.S. companies and supported in the published NACA proto­ col. Finally, directions for future research in field worker expo­ sure technology will be considered. A H i s t o r i c a l Perspective sure Research

o f NACA Involvement i n F i e l d Worker Expo-

In September 1983, NACA formed an ad hoc subcommittee on worker exposure assessment. The g o a l o f t h i s subcommittee was t o b r i n g t o g e t h e r t e c h n i c a l l e v e l e x p e r t s from s e v e r a l member companies i n v o l v e d d i r e c t l y i n p e r f o r m i n g f i e l d w o r k e r exposure s t u d i e s t o a s s e s s and advance the s t a t e o f the a r t i n m i x e r - l o a d e r - a p p l i c a t o r exposure t o p e s t i c i d e s . T h i s subcommittee ( p a r t o f t h e NACA P u b l i c H e a l t h and T o x i c o l o g y Committee) has a c c o m p l i s h e d t h e f o l l o w i n g object ives: 0097-6156/89/0382-0368$06.00/0 ° 1989 American Chemical Society

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

28. HONEYCUTT 1. 2. 3.

4.

369

Mixer—Loader—Applicator Exposure

Developed a NACA p r o t o c o l f o r p e r f o r m i n g f i e l d worker e x p o s u r e studies. T h i s p r o t o c o l was p u b l i s h e d i n 1985. Supported and a s s i s t e d EPA i n t h e development o f a j o i n t NACA EPA - p u b l i c g e n e r i c d a t a b a s e . Developed and c o o r d i n a t e d r e s e a r c h t o improve e x p o s u r e moni­ t o r i n g t e c h n o l o g y (e.g., R. Fenske's r e s e a r c h on F l u o r e s c e n t Tracer Technology). I n t e r a c t e d w i t h s t a t e and f e d e r a l r e g u l a t o r y a g e n c i e s t o r e s o l v e i s s u e s o f mutual i n t e r e s t on m i x e r - l o a d e r - a p p l i c a t o r exposure assessment.

The communication w i t h EPA i n t h e development o f t h e worker-exposure p r o t o c o l and t h e g e n e r i c d a t a b a s e have been t i m e l y and have had some impact on r e g u l a t o r y p o l i c y a t EPA i n r e c e n t months. This i s r e f l e c t e d i n t h e f a c t t h a t t h e NACA p r o t o c o l and EPA Subpart U guidelines are quite s i m i l a r I additio t h EPA-NACA publi d a t a base w i l l save i n d u s t r money on f u t u r e exposur Development o f t h e NACA P r o t o c o l c a t o r Exposure S t u d i e s

for Conducting

Mixer-Loader-Appli­

The NACA p r o t o c o l on worker exposure was d e v e l o p e d between 1983-1985 by t h e NACA submcommittee. The o b j e c t i v e s o f t h e p r o t o c o l were t o s t a n d a r d i z e the methodology f o r f i e l d exposure s t u d i e s w i t h i n i n d u s ­ t r y and g i v e d i r e c t i o n t o new r e s e a r c h e r s i n t h i s f i e l d . The NACA p r o t o c o l i s c o n s i s t e n t w i t h t h e WHO p r o t o c o l on worker exposure. Comments on t h e NACA p r o t o c o l were a l s o s o l i c i t e d from EPA and i t i s g e n e r a l l y f e l t among the subcommittee members t h a t t h e A p p l i c a t o r E x p o s u r e M o n i t o r i n g G u i d e l i n e s ( S u b p a r t U) p u b l i s h e d by EPA i n 1987 (1) r e f l e c t many methods s i m i l a r t o t h o s e i n t h e NACA protocol. A listing 1. 2. 3.

4.

5.

o f the g e n e r a l

c o n t e n t s o f t h e NACA p r o t o c o l

follows:

E v a l u a t i o n o f the need f o r a f i e l d s t u d y . P r o c e d u r e s f o r a d m i n i s t r a t i o n o f a f i e l d e x p o s u r e s t u d y , e.g., s e l e c t i o n o f the work crew. While t h e g e n e r a l n a t u r e o f t h e p r o t o c o l i s s t a n d a r d i z a t i o n , exposure assessment t e c h n i q u e s a r e f l e x i b l e g i v i n g the r e s e a r c h e r some l a t i t u d e i n methodology. F i e l d d a t a c o l l e c t i o n forms a r e p r e s e n t e d i n o r d e r t o s t a n d a r d ­ i z e r e p o r t i n g and add q u a l i t y a s s u r a n c e . C a l c u l a t i o n methods are a l s o i n c l u d e d . Appendices a r e a t t a c h e d which p r o v i d e d e t a i l e d methodology f o r researchers with l i t t l e experience i n t h i s f i e l d .

These NACA g u i d e l i n e s p o i n t out t h a t t h e e x i s t i n g p a t c h method o f exposure assessment, w h i l e t h e o r e t i c a l l y n o t t h e most r e l i a b l e meth­ od, i s the method o f c h o i c e i n the U.S. The NACA p r o t o c o l r e c o g ­ n i z e s t h e s h o r t comings o f such an e m p i r i c a l method. However, t h e only current a l t e r n a t i v e , B i o l o g i c a l Monitoring, while t h e o r e t i c a l l y the s u p e r i o r method, has n o t been d e v e l o p e d t o a p o i n t o f g e n e r a l implementation o r t o a p o i n t o f r e g u l a t o r y impact. This i n d i r e c t

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

370

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

method r e q u i r e s c o n s i d e r a b l e s u p p o r t s t u d i e s t o make t h e d a t a i n t e r pretable. Such s t u d i e s are d i s c u s s e d below and as you w i l l see a r e complex and e x p e n s i v e . A D i s c u s s i o n of S p e c i f i c F i e l d Methods as Recommended by the NACA P r o t o c o l f o r M i x e r - L o a d e r - A p p l i c a t o r Exposure Assessment Measuring I n h a l a t i o n Exposure: C u r r e n t l y i n the U.S. i n d u s t r y i t i s r e c o g n i z e d t h a t i n h a l a t i o n exposure t o p e s t i c i d e s i s o n l y a s m a l l f r a c t i o n ( 1%) o f dermal exposure. There a r e , however, a few i n s t a n c e s when i n h a l a t i o n exposure can be c r i t i c a l such as a Category I p e s t i c i d e t h a t may be h i g h l y v o l a t i l e . In t h i s case r e s p i r a t o r p r o t e c t i o n w i l l be r e q u i r e d . I n h a l a t i o n exposure i s somewhat d i f f i c u l t t o measure i n a f i e l d study. The NACA p r o t o c o l p r e f e r r e d method i s t o use p e r s o n a l samp l i n g d e v i c e s w h i c h a r e c a l i b r a t e d t o an a i r f l o w o f 4 1 i t e r s / m i n u t e Appropriate c a l i b r a t i o n v a l i d a t e t h i s method. M o d i f i e d e s c r i b e d by Duhram and Wolf a r e a c c e p t a b l e but have some d i s a d v a n t ages, such as ease o f c o n t a m i n a t i o n and i n c o n v e n i e n c e t o the t e s t s ub j e c t . Measuring Dermal Exposure: The NACA p r o t o c o l c a l l s f o r use o f t h e c l a s s i c a l p a t c h method t o measure dermal exposure. Gauze pads o r - c e l l u l o s e pads are commonly used w i t h a b a c k i n g m a t e r i a l such as aluminum f o i l o r a p l a s t i c f i l m . Patches a r e g e n e r a l l y p l a c e d i n s i d e or o u t s i d e the c l o t h i n g . Recent advances i n t e c h n o l o g y have shown t h a t patches can be c o v e r e d w i t h a chambray o r denim c o v e r t o s i m u l a t e i n s i d e p a t c h e s . These " i n s i d e " patches as w e l l as uncovered patches can a l l be worn on t h e o u t s i d e o f work c l o t h i n g f o r convenience. I t i s now r e c o g n i z e d t h a t hand exposure i s the major component of t o t a l exposure f o r m i x e r - l o a d e r s and i s f a r g r e a t e r than f o r any o t h e r t y p e o f exposure i n v o l v i n g p e s t i c i d e s . Hand wash p r o c e d u r e s f o r measuring hand exposure a r e commonly used i n the i n d u s t r y . Hand exposure can a l s o be measured by u s i n g adsorbent c o t t o n g l o v e s , a l t h o u g h t h i s t e c h n i q u e may o v e r e s t i m a t e exposure. C a l c u l a t i n g T o t a l Dermal Exposure from P a t c h Data: T o t a l dermal exposure i s g e n e r a l l y c a l c u l a t e d u s i n g i n d i v i d u a l p a t c h d a t a . The amount o f p e s t i c i d e per cm^ on a p a t c h i s m u l t i p l i e d by the s p e c i f i c body a r e a i n q u e s t i o n ( e . g . , c h e s t , neck, arm, l e g , e t c . ) . T h i s procedure may l e a d t o c o n s i d e r a b l e e r r o r i f patches a r e s m a l l . Thus, i t i s d e s i r a b l e t o maximize t h e s i z e o f t h e p a t c h e s used. These e l e m e n t a l exposures are then summed t o g i v e t o t a l body exposure. I t i s p r e f e r a b l e t o e x p r e s s r e s u l t s i n terms o f amount o f exposure per pound a i handled o r per l o a d h a n d l e d . Maximum exposure v a l u e s a r e c a l c u l a t e d by d e t e r m i n i n g t h e t o t a l pounds handled/day o r the number of loads/day. Measuring Exposure U s i n g B i o l o g i c a l M o n i t o r i n g : The term b i o l o g i c a l m o n i t o r i n g r e f e r s t o any method w h i c h d e r i v e s exposure from meas u r i n g parent or m e t a b o l i t e s i n body f l u i d s . The use o f b i o l o g i c a l m o n i t o r i n g i s the most e f f e c t i v e means f o r measuring t o t a l dose o r

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

28. HONEYCUTT

Mixer-Loader-Applicator Exposure

371

body burden. Use o f t h i s t e c h n i q u e r e s o l v e s many problems a s s o ­ c i a t e d w i t h the patch t e c h n i q u e i n c l u d i n g o v e r e s t i m a t i o n o f dermal exposure. The NACA p r o t o c o l c o n s i d e r s B i o l o g i c a l M o n i t o r i n g t o be an i n d i r e c t method and must be accompanied by adequate s u p p o r t i n g s t u d i e s t o d e f i n e the a b s o r p t i o n , metabolism, p h a r m a c o k i n e t i c s and e x c r e t i o n o f the p a r e n t / m e t a b o l i t e s i n humans o r an a p p r o p r i a t e s u r r o g a t e animal. These s t u d i e s d e f i n e t h e q u a n t i t a t i v e r e l a t i o n ­ s h i p between dose and e x c r e t i o n o f the p e s t i c i d e and i t s meta­ bolites. I w i l l b r i e f l y d e s c r i b e t h e s e r e q u i r e m e n t s below: Criteria

f o r U t i l i z a t i o n of B i o l o g i c a l

Monitoring

A.

