Environmental chemistry

Envi ronmenta I Chemist ry Volume 3

A Specialist Periodical Report

Environmental Chemistry Volume 3 A Review of the Literature published up to end 1982

Senior Reporter H. J. M. Bowen Department of Chemistry, University of Reading Reporters

S. J. Blunden International Tin Research Institute, Greenford 1. Colbeck University of Lancaster R. M. Harrison University of Lancaster L. A. Hobbs International Tin Research Institute, Greenford S. A. Katz Rutgers University, NJ, U.S.A. K. Simkiss University of Reading P. J. Smith International Tin Research Institute, Greenford M. G. Taylor Robens Institute, University of Surrey

The Royal Society of Chemistry Burlington House, London, WIV OBN

ISBN 0-85186-775-8 ISSN 0305-7712

Copyright 01984 The Royal Society of Chemistry

A fI Rights Reserved No part o f this book may be reproduced or transmitted in any form or by any means -graphic, electronic, includlng photocopying, recording, taping, or informotlon storage and retrieval systems - without written permission from The Royal Society of Chemistry

Set by Unicus Graphics Ltd, Horsham and printed in Great Britain by Whitstable Litho Ltd, Whitstable, Kent

Preface Research in environmental chemistry continues t o proliferate at such a rate that it is often difficult t o see the overall trends in particular topics. The subject is advancing hand-in-hand with the remarkable developments in analytical chemistry which have occurred during the past 3 0 years. It is probably fair to say that improvements in analytical sensitivity have exceeded our capacity to interpret the effects of environmental contaminants, so that legally defined limits of concentration are often based on subjective interpretations. Nevertheless the potential consequences of both local and global pollution are so serious that failure to understand them could affect large parts of the human race. The importance of the ozone layer as a radiation shield in the stratosphere has long been known. I t is less widely realized that measurable quantities of ozone also occur in the troposphere, where its fairly long lifetime together with its toxic and corrosive properties make it worthy of study as a pollutant. Concentrations of ozone capable of causing biological damage occur more often than is desirable at ground level. Indeed ozone, like lead, is a contaminant which exists close to or exceeding toxic levels in our environment. Colbeck and Harrison’s review covers these points and shows how the complexities of the sources and sinks of tropospheric ozone are slowly being unravelled. Unlike ozone, tin has such low toxicity that its environmental chemistry has been neglected. Trialkyl-tin species have much greater toxicity and are used as biocides, so their environmental degradation needs to be studied with care. Blunden, Hobbs, and Smith review the current situation with regard t o organotin compounds. They conclude that toxic effects are essentially local and fairly short-lived, and that no long-term hazards need arise from their use. The question of the natural biomethylation of tin is also discussed, as it is only recently that such environmental reactions have been shown to occur. Other heavy metals, notably cadmium, lead, and zinc, are known to accumulate in sewage sludges whose disposal has caused concern. Sidney Katz describes how determinations of heavy metals in sludges can now be made with reasonable precision and accuracy. Such determinations are vital t o decision-makers in assessing the risks of sludge disposal, whether by application as a fertilizer or landfill, by incineration or by burial at sea. Organisms themselves can become adapted t o high concentrations of metals. One of the ways in which they do this is by forming solid granules of inorganic material within their cells. The structure of these granules, which are usually amorphous, is becoming clearer as analyses by electron probe XRF, EXAFS, and infrared spectrometry improve. Taylor and Simkiss propose a preliminary classification of granules into calcareous, phosphatic, siliceous, and sulphur-rich categories which makes sense in terms of the likely functions of the

vi

Environmental Chemistry

granules. Their observation that the formation of calcareous granules is a normal rather than a pathological cell response seems justified, while the formation of phosphatic and sulphur-rich granules appear to be mechanisms of detoxification or excretion. H. J . M. BOWEN

Contents Chapter 1 Tropospheric Ozone

1

By I. Colbeck and R. M. Harrison

1 Introduction

1

2 Ozone Sources in the Unpolluted Troposphere

3 3

Stratosphere-Troposphere Exchange

3 Photochemistry of the Clean Troposphere

7

4 Ozone Distribution in the Troposphere Latitudinal Distribution of Ozone

10 13

5 Sinks of Ozone in the Unpolluted Troposphere

17

6 Tropospheric Ozone Budget

18

7 Ozone Formation and Destruction in Polluted Air Smog-chamber Studies Modelling of Photochemical Pollution Urban Ozone Rural Ozone

24 27 30 32 33

8 Elevated Ozone Levels

35 40 42

Meteorological Conditions Associated with Ozone Pollution Weekday-Weekend Variations

9 Biological Effects of Ozone Effects on Humans Effects on Vegetation Environmental Health Guidelines 10 Analytical Techniques

43 43 45 46 47

Chapter 2 The Environmental Chemistry of Organotin

Compounds

49

By S. J. Blunden, L. A. Hobbs, and P. J. Smith

1 Introduction

49

2 Toxicological Patterns of Organotins Toxicology and Mode of Action Metabolism

56 56 59

...

Envir o nmen tal Chemistry

Vlll

3 Analysis of Organotins at Environmental Levels

60

4 Modes of Entry into the Environment

61

5 Aqueous Chemistry

65

6 Transformations in the Environment

70

7 Degradation of Organotin Compounds

71

U.V. Irradiation Biological Cleavage Chemical Cleavage

8 Summary Chapter 3 Determination of Heavy Metals in Sewage Sludge By S.A. Katz

72 74 75 76

78

1 Introduction

78

2 Analysis of Sewage Sludge

79 79 82 83 83 83 89 89 91 91

Sample Collection Sample Preservation Sample Preparation Drying Dissolution Elemental Determinations Atomic Absorption/Atomic Emission Spectrometry Neutron Activation/Photon Activation Analysis Other Methodologies

3 Selected Procedures for Sludge Analysis US EPA Procedures UK DOE Procedures Other Procedures

92 92 94 96

4 Disposal and Utilization of Sewage Sludge

96 97 97 97 97 98

Incineration Land Disposal Ocean Dumping Cropland Applications Miscellaneous Applications 5 Possible Consequences of Sewage Sludge Disposal and Utilization and the Need for Monitoring

Soil and Water Contamination Contamination of Plants Contamination of Animals 6 Conclusions

98 98 99 100

101

Contents Chapter 4

ix

Inorganic Deposits in Invertebrate Tissues G. Taylor and K. Simkiss

102

By M.

1 Introduction

Basic Approaches

102 103

2 Metal Deposits Potassium Magnesium, Calcium, Strontium, and Barium Aluminium and Silicon Vanadium Chromium and Molybdenum Manganese Iron Cobalt Nickel Copper Zinc, Cadmium, and Mercury Lead

105 106 107 108 108 109 110 111 113 113 113 116 117

3 Ligand Binding

118

Oxygen Donor Ligands - Carbonate, Phosphate, Sulphate, and Oxalate Granules Oxygen Donor Ligands - Analyses and Structure The Formation of Inorganic Granules Sulphur Donor Ligands