Dermal A b s o r p t i o n - Dermal a b s o r p t i o n s t u d i e s s h o u l d be p e r ­ formed on a p p r o p r i a t e s p e c i e s ( t h e use o f r a t s , monkeys o r p i g s are common). In v i t r o dermal a b s o r p t i o n s t u d i e s a r e g a i n i n g i n popularity. The p e s t i c i d e i n q u e s t i o n must be absorbed t o an appropriate extent ove m o n i t o r i n g can be use

B.

P h a r m a c o k i n e t i c s - P h a r m a c o k i n e t i c and e x c r e t i o n k i n e t i c s i n a p p r o p r i a t e s u r r o g a t e a n i m a l s o r man must be u n d e r s t o o d . Multi­ p l e s p e c i e s s h o u l d be t e s t e d s i n c e the chance o f one s p e c i e s e x c r e t i o n p a t t e r n b e i n g d i f f e r e n t from the o t h e r i s c o n s i d e r ­ able. I d e a l l y , e x c r e t i o n of parent/metabolites should occur o v e r the f i r s t 24 hours a f t e r o r a l d o s i n g and w i t h i n a few days a f t e r dermal d o s i n g . In a d d i t i o n , a l a r g e p e r c e n t a g e o f the dose s h o u l d be e x c r e t e d i n t o u r i n e .

C.

A n a l y t i c a l Method - The a n a l y t i c a l method must measure e x c r e t e d p a r e n t and m e t a b o l i t e s and account f o r a l a r g e p e r c e n t a g e o f the e x c r e t e d compounds. The s e n s i t i v i t y o f the a n a l y t i c a l method must be enough t o d e t e c t exposure a t and below t h e t o x i c o l o g i c a l end p o i n t c o n c e n t r a t i o n . E x t e n s i v e m e t a b o l i s m makes b i o l o g i c a l m o n i t o r i n g e x t r e m e l y d i f f i c u l t and makes i t e x p e n s i v e t o d e v e l o p s u p p o r t i n g p h a r m a c o k i n e t i c and a n a l y t i c a l methods.

D.

Convenience t o and C o o p e r a t i o n o f F i e l d Workers - U r i n e m o n i ­ t o r i n g o f a g r i c u l t u r a l workers i s d i f f i c u l t . C o l l e c t i n g 24-hour u r i n e s c a r r i e s w i t h i t c o n s i d e r a b l e l o s s o f c o n t r o l o f the experiment s i n c e workers g e n e r a l l y t a k e a c a s u a l a t t i t u d e toward their responsibility. A c t i v i t y p a t t e r n s may a l s o a p p r e c i a b l y change u r i n a r y o u t p u t by a l t e r i n g p e r s p i r a t i o n r a t e s . A l s o , l i q u i d i n t a k e can a l t e r u r i n e o u t p u t . Measurement o f c r e a t i n i n e (WHO p r o t o c o l ) can overcome some o f t h e s e p o t e n t i a l problems s i n c e c r e a t i n i n e e x c r e t i o n i s c o n s t a n t over time and measurement a l l o w s one t o e s t i m a t e t o t a l e x c r e t i o n volume. A d d i t i o n a l problems may a r i s e i f u r i n e samples a r e t o be t a k e n on a l o n g - t e r m b a s i s . Of c o u r s e , B i o l o g i c a l M o n i t o r i n g o f body t i s s u e s such as b l o o d i s an i n t r u s i v e method and s h o u l d always be accompanied by m e d i c a l s u r v e i l l a n c e and s u p e r v i s i o n t o a v o i d p o t e n t i a l problems.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

372 Costs 1.

for Biological

Monitoring:

Dermal A b s o r p t i o n

Studies

Rat Model $70,000 Monkey Model - $100,000 In V i t r o Models $25,000 2.

3.

Pharmacokinetic

Excretion Studies

Rat Model Monkey Model

-

$65,000 $20,000

A n a l y t i c a l Method Development M e t a b o l i s m S t u d i e s (2 s p e c i e s ) - $200,000 Method Development 2 Major M e t a b o l i t e M u l t i p l e M e t a b o l i t e s - $100,000 Method V a l i d a t i o n $10,000

Examples o f Uses o f B i o l o g i c a l M o n i t o r i n g o f P e s t i c i d e s by I n d u s t r y : T h e r e a r e a few companies w h i c h have d e v e l o p e d b i o l o g i c a l m o n i t o r i n g f o r s p e c i f i c p e s t i c i d e s and f o r s p e c i f i c p u r p o s e s . Dow Chemical - S c i e n t i s t s a t Dow Chemical were some o f the f i r s t t o d e v e l o p u r i n e m o n i t o r i n g as a t o o l f o r e x p o s u r e assessment. In a 1982 r e v i e w a r t i c l e by Leng, Levy and o t h e r s ( 2 ) , i t was shown t h a t exposure t o 2.4,5T c o u l d be measured r e l i a b l y and i n f a c t u r i n a r y e x c r e t i o n p r o v i d e d a more r e l i a b l e measure o f dose than a n a l y s i s o f patch data. Monsanto - S c i e n t i s t s at Monsanto have r e c e n t l y performed s u p p o r t i n g s t u d i e s f o r b i o l o g i c a l m o n i t o r i n g o f Lasso® f o r m i x e r - l o a d e r a p p l i ­ cators. These s u p p o r t i n g s t u d i e s meet many o f the c r i t e r i a l i s t e d above. R e s e a r c h e r s a t Monsanto p o i n t out t h a t m u l t i p l e s p e c i e s s t u d i e s a r e o f g r e a t importance i n t h e i r e x p e r i e n c e and t h a t when a p p r o p r i a t e s u p p o r t i n g s t u d i e s a r e performed, p a t c h and u r i n e d a t a are q u i t e c o m p a t i b l e . CIBA-GEIGY - S c i e n t i s t s at CIBA-GEIGY have u t i l i z e d b i o l o g i c a l m o n i ­ t o r i n g i n a h i g h l y p r a c t i c a l manner. Over the p a s t s e v e n y e a r s , some 50,000 u r i n e samples from a e r i a l a p p l i c a t o r s / m i x e r - l o a d e r s have been a n a l y z e d t o d e t e r m i n e c h l o r d i m e f o r m exposure. A discussion of the r e s u l t s o f these m o n i t o r i n g s t u d i e s a r e beyond the scope o f t h i s presentation. However, the s u p p o r t i n g s t u d i e s w h i c h were used t o d e v e l o p the b i o l o g i c a l m o n i t o r i n g system meet many o f the c r i t e r i a l i s t e d above and the program i s one o f the few t h a t has been u s e d t o study an e n t i r e p o p u l a t i o n o f workers u s i n g b i o l o g i c a l m o n i t o r i n g . T h i s e x t e n s i v e program has overcome one s e r i o u s problem o f the p a t c h method, i . e . , d o i n g enough r e p l i c a t i o n s t o a c h i e v e a r e p r e s e n t a t i v e p o r t i o n o f the p o p u l a t i o n o f workers and t h e i r work h a b i t s .

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

28. HONEYCUTT

Mixer-Loader-Applicator Exposure

Development o f a G e n e r i c Data Base t o E s t i m a t e t o r Exposure

373

Mixer-Loader-Applica­

Development o f a g e n e r i c exposure d a t a base i s one o f t h e major p r o j e c t s which t h e NACA subcommittee on exposure assessment h a s been working on f o r 2 y e a r s . T h i s d a t a base was f i r s t proposed d u r i n g a NACA subcommittee m e e t i n g and p u t f o r t h a t a p e s t i c i d e d i v i s i o n symposium a t the ACS meeting i n S t . L o u i s , M i s s o u r i i n 1983 ( 3 ) . L a t e r , NACA and EPA agreed t o j o i n t l y d e v e l o p t h i s d a t a base. P u b l i c groups were i n v i t e d t o p a r t i c i p a t e i n t h i s p r o j e c t . The purpose o f t h e g e n e r i c d a t a b a s e i s t o p r o v i d e e q u i v a l e n t m i x e r - l o a d e r - a p p l i c a t o r exposure d a t a t o EPA, i n d u s t r y and t h e pub­ l i c i n o r d e r t o s t a n d a r d i z e exposure e s t i m a t e s . It i s w e l l r e c o g ­ n i z e d t h a t m i x e r - l o a d e r - a p p l i c a t o r exposure i s a f u n c t i o n o f formu­ l a t i o n and t y p e o f a p p l i c a t o r equipment and t h a t a d a t a b a s e c o u l d be developed t o c o o r d i n a t e a l l a v a i l a b l e exposure d a t a f o r worker exposure and r i s k assessment ty t o s m a l l pockets o f d a t b i l i t y m a i n l y due t o t h e number o f r e p l i c a t e s performed. Ground r u l e s f o r development o f the d a t a base have been l a i d out o v e r t h e p a s t y e a r i n s e v e r a l meetings between EPA and NACA representatives. EPA has c o n t r i b u t e d $145,000 and Canada r e g u l a t o r y o f f i c i a l s $40,000 ( C a n a d i a n ) . EPA i s committed t o t h e d a t a base development and w i l l r e g u l a t e w i t h t h e d a t a . C r i t e r i a have been selected to evaluate a l l studies f o r a c c e p t a b i l i t y i n t o the data base. Both i n d u s t r y and academic s t u d i e s w i l l be e v a l u a t e d . Data w i l l be e n t e r e d and r e t r i e v e d on a p a t c h by p a t c h b a s i s . The u s e r can b u i l d a "Body P a r t s " s u r r o g a t e model f o r any s c e n a r i o d e s i r e d (e.g., protected vs. nonprotected). There a r e s e v e r a l advantages f o r EPA, the p u b l i c and i n d u s t r y t o have t h i s d a t a base i n p l a c e . 1.

2.

3. 4.

Data at EPA on which r e g u l a t o r y d e c i s i o n s a r e based w i l l be expanded and i t s c r e d i b i l i t y s t r e n g t h e n e d . Also, the r e l i a b i l i ­ ty and s t a t i s t i c a l power o f the Agency's r i s k assessments f o r workers w i l l be improved. I n d u s t r y w i l l i n c u r c o s t s a v i n g s o f $200,000-$500,000 p e r y e a r s i n c e EPA w i l l not r e q u i r e f i e l d s t u d i e s where s u f f i c i e n t d a t a e x i s t s t o perform a surrogate estimate. Use o f a m u t u a l l y agreed upon d a t a base w i l l reduce c o n t e n t i o n i n t h e exposure o f t h e r i s k assessment p r o c e s s . P u b l i c use o f the d a t a base w i l l r e s u l t i n a b e t t e r u n d e r s t a n d ­ i n g o f t h e r i s k assessment p r o c e s s f o r a g r i c u l t u r a l c h e m i c a l s .

The NACA - EPA j o i n t committee has made s i g n i f i c a n t p r o g r e s s o v e r the p a s t few months i n i m p l e m e n t a t i o n o f t h e d a t a base. VERSAR, Inc. has agreed t o r e v i e w s t u d i e s w i t h d e v e l o p e d c r i t e r i a o f v a l i d i ­ t y and t o i n p u t t h e d a t a i n t o s t o r a g e . R e t r i e v a l by i n d u s t r y w i l l be through computer hookup. R e t r i e v a l by EPA and t h e p u b l i c w i l l be through t h e NTIS.

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

374

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Future Research I s s u e s f o r

the NACA

Subcommittee

The NACA subcommittee on exposure w i l l c o n t i n u e t o m o n i t o r the p r o ­ g r e s s o f t h e d a t a base as w e l l as l o o k a t o t h e r e x p o s u r e i s s u e s ; areas such as B i o l o g i c a l M o n i t o r i n g w i l l be h i g h on our l i s t o f activities. Other a c t i v i t i e s w i l l be t o a d d r e s s r e e n t r y r e s e a r c h as w e l l as r e s e a r c h i n dermal p e n e t r a t i o n , improvement i n exposure assessment t e c h n o l o g y (Fenske) and r e s e a r c h i n e x p o s u r e r e d u c t i o n technology. Conclusions: 1.

2. 3. 4.

NACA ad hoc subcommittee on p e s t i c i d e worker exposure i s and w i l l c o n t i n u e t o a d d r e s s p r o c e d u r e s f o r m e a s u r i n g worker expo­ sure . NACA g u i d e l i n e s f o t hav bee developed d agree i n p r i n c i p l e w i t A g e n e r i c exposure d a t p l a c e by mid 1988. In t h e f u t u r e t h e NACA subcommittee w i l l a d d r e s s ; r e e n t r y ; B i o ­ l o g i c a l M o n i t o r i n g ; improved exposure assessment t e c h n o l o g y and exposure r e d u c t i o n t e c h n o l o g y .

REFERENCES 1.

Reinert, J. C. et. a l . , Pesticide Assessment Guidelines, Sub­ division U, Applicator Exposure Monitoring, U.S. Environmental Protection Agency, October 1986.

2.

Leng, M. L . , Ramsey, J. C., Braun, W. H., Lavy, T. L. "Review of Studies with 2,4,5-Trichlorophenoxyacetic acid in Humans Including Applicators under Field Conditions," American Chemical Symposium Series 182, p. 133-156, Editor: J. Plimmer, American Chemical Society, Washington, D.C. (1982).

3.

Hackathorn, D. R., Eberhart, D. C., "Data Base Proposal for Use in Predicting Mixer-Loader-Applicator Exposure," American Chemical Society Symposium Series 273, p. 341-351, Editors: Honeycutt, R. C., Zweig, G., Ragsdale, N. N., American Chemical Society, Washington, D.C. (1985).

RECEIVED

June 7, 1988

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

Author Index A d a m s , S. M . , 85 A l - B a y a t i , M . A . , 288 B a l u , K , 47 B a r t e l s , M . J . , 251 B e l l e t , E u g e n e M . , 152 B e r t e a u , P e t e r E., 315 Bisbiglia, M . , 338

Maddy, K e i t h T., 215, 338 M a i b a c h , H o w a r d I., 1 3 1 , 1 3 7 , 1 5 2

Blair, A a r o n , 38

M o r g a n , M i c h a e l S., 56

B r a d y , James F., 2 6 2

M u m m a , R a l p h O., 262

B u c k s , D a n i e l A . W . , 152 B u r i n , G a r y , 327 C a m p b e l l , R . A . , 251

Nigg, H . N., 6 N i x o n W B . 231

Cantor, Kenneth P., 38 C h e u n g , M . W . , 231 C o s t l o w , R i c h a r d D . , 137 C o w e l l , J . E., 28, 240 D i D o n a t o , L a u r a J . , 137 D u b e l m a n , S . , 2 8 , 240 D u n n , Bruce P., 98 F e n s k e , R i c h a r d A . , 70 F i s h e r , H . L., 169 F l e e k e r , James R . , 2 6 2 F r a n k l i n , C l a i r e A . , 2, 126, 354 H a l l , L. L., 169 H i x o n , E . J . , 304 H o n e y c u t t , R i c h a r d C , 190, 3 6 8 H u r t , S u s a n S . , 137 J a c o b , P e y t o n , III, 215 J i m i n e z , B . D . , 85 Jones, P., 288 K a h r s , R . A . , 231 K a s t l , P. E . , 251 Kirkpatrick, Eileen M . , 56 K l e i n , A . J . , 28 K n a a k , James B . , 288, 315 K r i e g e r , R . I., 3 3 8 K r o p s c o t t , B . E., 251 L a v y , T . L., 192

Margetich, S., 338 M a t t i c e , J . D . , 192 M c C a r t h y , J . F., 85 M c M a s t e r , James, 152 M e n g l e , D o n a l d G , 315

O s t e r l o h , J o h n D . , 215 Pensyl, J . W . , 288 Phillips, G a r y S., 56 R a a b e , O. G . , 288 R e i n e r t , J o s e p h C , 286, 327 R i t t e r , L e o n a r d , 354 R o s i n , M i r i a m P., 98 R o s s , J . A . , 231 R o s s , J . R , 288 San, Richard H . C , 98 S c h r e i d e r , Jay B . , 315 S c o t t , R . C , 158 See, R a y m o n d H . , 9 8 S e i b e r , J . N . , 206 S h a h , P. V . , 169 S h u g a r t , L. R . , 85 Stamper, J . H . , 6 Stewart, P a t r i c i a A . , 3 8 Stich, Hans F , 98 S u m l e r , M . R . , 169 T a l m a g e , S. S . , 85 T w e e d y , B . G . , 231 W a n g , R h o d a , 215 W a r k e n t i e n , K a r e n E . , 327 Weisskopf, C P., 206 W e s t e r , R o n a l d C , 131, 1 3 7 , 1 5 2 W i e d m a n n , J . L., 2 8 8 W i l s o n , R i c h a r d A . , 262 Z a h m , Shelia H o a r , 38

Leber, A . P., 288 L o n g a c r e , S t e p h e n L., 137 L o t t i , M a r c e l l o , 117 L u n c h i c k , C u r t , 327

375 In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

376

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Affiliation Index British Columbia Cancer Research Centre, 98 California Department of F o o d and Agriculture, 215, 315, 338 C a l i f o r n i a D e p a r t m e n t o f H e a l t h Services, 288,315 C h e m i c a l C o n s u l t a n t s I n t e r n a t i o n a l , 152 C i b a - G e i g y C o r p o r a t i o n , 4 7 , 1 9 0 , 231 D o w C h e m i c a l C o m p a n y , 251 E n v i r o n m e n t a l H e a l t h Directorate, 2 , 1 2 6 , 354 I m p e r i a l C h e m i c a l Industries P L C , 158 M o b a y C o r p o r a t i o n , 304 M o n s a n t o Agricultural Company, 28, 240 N a t i o n a l A g r i c u l t u r a l Chemicals Association, 368 N a t i o n a l C a n c e r Institute, 3 8 N o r t h D a k o t a State U n i v e r s i t y , 262 N o r t h r o p C o r p o r a t i o n , 169

O a k R i d g e N a t i o n a l L a b o r a t o r y , 85 P a n - A g , 288 P e n n s y l v a n i a State U n i v e r s i t y , 262 P P G Industries, 2 8 8 R o h m a n d H a a s C o m p a n y , 137 Rutgers University, 70 S i m o n Fraser University, 98 U n i v e r s i t a ' d e g l i S t u d i d i P a d o v a , 117 U n i v e r s i t y o f A r k a n s a s , 192 University of California, 131,137, 152, 2 0 6 , 2 1 5 , 288 University o f Florida, 6 University o f Washington, 56 U.S. Environmental Protection

Subject Index Alachlor—Continued m e t a b o l i s m a n d e x c r e t i o n , 2 4 2 - 2 5 0 , 242/ o n c o g e n i c p o t e n t i a l , 331 A b s o r b e d dose m i n i m i z i n g , 203 m i x i n g - l o a d i n g a n d a p p l i c a t i o n , 302/ A b s o r b e d dose studies, c o n t r o l l e d , 204 A b s o r p t i o n , p e r c e n t , 241 A b s o r p t i o n - e x c r e t i o n study, G a l e c r o n 4 E , 51 A c c e p t a b l e d a i l y e x p o s u r e , pesticide residue, 355 N-Acetylglucoseamidase elevated activity, 2 2 9 injured tubular epithelium i n the k i d n e y , 216 Agricultural workers e x p o s u r e to pesticides, 3 6 8 - 3 7 4 factors affecting e x p o s u r e , 7 A i r - p u r i f y i n g respirator description, 56,59-61 performance i n controlled human e x p o s u r e , 61 p e r f o r m a n c e i n i n d u s t r i a l settings, 6 1 - 6 2 A i r a n d surface residues, pesticide a p p l i c a t i o n , 325 A i r s a m p l e s , analysis, 2 9 4 - 2 9 5 Alachlor a n i m a l m e t a b o l i s m studies, 243 a p p l i c a t o r study, 244 body dose estimates, 247/ excretion profiles and metabolite c l a s s i f i c a t i o n , 243/ gauze p a d recovery studies, 246/

s p e c i a l review, 3 2 7 - 3 3 7 u r i n e recovery studies, 245/ A l a c h l o r metabolites a m o u n t excreted, 244 c a l c u l a t i o n o f body d o s e , 2 4 8 hydrolysis, 2 4 9 / Aldicarb analytical techniques, 263 antialdicarb antibody, 269 antibody formation, 264 a p p l i e d t o t h e s o i l , 275 characteristics, 2 6 2 enzyme immunoassay, 2 6 2 - 2 8 4 i n w a t e r , 275 metabolites, 2 7 1 - 2 7 4 p o t e n t i a l l y cross-reactive c h e m i c a l s , 2 6 9 p r e p a r a t i o n o f i m m u n o g e n , 264 s t r u c t u r a l change, 274 synthesis ligand 1 immunogen, 265/ l i g a n d 2 a n d e n z y m e tracer, 2 6 6 / ligands, 2 6 3 t o t a l toxic r e s i d u e , 274 A l d i c a r b enzyme immunoassay applications, 2 7 5 - 2 8 2 i n h i b i t i o n o f selected c h e m i c a l s , 273/ A l k a l i n e - i n d u c e d strand separation, 90 A l t e r e d bases, 8 8 Alveolar air a n d m i x e d e x p i r e d a i r , 57—58

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

376

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

Affiliation Index British Columbia Cancer Research Centre, 98 California Department of F o o d and Agriculture, 215, 315, 338 C a l i f o r n i a D e p a r t m e n t o f H e a l t h Services, 288,315 C h e m i c a l C o n s u l t a n t s I n t e r n a t i o n a l , 152 C i b a - G e i g y C o r p o r a t i o n , 4 7 , 1 9 0 , 231 D o w C h e m i c a l C o m p a n y , 251 E n v i r o n m e n t a l H e a l t h Directorate, 2 , 1 2 6 , 354 I m p e r i a l C h e m i c a l Industries P L C , 158 M o b a y C o r p o r a t i o n , 304 M o n s a n t o Agricultural Company, 28, 240 N a t i o n a l A g r i c u l t u r a l Chemicals Association, 368 N a t i o n a l C a n c e r Institute, 3 8 N o r t h D a k o t a State U n i v e r s i t y , 262 N o r t h r o p C o r p o r a t i o n , 169