118 122 127 129

4 Silica Deposition

132

5 Urates

133

6 Conclusions The Penetration of Metals into Organisms and Cells Precipitation or Binding Accumulation Sites

135 137 137 138

I Tropospheric Ozone B Y I. C O L B E C K A N D R. M. H A R R I S O N

1 Introduction

There has been an enormous growth in our understanding of the chemistry of atmospheric ozone since the early 1970's. It was Schoenbein' who first suggested that there existed in the atmosphere a constituent which had a particular odour and named it ozone. In 1858 Houzeau2 chemically proved that ozone existed at ground level and in 1880 Chappuis3 made the first spectroscopic detection of atmospheric ozone. Since the measurements reported by Dobson4 in 1926, routine observations of total column ozone have been conducted a t a large number of places all over the world. Penndorf5 and Dutsch6 summarize the measurements of the vertical ozone distributions and of the distribution of total column ozone with latitude and season. Figure 1 shows the variation of ozone concentration' and atmospheric temperature with height. Ozone absorbs solar ultraviolet radiation in the Hartley band (200-300 nm) and at mid-latitudes, the heating of the atmosphere due to ozone absorption peaks at an altitude of approximately 50 km. When integrated over a day, it would amount to an increase of temperature of 8 K, which is balanced mainly by emission of infrared radiation by carbon dioxide. Owing t o strong mixing, the ozone mixing ratio is approximately constant in the troposphere and then increases to a maximum of about 10 p.p.m. ( V / V )at a height of 30 to 3 5 km before decreasing with altitude above this. Above 3 5 km the ozone is in photochemical equilibrium. However, below this altitude the photochemical relaxation time for ozone is of the order of a few days, increasing t o several years at the tropopause. Hence transport processes determine the ozone distribution below 35 km and photochemistry above that height. Since ozone plays an important role in determining the temperature structure of the stratosphere, the most important photochemical processes in the annosphere are those leading t o the formation and destruction of ozone. Chapmans

' C. F. Schoenbein, C. R. Acad. Sci. Paris, 1840, 10, 706. ' A. Houzeau, C. R . Acad. Sci. Paris, 1858,46, 89. J. Chappuis, C. R . Acad. Sci, Paris, 1880, 91, 985. G. M. B. Dobson and D. N. Harrison, Proc. R . SOC.London, Ser. A , 1926, 110,660. R. Penndorf, U.S. Department of Transportation, 1978, Federal Aviation Administration

Report No. FAA-EE-78-29. H . U . Dutsch,Can. J . Chem., 1974, 52, 1491. 'A. J. Krueger and R. A. Minzer, J. Geophys. Res., 1976, 81, 4477. a S. Chapman, Mem. R . Met. SOC.,1930, 3 , 103.

Environmental Chemistry

2

TEMPERATURE PC)

MIXING RATIO (PPm)

Figure 1 Variation of ozone mixing ratio and atmospheric temperature with height at mid-latitudes

established the basic photochemistry of an oxygen atmosphere [equations (1)(4)J.In the stratosphere ozone absorbs sunlight much more strongly than does oxygen. Any depletion of stratospheric ozone would lead t o an increase in the amount of harmful ultraviolet light, of wavelength between 290 and 320 nm,

o2+ h~ -+ ~ o ( ~ P )

A < 242 nm

(2 1

02+ O+ M+03+M 03+hv+02+O('~)

0 3 +0 -+ O2

+ O2

(1)

A < 310nm

(3 1 (4)

reaching the Earth's surface. Light of such a wavelength may cause an increase in skin cancer in humansg and have adverse influences on plant sysrems. Over the past few years there has been considerable concern over possible changes in concentrations of stratospheric ozone due to a variety of human influences including oxides of nitrogen from supersonic aircraft, and chlorofluorocarbon aerosol propellants." The resultant research has led to a great improvement in

lo

H. G . Booker, Environmental Impact of Stratospheric Flight, Washington, D.C., Climatic Impact Committee, 1975. J. A. Logan, M. J. F'rather, S. C. Wofsy, and M. B. McElroy, J. Geophys. Res., 1981,86, 7210.

Tropospheric Ozone

3

our knowledge of the causes and effects of a stratospheric ozone reduction,llS l2 although accurate quantitative information is still lacking. Ozone is a major component of the stratospheric trace gas system whose concentration is controlled by the very complex series of homogeneous chemical processes in which it takes part. 2 Ozone Sources in the Unpolluted Troposphere