O a k R i d g e N a t i o n a l L a b o r a t o r y , 85 P a n - A g , 288 P e n n s y l v a n i a State U n i v e r s i t y , 262 P P G Industries, 2 8 8 R o h m a n d H a a s C o m p a n y , 137 Rutgers University, 70 S i m o n Fraser University, 98 U n i v e r s i t a ' d e g l i S t u d i d i P a d o v a , 117 U n i v e r s i t y o f A r k a n s a s , 192 University of California, 131,137, 152, 2 0 6 , 2 1 5 , 288 University o f Florida, 6 University o f Washington, 56 U.S. Environmental Protection

Subject Index Alachlor—Continued m e t a b o l i s m a n d e x c r e t i o n , 2 4 2 - 2 5 0 , 242/ o n c o g e n i c p o t e n t i a l , 331 A b s o r b e d dose m i n i m i z i n g , 203 m i x i n g - l o a d i n g a n d a p p l i c a t i o n , 302/ A b s o r b e d dose studies, c o n t r o l l e d , 204 A b s o r p t i o n , p e r c e n t , 241 A b s o r p t i o n - e x c r e t i o n study, G a l e c r o n 4 E , 51 A c c e p t a b l e d a i l y e x p o s u r e , pesticide residue, 355 N-Acetylglucoseamidase elevated activity, 2 2 9 injured tubular epithelium i n the k i d n e y , 216 Agricultural workers e x p o s u r e to pesticides, 3 6 8 - 3 7 4 factors affecting e x p o s u r e , 7 A i r - p u r i f y i n g respirator description, 56,59-61 performance i n controlled human e x p o s u r e , 61 p e r f o r m a n c e i n i n d u s t r i a l settings, 6 1 - 6 2 A i r a n d surface residues, pesticide a p p l i c a t i o n , 325 A i r s a m p l e s , analysis, 2 9 4 - 2 9 5 Alachlor a n i m a l m e t a b o l i s m studies, 243 a p p l i c a t o r study, 244 body dose estimates, 247/ excretion profiles and metabolite c l a s s i f i c a t i o n , 243/ gauze p a d recovery studies, 246/

s p e c i a l review, 3 2 7 - 3 3 7 u r i n e recovery studies, 245/ A l a c h l o r metabolites a m o u n t excreted, 244 c a l c u l a t i o n o f body d o s e , 2 4 8 hydrolysis, 2 4 9 / Aldicarb analytical techniques, 263 antialdicarb antibody, 269 antibody formation, 264 a p p l i e d t o t h e s o i l , 275 characteristics, 2 6 2 enzyme immunoassay, 2 6 2 - 2 8 4 i n w a t e r , 275 metabolites, 2 7 1 - 2 7 4 p o t e n t i a l l y cross-reactive c h e m i c a l s , 2 6 9 p r e p a r a t i o n o f i m m u n o g e n , 264 s t r u c t u r a l change, 274 synthesis ligand 1 immunogen, 265/ l i g a n d 2 a n d e n z y m e tracer, 2 6 6 / ligands, 2 6 3 t o t a l toxic r e s i d u e , 274 A l d i c a r b enzyme immunoassay applications, 2 7 5 - 2 8 2 i n h i b i t i o n o f selected c h e m i c a l s , 273/ A l k a l i n e - i n d u c e d strand separation, 90 A l t e r e d bases, 8 8 Alveolar air a n d m i x e d e x p i r e d a i r , 57—58

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

377

A l v e o l a r air—Continued effect o f t i m e , 58 A l v e o l a r concentration, 68 A n a l y t i c a l methods

Baygon, propoxur, 317-319 Bayleton

development, 198-200

dose d e t e r m i n a t i o n , 2 0 2 - 2 0 3

sensitivity, 371 trace elements o f a substance, 190 A n a l y t i c a l p r o c e d u r e , c h l o r d i m e f o r m , 51 A n i m a l d a t a , equivalent d o s e , 7 A n i m a l models e n v i r o n m e n t a l c o n t a m i n a n t s , 85 extrapolation of health hazard effects, 156

u r i n a r y m e t a b o l i t e s , 200 B e l u g a w h a l e , field studies, 8 8 - 9 0 B e n d i o c a r b , i n structures, 316/ B e n o m y l , u r i n a r y m e t a b o l i t e s , 200 Benzo[fl]pyrene b i n d i n g to m a c r o m o l e c u l e s , 134 b r a i n tissue, 8 9 - 9 0 cigarettes, 92

p e r c u t a n e o u s a b s o r p t i o n , 128 pesticides i n h o u s e h o l d setting, 324 relevance to h u m a n s , 1 5 2 - 1 5 7 A n i m a l studies

detection, 88 d i o l e p o x i d e a d d u c t , 88 i n environment, 92 m e t a b o l i c r e a c t i o n , 134

g a s t r o i n t e s t i n a l exposure, 3 to c a l c u l a t e p r o b a b l e h u m a n exposure A n i m a l toxicity database, largely feedin studies, 304 A n i m a l toxicology data, 3 0 4 - 3 1 4 A n i m a l u r i n a r y e x c r e t i o n , relevance to h u m a n studies, 200 Antialdicarb antibody, 2 6 4 - 2 6 9 A n t i b o d y b i n d i n g , enzyne tracer, 271 A n t i b o d y i n h i b i t i o n , 270 A p p l i c a t i o n equipment

d e t e c t i o n i n D N A o f B e l u g a whales, 89/ h e m o g l o b i n o f a n i m a l s , 92/ in hemoglobin of smokers and n o n s m o k e r s , 93/ in humans, 9 2 - 9 3 Benzo[a]pyrene diolepoxide

a e r i a l a p p l i c a t i o n o f c h l o r d i m e f o r m , 50 c o m p a r i s o n , 33/

bladder tumors, 89 carcinogen, 88 B i l h a r z i a l patients, m i c r o n u c l e u s test, 99 Bioavailability e n v i r o n m e n t a l c o n t a m i n a n t s , 85

l o w - p r e s s u r e sprayers, 335 s h i e l d e d sprayers, 33 t r a c t o r cabs c l o s e d , 3 3 , 333 c o m p a r i s o n study, 33/, 335 A p p l i c a t i o n r e c o r d s , 43 A p p l i c a t o r exposure

techniques f o r m e a s u r i n g , 323 Biological endpoints need for c l e a n u p , 94

measurement, 2 4 0 - 2 5 0 studies, 328 ways to r e d u c e , 28—37 A p p l i c a t o r s , risk of cancer, 39 A p p l i e d d o s e , percent absorbed i n v i v o a n d i n v i t r o , 166/ A q u a t i c o r g a n i s m s , f i e l d studies, 9 3 - 9 4 Arsenic

sweat, 16 u r i n e , 8—14 vagal t o n e , 14 Biological markers D N A damage, 90 exposure a n d b i o a v a i l a b i l i t y o f e n v i r o n m e n t a l c o n t a m i n a n t s , 87/ impact o n health, 86 in animals, 8 5 - 9 7 pesticides a n d m e t a b o l i t e s i n body tissues, 3 Biological monitoring

exposure—urine-level r e l a t i o n s h i p , u r i n a r y m e t a b o l i t e s , 10 Assay specificity, aldicarb, 2 6 9 - 2 7 1 A t r o p i n e , vagal t o n e , 14 Azinphos-methyl analysis by H P L C , 3 6 3 / e x c r e t i o n o f D M T P , 361/, 362/

i n w i l d a n i m a l s , 86 Biological indicators blood,

10—11

a b s o r p t i o n a n d m e t a b o l i s m data, 350 advantages a n d disadvantages, 3 2 8 - 3 3 0 , 355

exposure—urine-level r e l a t i o n s h i p , 12—13 m e t a b o l i t e e x c r e t i o n , 363/ o r c h a r d insects, 3 6 0 u r i n a r y clastogenic activity, 107 A z i n p h o s - m e t h y l metabolites exposure—urine-level r e l a t i o n s h i p , m i x e r - a p p l i c a t o r s , 72

7-9

s a l i v a , 14

10—11

a l a c h l o r average b o d y d o s e , 250/ a n a l y t i c a l t e c h n i q u e s , 190 a p p r o p r i a t e c h e m i c a l s , 356 best use, 241/ c o l l e c t i n g u r i n e v o i d s , 333 c o m p u t e r system, 239 costs, 372 c r i t e r i a for u t i l i z a t i o n , 371

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

378

BIOLOGICAL MONITORING FOR PESTICIDE

B i o l o g i c a l monitoring—Continued data f r o m pesticide users, 3 3 8 - 3 5 3 definition, 2 8 - 2 9 disadvantages, 242 E P T C , 293 h e a l t h related t o exposure, 4 l e v e l o f i n t e r n a l dosage, 328 l i m i t a t i o n s , 355 m e t a b o l i t e s i n t h e u r i n e , 242 methods, 6 - 2 7 p e r s o n a l a i r c o n c e n t r a t i o n s , 227

EXPOSURE

Captan—Continued metabolites, 349 statistics, 346 strawberry p i c k e r s , 3 3 8 - 3 5 3 C a r b a m a t e pesticides, u r i n e concentrations, 8 Carbaryl agreement between i n v i v o a n d i n v i t r o d a t a , 162 e l i m i n a t i o n rates, 17 in vivo and i n vitro

pesticide-exposed h u m a n s , 2 - 5 , 9 8 - 1 1 6 p r o d u c t s i n body tissues, 3 rationale, 6 - 7

a b s o r p t i o n , 164/, 166/ percutaneous a b s o r p t i o n , 163/ percutaneous a b s o r p t i o n a n d

shortcomings, 3 6 9 - 3 7 0

d i s p o s i t i o n , 170 p r e d i c t i o n o f i n v i v o a b s o r p t i o n , 162 s a l i v a , 15

study design f o r v a l i d a t i o n , 73 s u p p o r t i n g studies, 3 7 0 - 3 7 1 u r i n a r y m e t a b o l i t e levels, 3 2 urine, 4 7 - 5 5 use b y industry, 3 7 2 use i n t h e regulatory process, 3 5 4 - 3 6 7 U . S . E P A use o f d a t a , 3 2 7 - 3 3 7 v a l i d a t i o n o f e n v i r o n m e n t a l studies, 7 0 - 8 4 value o f data, 66, 3 4 3 - 3 4 9 B i o l o g i c a l m o n i t o r i n g techniques, h u m a n s exposed t o pesticides, 1 9 2 - 2 0 5 , 2 4 0 - 2 5 0 Biological samples, 6 Blood monitoring erythrocyte p r o t e i n conjugates, 8 problems, 8 B o d y b u r d e n o f c h e m i c a l , 355 Body fluids, sampling, 2 7 5 - 2 7 7 B r e a t h - s a m p l i n g cartridge advantages, 6 6 construction, 59 cutaway v i e w , 6 0 f problems, 66 q u a n t i t a t i o n range, 6 5 recovery efficiency, 5 9 - 6 1 B r e a t h analysis m o n i t o r i n g pesticide exposure, 5 8 p r a c t i c a l p r o b l e m s , 57 B r e a t h l e v e l o f solvent, time-weighted-average concentration, 65

exposure

C Cancer e p i d e m i o l o g i c a l studies, 3 8 - 4 6 p o p u l a t i o n s at r i s k , 113 Captan avenue o f entry i n t o the body, 202 estimates o f exposure, 3 4 9 exposure-urine-level relationship, 10-11 location o f label, 200-201 m e t a b o l i c studies, 347

l i n e a c o m p a r t m e n t a f l o m o d e l , 183 parameters i n p h y s i o l o g i c a l m o d e l , 182/ percutaneous a b s o r p t i o n a n d disposition, 1 7 0 , 1 7 7 - 1 8 3 physiological model for dermal a b s o r p t i o n , 180f s a l i v a , 15 skin absorption, 173,174/ C a r b o n - 1 4 analysis, 2 9 2 Carcinogenic risk, a i r concentrations o f D C P , 216 Carcinogenicity, 38 C a s e - c o n t r o l studies biochemical monitoring, 44 d i f f e r e n t i a l r e c a l l , 41 l i f e t i m e histories o f s p e c i f i c pesticides, 41 Cellular biomarkers, 87 C h e c k e r b o a r d assay, 271 C h i l d r e n , pesticide p o i s o n i n g , 3 1 6 Chlordane, carcinogenicity, 38 Chlordecone carcinogenicity, 38 i n v i t r o a b s o r p t i o n , 185 intravenous d o s i n g m o d e l , 183 percutaneous a b s o r p t i o n a n d d i s p o s i t i o n , 170 s a l i v a , 15 skin absorption, 177,179/ Chlordimeform c a l c u l a t i o n o f equivalents i n u r i n e , 235 c h r o m a t o g r a m s o f u r i n e samples, 2 3 6 / concentration i n urine, 48 d e r m a l a b s o r p t i o n studies, 4 8 d e t e r m i n a t i o n o f residue c o n c e n t r a t i o n s , 235 excretion curve, 4 9 / exposure—urine-level r e l a t i o n s h i p , 4 8 field w o r k e r exposure, 4 7 - 5 5

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX Chlordimeform—Continued H P L C analytical procedures, 236/ information flow via I B M computer, 238/ insect c o n t r o l i n c o t t o n , 231 p r o c e d u r a l recoveries, 239r structure, 2 3 2 f t o t a l residues, 2 3 1 - 2 3 9 worker exposure, 2 3 1 - 2 3 2 C h l o r d i m e f o r m d i s t r i b u t i o n profiles f i e l d w o r k e r s , 54/ m i x e r - l o a d e r s , 52/ pilots a n d o t h e r w o r k e r s , 53/ C h l o r d i m e f o r m residues, storage stability, 237/ C h l o r i n a t e d h y d r o c a r b o n insecticides, stored i n fatty tissues, 197 4-Chloro-o-toluidine analysis o f residues i n h u m a n u r i n e c a l i b r a t i o n d a t a , 234/ U V - v i s i b l e absorption spectrum, 238/ Chlorobenzilate sweat a n d u r i n e , 16 urinary m e t a b o l i t e s , 10 Chlorpyrifos d e r m a l a b s o r p t i o n , 321 exposure o f infants, 3 2 1 , 322/ e x p o s u r e - u r i n e - l e v e l r e l a t i o n s h i p , 12 h a l f - l i f e f o r a b s o r p t i o n , 321 i n structures, 316/ l y m p h o c y t i c N T E , 121 m o s q u i t o e s a n d o t h e r h o u s e h o l d pests, 321 O P I D P , 118,121 p e r i p h e r a l nerve, 119 p l a s m a cholinesterase, 321 structure, 321 Cholinesterase aerial applicators, 7 carbonate pesticides, 8 orchard parathion applicators, 7 - 8 o r g a n o p h o s p h o r o u s pesticides, 8 C h o l i n e s t e r a s e - i n h i b i t i n g pesticides, p o t e n t i a l h u m a n h e a l t h h a z a r d , 315 C h o l i n e s t e r a s e i n h i b i t o r s , i n the h o m e , 316 C h r o m a t i d a b e r r a t i o n s , 101, 1 0 7 , 1 0 8 Cinnamyl anthranilate, maximum a b s o r p t i o n , 185 C l a s t o g e n i c activity i n u r i n e a g r i c u l t u r a l research s t a t i o n p e r s o n n e l , 104 assay f o r , 100 dose-related increases, 100 dose—response curves, 105/ effect o f pesticide exposure, 101/ i n d i v i d u a l v a r i a t i o n s , 1 0 3 / 109 orchardists, 102f, 1 0 4 - 1 0 7 pesticide exposure, 1 0 8 - 1 0 9 t i m e d e p e n d e n c e , 109 C l o s e d - c a b tractor, 33

C l o s e d - t r a n s f e r m i n i b u l k package system, 32 C o f f e e d r i n k e r s , m i c r o n u c l e u s test, 9 9 C o h o r t studies, 4 0 C o m p l a c e n c y factor, 3 3 0 - 3 3 1 C o m p u t e r system biological monitoring, 239 peak area o f c o m p o u n d , 2 3 4 - 2 3 5 C o s m e t i c c h e m i c a l s , toxicity, 135 Cotton, chlordimeform, 4 7 - 5 5 Creatinine c a l c u l a t i o n o f a m o u n t excreted, 195 n o r m a l i z e differences i n e x c r e t i o n , 9 Cross-reactivity, 2 7 4 - 2 7 5 C u r r e n t practices, 6 C u t a n e o u s carcinogens, m e t a b o l i c production, 132-133 Cypermethrin

i n v i v o a n d i n v i t r o a b s o r p t i o n , 159 m a x i m u m a b s o r p t i o n , 185 C y s t i c f i b r o s i s , sweat test, 1 6 - 1 7

D 2,4-D a b s o r p t i o n a n d excretion d e r m a l l y a p p l i e d dose, 3 5 6 intravenous dose, 3 5 6 n o r m a l spray a p p l i c a t i o n s , 3 5 6 oral dose, 356 applicators, 7 2 - 7 3 dermal absorption, 195-197 dermal deposition and urinary e x c r e t i o n , 359/ e l i m i n a t i o n rates, 17 e x c r e t i o n rate, 1 9 5 - 1 9 7 exposure-urine-level relationship, 10-11 forest w o r k e r s , 195 m g herbicide/kg b o d y weight, 196/ percent recovery i n m o n k e y s , 3 5 8 / percentage excreted i n u r i n e , 357/ sweat a n d u r i n e , 16 u r i n e o f exposed w o r k e r s , 196/ 2 , 4 - D a m i n e , farmers, 77 D a i l y exposure values, f i e l d w o r k e r s , 48 D a i l y u r i n a r y e x c r e t i o n , quantitative studies, 195 D e l i v e r e d dose, 41 D e r i v a t i z a t i o n , 1 9 9 - 2 0 0 , 210 D e r m a l , see also P e r c u t a n e o u s D e r m a l exposure assessment m e t h o d s , 8 1 - 8 2 l o n g underwear as w h o l e - b o d y d o s i m e t e r , 347 monitoring, 29 patches o r c l o t h i n g , 2

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

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BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

D e r m a l exposure—Continued v a l i d a t i o n studies, 7 2 Dermal fluorescence measurement, 78 quenching, 82 D e r m a l - o r a l a b s o r p t i o n studies, 1 3 8 - 1 5 1 D e r m a l study, G a l e c r o n 4 E , 51 D i a l k y l p h o s p h a t e metabolites effect o f v a r i o u s salts, 2 0 8 u r i n a r y analysis, 2 0 6 D i a l k y l phosphates c o m m o n , 207/ f l o w d i a g r a m f o r u r i n a r y analysis, 2 0 9 / gas c h r o m a t o g r a m , 2 1 1 / i n a d e q u a t e analysis m e t h o d s , 213 measurement problems, 2 0 6 - 2 0 7 recovery f r o m f o r t i f i e d u r i n e s a m p l e s , 212/ D i a l k y l thiophosphate metabolites analysis, 2 0 6 Diallate, carcinogenicity, 38 D i a z i n o n , i n structures, 316/ Dichloropropene air concentrations, 221, 223/-226/ biological monitoring, 215-230 e x c r e t i o n patterns, 2 1 5 - 2 3 0 kidney sulfhydryl content, 216 k i d n e y toxicity, 2 2 8 respiratory neoplasia, 216 thiol depletion, 228 t r a n s i t i o n a l c e l l cancers, 215 Dichlorvos exposure dose f o r a n infant, 320/ exposure l i m i t s , 3 1 9 metabolic breakdown, 320 structure, 319 surface residues, 3 1 9 - 3 2 0 vagal t o n e , 14 v a p o r pressure, 3 1 9 ventilation, 316 D i f f u s i o n b a r r i e r , glass d i f f u s i o n c e l l , 160 D i f f u s i o n cells e p i d e r m a l m e m b r a n e s , 160 systems, 184 Dinocap cumulative excretion o f 1 4 C l a b e l , 146/, 148/ dermal absorption, 137-151 d e r m a l exposure, 147 d e v e l o p m e n t a l toxicity, 1 3 8 , 1 4 7 i n excreta, 141 i n t r a v e n o u s study, 145 m a t e r n a l toxicity, 147 peak p l a s m a 1 4 C c o n c e n t r a t i o n , 143/ percent a b s o r p t i o n , 142/ recovery o f 1 4 C l a b e l , 144/ residues o n c r o p s , 138 r i s k e s t i m a t i o n , 149

Dinocap—Continued s t r u c t u r a l f o r m u l a , 139/ terata, 138 Dinoseb b i p h a s i c a b s o r p t i o n m o d e l , 183 percutaneous a b s o r p t i o n , 155/ percutaneous absorption a n d d i s p o s i t i o n , 170 skin absorption, 173,176/ D i s l o d g e a b l e f o l i a r residues, 3 4 3 - 3 4 4 , 347 D M P T , exposure-urine-level relationship, 1 0 - 1 1 D N A adducts b r a i n tissue, 9 0 detection and quantitation, 88 f o r m a t i o n after acute exposure t o B a P , 89/ D N A damage

nonadductive, 9 0 D N A modification, 87 Dose a c c o u n t a b i l i t y , d i n o s e b , 155 administration method, 11-13 c a l c u l a t i o n s , l i m i t a t i o n s , 322 e s t i m a t i o n , b i o l o g i c a l f l u i d s , 22 estimation methods, nonbiological and biological, 1 9 - 2 1 exposure studies, 193 t i m e effect, 3 1 1 - 3 1 2 D o s e - r e s p o n s e c u r v e , p r e d i c t i n g adverse h e a l t h effects, 126 D r u g s , s a l i v a , 15 D u p l i c a t e assessments o f exposure, 4 2 - 4 4 Dursban, chlorpyrifos, 3 2 1 - 3 2 2

E l i m i n a t i o n pathways, 17 E m p l o y m e n t records, C h e m L a w n , 40 Environmental contaminants exposure a n d b i o a v a i l a b i l i t y , 8 5 - 9 7 sources o f exposure, 85 E n v i r o n m e n t a l m o n i t o r i n g , v a l i d a t i o n by b i o l o g i c a l m o n i t o r i n g , 70—84 E n z y m e immunoassay aldicarb, 2 6 2 - 2 8 4 aldicarb-fortified h u m a n saliva, 279/ aldicarb-fortified h u m a n urine, 279/ aldicarb-fortified lemonade, 280f aldicarb-fortified limeade, 280f a l d i c a r b - f o r t i f i e d orange j u i c e , 2 8 1 / a l d i c a r b - f o r t i f i e d rabbit s e r u m , 278/ a l d i c a r b - f o r t i f i e d sheep p l a s m a , 2 7 8 / a l d i c a r b - f o r t i f i e d stream water, 2 7 6 / aldicarb-fortified watermelon juice, 281/