The origin of the natural background level of tropospheric ozone has become a controversial subject. The major possibilities are stratosphere-troposphere exchange and photochemical processes: much work has been published in favour of each mechanism. According to the classical view ozone was inserted through the tropopause, mixed downwards and destroyed at the surface. Since molecular oxygen is not photodissociated in the troposphere, the mixing ratio of ozone thus decreases towards the surface. This mechanism has been studied by several worker^;'^-^^ the possibility of local synthesis of ozone making the major contribution to tropospheric ozone is favoured by other^.^*-^' Stratosphere-Troposphere Exchange. - The large amounts of ozone produced in the stratosphere are inhibited from entering the troposphere by a sharp increase in atmospheric stability at the tropopause. Small quantities of ozone-rich stratospheric air are transported to the troposphere by one of several stratospheretroposphere exchange processes, often operating simultaneously, but of differing magnitude. Reiter28lists these processes as: (i) seasonal adjustments in tropopause levels, (ii) mean meridional circulation transport, (iii) mesoscale and small scale eddy transport across the tropopause, and (iv) large scale eddy transports (tropopause folding). R. D. Hudson and E. 1. Reed eds., ‘The Stratosphere: Present and Future’, NASA Reference Publication 1049, 1979. l 2 World Meteorological Organisation, The Stratosphere 1981: Theory and Measurements, WMO Report No. 11,1981. I ’ H. B. Singh, F. L. Ludwig, and W. B. Johnson, Atmos. Environ., 1978,12, 2185. l4 H. B. Singh, W. Viezee, W. B. Johnson, and F. L. Ludwig, J. Air Pollut. Control ASSOC., 1980,30,1009. l 5 C. E. Junge and G . Czeplak, Tellus, 1968,20,422. l6 P. Fabian and P. G. Pruchniewicz, J. Geophys. Res., 1977,85,2063. R. Chatfield and H. Harrison, J. Geophys Res., 1977,82, 5969. I s L. Husain, P. E. Coffey, R. E. Meyers, and R. T. Cederwall, Geophys. Res. Lett., 1977, 4,363. 19 W. B. Johnson and W. Viezee, Atmos. Environ., 1981,15, 1309. 2o P. J . Crutzen, Pugeoph, 1973,106,1385. W. L. Chameides and J . C. G. Walker, J. Geophys. Res., 1976,81,413. 2 2 J . Fishman and P. J. Crutzen, J. Geophys. Res., 1977,82, 5897. 23 J. Fishman and P. J. Crutzen, Nature, 1978,274, 855. 24 W. L. Chameides, Geophys. Res. Lett., 1978, 5 , 17. ” J.Fishman, S . Solomon, and P. J . Crutzen, Tellus, 1979,31,432. 26 J. Fishman, W. Seiler, and P. Haagenson, Tellus, 1980, 32,456. 27 W. Seiler and J . Fishman, J. Geophys. Res., 1981, 86, 7255. ‘I