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

381

E n z y m e immunoassay—Continued aldicarb oxime, choline, and acetylcholine, 276/ a l d i c a r b standard c u r v e , 2 7 2 / pesticide residue m e t h o d s , 2 7 0 reagent c o n c e n t r a t i o n , 2 6 7 - 2 6 8 E n z y m e tracer c h a r a c t e r i z a t i o n , 267 synthesis, 2 6 7 , 271 E p i d e m i o l o g i c a l studies, cancer, 3 8 - 4 6 E p i d e r m a l tumors percutaneous absorption, 134-135 s h a l e - o i l - i n d u c e d , 135/ EPTC adsorptive nature, 300 d o s e - r e l a t e d effect, 288

E x p o s u r e assessment methods b i o l o g i c a l a n d e n v i r o n m e n t a l m o n i t o r i n g , 71 h u m a n , 71 relative i n d i c a t o r s , 7 2 safety studies, 3 4 2 - 3 4 3 validation, 72 E x p o s u r e c o n d i t i o n s , average, 3 1 2 Exposure documentation, 38 E x p o s u r e estimates c a p t a n , 349 specific data r e q u i r e d , 339 u s i n g a n i m a l study d a t a , 313/ E x p o s u r e e v a l u a t i o n , i m m e d i a t e needs, 4 1 - 4 2 , 4 4 Exposure-excretion relationship, 227-228 E x p o s u r e m e a s u r e m e n t t e c h n i q u e s , 241/ E x p o s u r e t o pesticides

p e r c u t a n e o u s a b s o r p t i o n studies, 2 8 8 - 3 0 3

biological monitoring, 28

toxicity studies, 288 E r y t h e m a , o i l s used i n c o s m e t i c s , 13 E s t i m a t e d absorbed dosages

deposition,

a p p l i c a t i o n , 299r m i x i n g a n d a p p l i c a t i o n , 300/ m i x i n g a n d l o a d i n g , 299/ Ethion d i a l k y l p h o s p h a t e e x c r e t i o n , 76 protective clothing, 76 Excreted compounds, identification, 197-200 Excretion kinetics a p p l i c a t i o n m e t h o d , 17 b i o l o g i c a l f l u i d s , 18/ pesticide, 9 s a l i v a , 16 u r i n e , 11 u r i n e a n d sweat, 17 E x c r e t i o n p r o f i l e , a l a c h l o r , 248/ E x c r e t i o n rate h a l f - l i v e s , 19 Excretion route sex d e p e n d e n c e , 200 type o f test a n i m a l , 200 E x c r e t i o n study, h u m a n , 13 E x f o l i a t e d u r o t h e l i a l cells, analysis o f m i c r o n u c l e i , 100, 1 0 7 - 1 0 8 E x h a l e d b r e a t h , materials present, 57/ E x p e r i m e n t a l design a c t u a l exposure, 21 a l d i c a r b e n z y m e i m m u n o a s s a y , 270 a n i m a l m o d e l o f percutaneous a b s o r p t i o n , 152 c h l o r d i m e f o r m f i e l d use, 47 d e r m a l exposure a n d e x c r e t i o n rate, 13 development, 198-200 d i n o s e b rhesus m o n k e y study, 155 number o f replications, 20 protective clothing, 20 risk estimation, 3 3 2 - 3 3 3 s o u r c e o f differences i n results, 333 statistical c o n s i d e r a t i o n s , 1 9 - 2 1 E x p o s e d d e r m a l a r e a , w o r k e r , 193 E x p o s u r e , w o r k - t a s k - s p e c i f i c , 350

measurements, 2 8 - 3 7 m e t h o d s used t o evaluate, 3 9 Extraneous exposure, 73, 7 5 - 7 6

F Farmers, risk o f cancer, 39 F e d e r a l Insecticide, F u n g i c i d e , a n d R o d e n t i c i d e A c t , 328 Field monitoring, 50 F i e l d survey f o r m s , 244 F i e l d - w o r k e r e x p o s u r e , N A C A i n v o l v e m e n t , 368 Field workers activity patterns, 371 c o n v e n i e n c e t o a n d c o o p e r a t i o n , 371 F l o w - t h r o u g h d i f f u s i o n c e l l , 184 F l u o r e s c e n c e - i m a g i n g , 241/ F l u o r e s c e n t tracer t e c h n i q u e , 7 0 - 8 4 F l u o r e s c e n t w h i t e n i n g agent, 78 F o l i a r residue m o n i t o r i n g , 345 F o o d chain, Beluga whale, 89 F o r t i f i e d s a m p l e s , storage stability, 200

G Galecron 4 E a b s o r p t i o n - e x c r e t i o n study, 51 d e r m a l study, 51 worker exposure, 2 3 1 - 2 3 9 Genotoxic compounds biological monitoring, 9 8 - 1 1 6 D N A damage, 9 0 mode of action, 8 7 - 8 8 G e n o t o x i c damage ^ - c a r o t e n e , 113 cigarettes o r a l c o h o l , 112 Genotoxic metabolites, i n urine, 99

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

382

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G e n o t o x i c pesticides, used by orchardists, 106-107 Genotoxicity exposure to carcinogens, 1 0 9 - 1 1 1 markers, 4

PESTICIDE EXPOSURE

H e x a c h o r o b e n z e n e , c a r c i n o g e n i c i t y , 38 H i s t o r i c a l perspectives, 6 H u m a n exposure studies applicator, 192-205 e t h i c a l q u e s t i o n s , 159, 204

t i m e d e p e n d e n c e , 106—107 Genotoxins detectable l i m i t s , 111

pesticides, 194/ w o r k e r c o o p e r a t i o n , 195 H u m a n risk assessment, a n i m a l toxicity,

extraction and concentration p r o c e d u r e s , 111 G l a s s d i f f u s i o n c e l l , e p i d e r m a l surface, 160 G l o v e m a t e r i a l s , p e n e t r a t i o n studies, 31 G l u t a t h i o n e , m e t a b o l i t e l i n k a g e , 111 G r a b s a m p l e , 48

304-314 Hydrocephalus, Hydrocortisone

138

a p p l i c a t i o n frequency a n d percutaneous a b s o r p t i o n , 134/ c a r c i n o g e n i c assays, 134—135 toxicity, 1 3 4 - 1 3 5 H y g i e n e , c o m p a r i s o n , 34/

H Haloxyfop a n n u a l a n d p e r e n n i a l grasses, 25 calculation of concentration, 254-25 e x t r a c t i o n m e t h o d , 253 H P L C p u r i f i c a t i o n , 257 in human urine, 251-261 ion chromatograms, 259/ mass s p e c t r a , 256/ quantitative determination, 2 5 1 - 2 6 1 recovery f r o m u r i n e , 2 5 5 - 2 5 7 , 258/ reduced-pressure G C - M S interface, 2 5 8 / stability i n u r i n e s a m p l e s , 260 structure, 252f H a n d d e p o s i t i o n , 31/ H a n d washes analysis, 294 c a p t a n , 345 i m p a c t o n b i o l o g i c a l m o n i t o r i n g , 29 m i x e r - l o a d e r - a p p l i c a t o r , 298/ H a z a r d assessment insecticides o n i n d o o r surfaces, 3 1 5 - 3 2 6 pesticide a p p l i c a t i o n , 339 process, 3 4 1 - 3 4 3 H a z a r d o u s substances a m o u n t s released to the e n v i r o n m e n t , 86 b i o l o g i c a l a v a i l a b i l i t y , 86 p r o c e d u r e s for m o n i t o r i n g exposure, 86 H e m o g l o b i n - b o u n d m e t a b o l i t e s , exposure to h a r m f u l c h e m i c a l s , 91 Hemoglobin m o d i f i c a t i o n , 9 0 - 9 1 , 94 mutagenic or cancer-initiating c o m p o u n d s , 91 H e p t a c h l o r , c a r c i n o g e n i c i t y , 38 Hexachlorobiphenyl i n v i t r o a b s o r p t i o n , 185 methods c o m p a r i s o n o f s k i n a b s o r p t i o n , 178/ m o d e l e d w i t h parent a n d m e t a b o l i t e , 183 percutaneous absorption and d i s p o s i t i o n , 170 s k i n a b s o r p t i o n , 177

I m m u n o a s s a y , p r i n c i p l e s , 268 I m m u n o c h e m i c a l t e c h n i q u e s , advantages, 262 In v i t r o t e c h n i q u e , advantages and p r o b l e m s , 159 In v i v o t i m e - c o u r s e e x p e r i m e n t , 162 I n d i v i d u a l exposure, u r i n a r y c o n c e n t r a t i o n s , 227 I n f o r m a t i o n sources, 42—44 I n h a l a t i o n a n d d e r m a l exposure, relative c o n t r i b u t i o n s , 330 I n h a l a t i o n exposure absorbent filter, 2 d a i l y , 298 E P T C , 293 m e a s u r e m e n t , 370 p e r s o n a l s a m p l i n g devices, 370 t o l u e n e , 66 Inherent toxicity, percutaneous absorption, 131-136 Injector b l o c k p r o p y l a t i n g reagent, 214 Insecticide a b s o r p t i o n , f r o m i n d o o r surfaces, 3 1 5 - 3 2 6 I n s t r u c t i o n l e v e l , c o m p a r i s o n , 34/ Interfering exposure, 7 3 - 7 4 Internal dosage alachlor, 3 3 1 - 3 3 5 c h e m i c a l s i n u r i n e , 341 e s t i m a t e d , 332 m i n i m u m l i k e l y , 336 Internal s t a n d a r d , s t a b l e - i s o t o p e l a b e l e d , 257 I s o p r o p y l myristate, e r y t h e m a , 135

J J u i c e p r o d u c t s , e n z y m e immunoassay,

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

277

INDEX

383

K Karathane, dermal absorption, 137-151 K e p o n e , s k i n a b s o r p t i o n , 177

M i c r o n u c l e a t e d cells elevated risk f o r c a n c e r , 108/ i n d i c a t i o n o f genotoxic damage, 1 0 7 - 1 0 8 M i c r o n u c l e u s test exfoliated h u m a n cells, 112, 9 9

L L a b e l requirements, 343 L a b o r a t o r y a n i m a l s , need f o r , 204 L a b o r a t o r y studies, c h l o r d i m e f o r m , 5 0 Lasso herbicide, 242-250, 3 3 2 - 3 3 5 Latent period, 3 8 - 3 9 L i p o p h i l i c m o l e c u l e , percutaneous a b s o r p t i o n , 160 L y m p h o c y t e N T E activity " f a l s e negative" results, 121/ " f a l s e p o s i t i v e " results, 120r Lymphocytes, 117-136

t i m e dependency, 113 M i g r a t i o n o f p e s t i c i d e , a l d i c a r b , 275 M i r e x , carcinogenicity, 38 M i t i g a t i o n measures, adequacy, 343 M i x e d - f u n c t i o n oxidases b i o l o g i c a l m a r k e r , 94 bluegills, 9 5 / carcinogenic metabolites, 93 i n d u s t r i a l p o l l u t i o n , 94 Mixer—loader—applicator exposure E P T C , 288-303 g e n e r i c database, 373

Models c a r b o f u r a n d e r m a l a b s o r p t i o n , 177—183 e x c r e t i o n process, 18 T H P I as m e t a b o l i c index, 3 5 0 M o n i t o r i n g equipment, air-purifying

M Malathion c o r r e l a t i o n o f exposure measurements w i t h m e t a b o l i t e e x c r e t i o n , 79/ d e r m a l fluorescence, 78 e l i m i n a t i o n rates, 17

respirator, 56, 5 9 - 6 1 M u l t i p l e dose a p p l i c a t i o n , 149 Mutagenicity, D C P impurities, 228

extraneous exposure, 79 r a p i d u r i n a r y e x c r e t i o n , 78 regression analysis o f fluorescent tracer exposure, 8Qf M a l e i c acid, renal tubular injury, 229 M a n u f a c t u r e r s , r i s k o f cancer, 3 9 M a r g i n o f safety, 3 0 0 M e r p h o s , l y m p h o c y t i c N T E i n h i b i t i o n , 120 M e t a b o l i c index, exposure assessments, 350 M e t a b o l i c studies, c a p t a n , 347 M e t a b o l i s m cage study, E P T C herbicide, 289, 290f M e t a b o l i s m d a t a , use, 341 M e t a b o l i t e recovery effect o f p H m o d i f i c a t i o n , 210/ effect o f salt a d d i t i o n , 210/ u r i n e c o n c e n t r a t i o n , 212/ M e t a b o l i t e - r e s p o n s e c u r v e , toxic effects, 4 M e t a l s i n t h e e n v i r o n m e n t , saliva, 15 M e t h a m i d o p h o s , O P I D P , 118 M e t h y l b r o m i d e , i n structures, 316/ M e t h y l c h l o r o p h e n o x y acetic a c i d , e x p o s u r e - u r i n e - l e v e l r e l a t i o n s h i p , 12 Methylene chloride delivered dose, 364 hepatocellular a d e n o m a s c a r c i n o m a s , 360—364 liver t u m o r s , 364 m e t a b o l i s m , 3 6 4 , 364/, 365/

N N A G u r i n a r y activity, 2 2 3 / - 2 2 5 / N e m a t o d e s , 215 N e u r o p a t h y target esterase, 1 1 7 - 1 3 6 h e n l y m p h a t i c system, 118 h u m a n s , 118 inhibition, 119-120 p e r i p h e r a l lymphocytes o f unexposed p o p u l a t i o n , 119 N e w approaches, 6 N i t r o p h e n y l p h o s p h a t e , hydrolysis by a l k a l i n e phosphatase, 272/ N o n v o l a t i l e pesticides, c r a c k a n d crevice t r e a t m e n t , 316 Nucleophilic atoms, 88

O O c c u p a t i o n a l exposure estimation methods, 328 legal s t a n d a r d , 2 passive d o s i m e t r y a n d b i o l o g i c a l m o n i t o r i n g , 329/ risk assessment, 2 8 6 r o u t e s , 169

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

384

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSl

O c c u p a t i o n a l illnesses, pesticider e l a t e d , 206 O i l s used i n c o s m e t i c s , percutaneous a b s o r p t i o n a n d e r y t h e m a , 135/ O r c h a r d i s t s , m i c r o n u c l e u s test, 9 9 Organochlorines e x c r e t i o n r o u t e s , 10 e x p o s u r e , 10 O r g a n o p h o s p h a t e m e t a b o l i t e s , analysis o f u r i n e o f field w o r k e r s , 2 0 6 - 2 1 4 Organophosphates, interaction with a primary target, 1 1 7 - 1 3 6 O r g a n o p h o s p h o r u s - i n d u c e d delayed polyneuropathy, mechanism o f i n i t i a t i o n , 118 Organophosphorus compounds e x p o s u r e - u r i n e - l e v e l r e l a t i o n s h i p , 12 r e s p i r a t o r y sinus a r r h y t h m i a , 1 u r i n a r y m e t a b o l i t e s , 10 vagal t o n e , 14 O r g a n o p h o s p h o r u s pesticides excretion o f alkyl phosphate m e t a b o l i t e s , 360 urine concentrations, 8

P a c k a g i n g a n d h a n d l i n g r e q u i r e m e n t s , 343 Paraquat u r i n a r y clastogenic activity, 107 u r i n a r y m e t a b o l i t e s , 10 P a r a q u a t d i c h l o r i d e , exposure—urine-level relationship, 10-11 Parathion cholinesterase, 7 - 8 exposure-urine-level relationship, 1 0 - 1 2 p a t c h t e c h n i q u e , 75 s a l i v a , 15 u r i n a r y m e t a b o l i t e s , 10 Passive d o s i m e t e r s , 244 Passive d o s i m e t r y advantages a n d disadvantages, 2 4 0 - 2 4 1 , 3 2 8 - 3 3 0 e x t r a p o l a t i o n f r o m d a t a , 330 large generic databases, 331 m e a s u r e m e n t m e t h o d s , 241/ p a t c h t e c h n i q u e , 328, 333 s a m p l i n g p u m p , 328 s u p e r v i s i o n , 330 P a t c h data a p p l i c a t o r , 297/ c a l c u l a t i n g t o t a l d e r m a l exposure, 3 7 0 e x t e r i o r a n d i n s i d e , 298/ m i x e r - l o a d e r , 297/ Patch technique analysis, 30/, 294 e v a l u a t i o n , 81

P a t c h technique—Continued exposure e s t i m a t i o n , 355 impact o n biological monitoring, 29 i n h e r e n t e r r o r , 241 m o n i t o r i n g sites, 293 q u e s t i o n a b l e accuracy, 7 5 s h o r t c o m i n g s , 369 systems, 132 v a l i d a t i o n , 7 4 - 7 8 , 75/ validation o f environmental monitoring, 7 0 - 8 4 Percutaneous absorption a c c o u n t a b i l i t y o f d i n o s e b , 156/ age-related differences, 184 a n i m a l data t o estimate h u m a n e x p o s u r e , 156 a n i m a l studies, 3 0 4 - 3 1 4

d i a g r a m o f i n v i t r o apparatus, 161/ dose a n d t i m e , 305 effect o f age, 1 7 0 , 1 7 2 E P T C h e r b i c i d e , 2 8 9 , 296/ e s t i m a t i o n based o n e x c r e t i o n o f r a d i o a c t i v i t y , 127 i n v i t r o d e r m a l a p p l i c a t i o n r e g i m e , 166/ in vitro methods, 1 2 8 , 1 5 8 - 1 6 8 i n h e r e n t toxicity, 1 3 1 - 1 3 6 k i n e t i c s o f e l i m i n a t i o n , 162 mass b a l a n c e study d e s i g n , 1 5 2 - 1 5 7 m a t h e m a t i c a l treatment o f results, 3 0 6 - 3 1 1 mechanism, 126-127 pesticides, 1 6 9 - 1 8 7 p r i m a r y r o u t e o f exposure, 126 process, 1 3 5 - 1 3 6 rat, r a b b i t , p i g , a n d m a n , 153/ R h e s u s m o n k e y a n d m a n , 154/ test d a t a , 306/ testosterone, 153/ t o x i c o l o g i c a l activity, 132/ Percutaneous absorption and excretion, o f f o u r pesticides, 74/ P e r c u t a n e o u s a b s o r p t i o n rate constant, related t o age, 181 P e r c u t a n e o u s a b s o r p t i o n study, p r o t o c o l s , 305 P e r c u t a n e o u s d e p o s i t i o n , c o n t a i n e r size, 32/ Percutaneous dosimetry c a p t a n exposure, 349 p r o t e c t i o n b y gloves, 345 P e r c u t a n e o u s exposure a m o u n t o f m e t a b o l i t e , 360 c a p t a n , 348/ E P T C , 293 g r o u n d b o o m a p p l i c a t i o n , 334/ h a n d exposure, 3 7 0 measurement, 370

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

385

P e r c u t a n e o u s exposure—Continued m i x i n g - l o a d i n g , 335 patch method, 370 range o f estimates, 335 strawberry harvesters, 344 P e r c u t a n e o u s toxicity d a t a , e x t r a p o l a t i o n t o humans, 13,162 P e r i p h e r a l p o l y n e u r o p a t h y , 118 Pesticide components, physicochemical properties, 58 P e s t i c i d e e x c r e t i o n , data f r o m s m a l l - a n i m a l

P h e n o x y herbicides e x c r e t i o n rate, 1 9 5 - 1 9 7 exposure c o r r e l a t e d w i t h e x c r e t i o n , 193 Phenoxyacetic a c i d h e r b i c i d e s , risk o f cancer, 39 /?-Phenylenediamine, skin absorption, 132,133/ P h o s a l o n e , u r i n a r y clastogenic activity, 107 Physiological m o d e l , carbaryl r a d i o a c t i v i t y , 185 Physiological modeling, 177-183 Picloram

studies, 197 P e s t i c i d e exposure

dermal absorption, 195-197 e x c r e t i o n rate, 1 9 5 - 1 9 7 forest w o r k e r s , 195

ambient monitoring, 2 biological indicators, 7 - 1 9 b i o l o g i c a l m o n i t o r i n g , 341 c o m m o n situations, 286 d e r m a l a b s o r p t i o n rate, 341 estimation, 6, 7 e v a l u a t i o n , 44 f i e l d reentry, 3 4 0 f i e l d w o r k e r s , 47 f o l i a r residue a n d passive d o s i m e t r y data, 351/ i n d o o r exposure, 3 3 9 - 3 4 0 infants, 2 8 7 , 317* metabolism data, 340

m g herbicide/kg b o d y weight, 196/ u r i n e o f exposed w o r k e r s , 196/ Plasma, 2,4-D, 8

c o n t a i n e r size, 31—33 dermal deposition, 32 P r o p e t a m p h o s , transfer f r o m f u r n i t u r e fabric to c l o t h i n g , 3 2 3 Propoxur acceptable d e r m a l c o n t a c t , 3 1 8 age a n d toxic r e a c t i o n , 3 1 9

m i x e r - l o a d e r - a p p l i c a t o r exposure, 3 4 0 pulmonary route, 228

exposure dose f o r a n infant, 318/ flea c o n t r o l i n d o o r s , 3 1 6 i n structures, 316/

use patterns, 3 4 2 workers, 287 P e s t i c i d e f o r m u l a t i o n s , dosage received, 335 P e s t i c i d e illnesses, f r o m s t r u c t u r a l a p p l i c a t i o n , 316r P e s t i c i d e regulatory d e c i s i o n s , 3 3 8 - 3 5 3 P e s t i c i d e regulatory p r o g r a m , California, 338-339 P e s t i c i d e residues i n f o o d , 355 i n h o u s e h o l d s , 324 P e s t i c i d e s e l e c t i o n , 74 Pesticides

o r a l toxicity, 3 1 8 rate o f a b s o r p t i o n t h r o u g h s k i n , 3 1 9 residual properties, 318 structure, 317 symptoms, 319 v a p o r toxicity, 3 1 8 v e c t o r c o n t r o l agent t o replace D D T , 317 P r o s p e c t i v e cancer studies, 44 Protective clothing alachlor, 248, 332 assumptions concerning value, 330 captan, 3 4 4 - 3 4 5 effectiveness, 3 5 , 2 4 1 , 3 5 0

genotoxic c o m p o u n d s , 106 need f o r p r o t e c t i o n , 192 specific activity a n d r a d i o l a b e l i n g , 171? t o x i c o l o g i c a l p r o p e r t i e s , 204

e x c r e t i o n studies, 2 0 - 2 1 experimental design, 20 Galecron 4 E , 50 gloves, 347

used b y o r c h a r d i s t s , 106/ Pharmacokinetic models p h y s i o l o g i c a l l y based, 185 u t i l i t y , 185 Pharmacokinetics alternate a n a l y t i c a l m e t h o d s , 2 8 b i o l o g i c a l m o n i t o r i n g study, 328 m u l t i p l e species, 371 pesticides, 1 6 9 - 1 8 7 P h e n o x y a c i d herbicides spot u r i n a r y e x c r e t i o n , 7 7 u r i n a r y m e t a b o l i t e s , 10

i m p r o v e m e n t , 204 oversleeves a n d leggings, 347 P y r i d o s t i g m i n e , vagal t o n e , 14 Q

Q u e n c h i n g , d e r m a l fluorescence, 8 4

R R a b b i t antibodies, aldicarb, 270

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

386

BIOLOGICAL MONITORING FOR PESTICIDE EXPOSURE

R a d i o l a b e l e d c o m p o u n d s , u r i n e analysis, 197 R a n g e o f exposure, 2 8 7 , 3 3 3 R a t e o f transfer, pesticide residue t o the skin o r clothing, 323 R e c a l l bias, 4 2 - 4 3 R e c e p t o r f l u i d , glass d i f f u s i o n c e l l , 160 R e c o r d - k e e p i n g system, county N o x i o u s W e e d Departments i n Kansas, 40, 43 Reentry interval basis f o r r e g u l a t i o n , 3 4 0 relevant c o n d i t i o n s , 3 4 0 R E G p r o c e d u r e , 307 R E G results, a c t u a l vs. p r e d i c t e d values, 310/, 31 If R e g r e s s i o n , 313 R e g u l a t o r y a c t i o n , insecticide c h e m i c a l s i n p r o d u c t s used i n d o o r s , 325 Regulatory considerations, 2 8 6 - 2 8 R e g u l a t o r y process, use o f b i o l o g i c a monitoring, 354-367 Regulatory requirements, 3 1 5 - 3 2 6 R e n a l i n j u r y , N A G e n z y m e activity, 2 2 8 - 2 2 9 R e s e a r c h m e t h o d s , n o n b i o l o g i c a l , 10 R e s e a r c h needs, b i o l o g i c a l m o n i t o r i n g , 21 R e s p i r a t o r y sinus a r r h y t h m i a , 14 R e t e s t r e l i a b i l i t y studies, 4 2 - 4 4 R i s k assessment

S k i n a b s o r p t i o n , e s t i m a t i o n , 152 Skin-depot, 291/ S k i n exposure, t o l u e n e , 6 6 S k i n permeability i n v i v o a n d i n v i t r o , 184 infants a n d a d u l t s , 1 8 3 - 1 8 4 S m o k e r s , m i c r o n u c l e u s test, 9 9 S p i n p e n e t r a t i o n o f pesticides, effect o f age, 1 7 0 , 1 7 2 Spray o p e r a t o r , d e r m a l exposure, 167 Static d i f f u s i o n c e l l , 184 Statistics calculations, 2 0 - 2 1 captan, 346 f o r three data sets, 312/ Storage stability, t o t a l c h l o r d i m e f o r m residues, 2 3 5 - 2 3 7

,

Stratum corneum m a j o r b a r r i e r t o p e n e t r a t i o n , 126 s k i n p e r m e a b i l i t y studies, 184 Sweat biological indicator, 1 6 - 1 8 quantifications, 1 6 - 1 7 Systemic h a z a r d , assessment, 165

basis, 3 5 8 / carcinogens, 39 e x t r a p o l a t i o n f r o m a n i m a l studies, 3 0 4 - 3 1 4 o c c u p a t i o n a l exposure to pesticides, 354 problems i n experimental design, 3 3 2 - 3 3 3 quantitative, 287, 350 regulatory c o n s i d e r a t i o n s , 2 8 6 - 2 8 7 small containers, 250 R o t a t i n g a p p l i c a t i o n sites, 149 R S Q U A R E p r o c e d u r e , 3 0 7 , 309/ R S R E G p r o c e d u r e , 3 0 6 - 3 1 1 , 308/

S Safety, related t o exposure, 2 8 8 - 3 0 3 Safety factor, 3 4 3 Safety i n s t r u c t i o n s , 3 3 , 3 4 2 Safety m a r g i n s , 295 S a f r o t i n , transfer f r o m f u r n i t u r e fabric to clothing, 323 Saliva mass b a l a n c e w o r k , 15—16 monitoring medium, 14-16 salivary esterases, 15 t o b a c c o - s p e c i f i c n i t r o s a m i n e s , 112 Salivary h o r m o n e s , used i n diagnosis, 15 S h i e l d e d sprayers, 3 3 S i m a z i n e , u r i n a r y clastogenic activity, 107 S i n g l e - e x p o s u r e d e s i g n , 73 S k i n , b a r r i e r p r o p e r t i e s , 131

T 2,4,5-T b a c k g r o u n d levels, 76 concentration i n urine, 193-194 exposure—urine-level r e l a t i o n s h i p , 10—11 single-exposure d e s i g n , 75 t i m e o f e x c r e t i o n , 76 T e r r e s t r i a l a n i m a l s , f i e l d studies, 91 T e s t o s t e r o n e , percutaneous a b s o r p t i o n i n s m a l l species, 153 Tetrachlorinphos, carcinogenicity, 38 Tetrachlorophenol, timber m i l l workers, 72 T e t r a h y d r o p h t h a l i m i d e , three-day c o m p o s i t e u r i n e s a m p l e s , 351/ T i s s u e - t o - b l o o d r a t i o s , 181 T o b a c c o - b e t e l - q u i d chewers, risk o f o r a l c a n c e r , 112 Toluene accuracy o f b r e a t h levels, 6 5 automobile body painters, 64/ concentration i n breath, 67/ field measurements, 66 f o u r - h o u r c o n t r o l l e d exposure, 63/ in human breath, 63 recovery f r o m c h a r c o a l c l o t h , 62/ variation with sampling time, 66 Total metabolite, i n urine during m i x i n g - l o a d i n g a n d a p p l i c a t i o n , 301/

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

INDEX

387

Toxaphene, carcinogenicity, 38 Toxicity l o c a l a n d systemic, 131

Urine collection

relationship to dose, 3 , 7 selectivity, 117 T o x i c i t y data f o r setting safe levels, 323 m e t h o d s f o r c o l l e c t i n g , 323

o p t i m u m p e r i o d , 195 U r i n e concentrations carbamate pesticides, 8 o r g a n o p h o s p h o r u s pesticides, 8 stability, 1 1 0 - 1 1 1

T o x i c i t y studies, i n v i v o percutaneous

U r i n e m o n i t o r i n g , E P T C , 293 U r i n e pesticide levels, v a r i a t i o n , 9 - 1 0 U r i n e s a m p l e s , analysis, 213/,295

a b s o r p t i o n , 158 T o x i c o l o g y assessment, toxicity a n d use p a t t e r n , 342 T o x i c o l o g y test standards, C a l i f o r n i a , 339 T r a c e r b i n d i n g , n o n s p e c i f i c , 271 T r a c e r results, l o c a t i o n o f l a b e l , 2 0 0 - 2 0 1 T r a c t o r , types, 33/ T r i c h l o r n a t e , O P I D P , 118 T r i c h l o r o p h o n , O P I D P , 118

U U r i n a r y clastogens, d e c o m p o s i t i o n , 110 Urinary C N A C 24-hour excretion, 226/ activity, 2 2 1 c h r o m a t o g r a m , 22% 222/ concentrations, 223/-226/ G C - M S d e t e r m i n a t i o n m e t h o d s , 221/ mass s p e c t r u m , 222/" separation procedures, 227 U r i n a r y e x c r e t i o n , p r o j e c t e d , 19/ U r i n a r y metabolites avenue o f entry i n t o the b o d y , 202 detectable levels, 300

m e t h o d , 346 m i s s e d p o r t i o n s , 229

V V a g a l t o n e , 14 V a l i d a t i o n study d e s i g n , 7 3 Verdict quantitative determination

n o n t o x i c fluorescent tracers, 2 4 1 - 2 4 2 s o u r c e o f d o s e , 242 Volatile compounds concentration, 60f field s a m p l i n g m e t h o d , 5 6 - 6 9 i n exhaled b r e a t h , 5 6 - 6 9 V o l a t i l e pesticide c o m p o n e n t s , 58/ W W o r k e r apparel i m p e r m e a b l e gloves, 31 inside-patch residue, 30 W o r k e r exposure a p p l i c a t i o n e q u i p m e n t , 33—34 clothing, 30 e s t i m a t i o n , 51—53 hygiene—work practices, 3 3 - 3 5

e x c r e t i o n as i n d i c a t o r o f relative dose, 73 Urine 2,4-D, 8 genotoxicity, 99 m o n i t o r i n g h u m a n exposure t o genotoxic agents, 9 8 - 1 1 6 2,4,5-T, 8 variability i n urinary output, 9 U r i n e analysis advantages, 110 alachlor, 331-335 b i o l o g i c a l m o n i t o r i n g , 197 captan, 347 e n v i r o n m e n t a l c o n t a m i n a n t s , 109 genotoxicity, 109 limitations, 110-111 m u l t i p l e agents, 110 t i m e d e p e n d e n c e , 110 U r i n e clastogencity assay cigarettes, 111 dietary factors, 1 1 1 - 1 1 2 e n v i r o n m e n t a l c o n t a m i n a n t s , 112 m e d i c a t i o n , 112

251-261

p r o d u c t p a c k a g i n g , 31—33 u n d e r field c o n d i t i o n s , 305 W o r k e r exposure assessment s u b c o m m i t t e e N A C A p r o t o c o l , 369 objectives, 3 6 8 - 3 6 9 W o r k e r exposure study chlordimeform, 52 E P T C h e r b i c i d e , 292 W o r k p l a c e hazards, p e s t i c i d e users, 339

X X e n o b i o t i c m e t a b o l i s m , 93

Z Z e n d z i a n protocol, 306 Z w e i g - P o p e n d o r f transfer c o e f f i c i e n t , first estimate o f d e r m a l exposure, 344

In Biological Monitoring for Pesticide Exposure; Wang, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.