4

Environmental Chemistry

The height of the tropopause varies with latitude and time of year. In the Northern hemisphere it is lowest in winter, highest in summer and exhibits a poleward decrease in height. The height variation is most pronounced North of latitude 30' and results in about 10%of the mass of the Northern hemisphere's stratosphere being exchanged annually with the troposphere. Some 4 to 8 X lo1' kg of ozone can be added to the Northern hemisphere troposphere by this process. The meridional circulation consists mainly of the Hadley cell circulation. This circulation pattern can be described as two cells on either side of the equator with converging equator-ward surface currents which merge at the equator into one upward current which splits in the low stratosphere into two diverging poleward currents. The upward flow of the Hadley cell transports large amounts of tropical tropospheric air into the stratosphere. This upward flux is largest in the winter and annually is equal to 38% of the mass of the stratosphere in the Northern hemisphere.28 The same amount of stratospheric air returns t o the troposphere in the middle and high latitudes at a rate dependent upon season. However, this process is slow with a mean residence time for air of 1-4 yr.29 The hemisphere asymmetries in the Hadley cells and tropical tropopause suggest that the flux is not evenly divided and more than half of it returns t o the Northern hemisphere troposphere. Mesoscale and smallscale eddy transport across the tropopause contributes about 1 to 5 % of the total flux from the stratosphere.28 Included in this process is the penetration of the tropopause by thunderstorms. This not only injects tropospheric air into the stratosphere, but causes downward mixing of stratospheric air into the troposphere. The dominant process for injecting stratospheric ozone into the troposphere is believed t o be large scale eddy transport. Reiter28 attributes 20%of the total flux from the stratosphere to this process, while Danielsen30 assumes that it accounts for 100%of the total flux. Reed3' proposed a mechanism whereby, during high-level cyclogenesis, stratospheric air may be injected into the troposphere and further studies have been made.32-34 A net outflow of stratospheric air occurs through the 'tropopause gaps' during the deformation of the tropopause in the jet stream. The process is shown schematically in Figure 2. Since the stratospheric air is moving faster than the frontal zone it is carried into the upper portion of the frontal zone, the region where the tropopause is steeply inclined on the cyclonic side of the jet maximum. This process where the tropopause deforms, becomes vertical in the core of the jet stream, and then folds beneath the jet core is known as tropopause folding. These -intrusions have been pbserved on occasions down to E. R. Reiter, R e v . G e o p h y s . Space Phys., 1975, 13,459. P. W. Krey, M. Schonberg, and L. Toonkel, Rep. HASL-281, U.S. Atom. Energy Comm., New York, 1974. 3 0 E. F. Danielsen,J. A m o s . Sci., 1968, 2 5 , 5 0 2 . 3 1 R.J . Reed, J. Meteorol., 1955, 12, 2 2 6 . 3 2 D. 0. Staley, J. Meteorol., 1960, 17, 591. 3 3 D. 0. Staley, J. Atmos. Sci., 1962, 19, 450. 34 E. F. Danielsen, J. Meteorol., 1961, 18, 479. 29

Tropospheric Ozone

5

TRAJECTORY

500 mb

\

FR3KTAL ZONE

Figure 2 Schematic three-dimensional view of mass flow from stratosphere t o troposphere near the j e t stream (Reproduced by permission from J. Appl. Meterorol., 1 9 6 3 , 2 , 6 9 1 )

ground leve1.35 Danielsenm first postulated this mechanism as a possible source of ozone in the troposphere. He found a correlation between ozone, radioactivity and potential vorticity in the upper troposphere and lower stratosphere; it had already been observed that relatively high concentrations of radioactive debris at ground-level appeared to be of stratospheric origin. This finding was later confirmed by many observational studies from aircraft, which found elevated levels of ozone in the free troposphere. On occasion high ground-level concentrations have been measured which, by isentropic analysis could be traced back t o the s t r a t o ~ p h e r e . ~ ~ Danielsen et aZ.36 presented one of the first cases of ozone of stratospheric origin to be observed in the troposphere. They found a positive correlation between ozone and radioactivity and three mesoscale folds in the boundary between the troposphere and stratosphere over the Rocky Mountains on 25 April 1969. Mohnen et aZ.37reported that measurements of ozone concentration at Whiteface Mountain (4860ft) exceeded that of surface stations and that the source level lay above 850 mb. Flight experiments found a significant downward transport of ozone from the stratosphere; Danielsen and Mohnen 38 analysed three intrusion cases (18, 26, and 27 April 1975) over Colorado, Oklahoma, and R. G . Derwent, A. E. J . Eggleton, M. L. Williams, and C. A. Bell, Atmos. Environ., 1978, 12,2173. 36 E. F. Danielsen, R. Bleck, J . Shedlovsky, A. Wartburg, P. Haagenson, and W. Pollock, J. Geophys. Res., 1970, 75,2353. 31 V. A. Mohnen, A. Hogan, E. F. Danielsen, and P. Coffey, Internat. Conf. on Photochemical Oxidant Pollution and its Control, EPA-600/3-77-001a, 1977. E. F. Danielsen and V. A. Mohnen, J. Geophys. Res., 1977,82, 5867. ”

’’

6

Environmental Chemistry

Texas. Estimates of the mass transfer were made based on radioisotope (strontium9 0 ) concentrations and observations of the rate of deposition of 90Sr. They predicted an annual outflow of 4.95 x 1014 g ozone in the Northern hemisphere. Concentrations of 1 0 0 to 2 1 0 p.p.b. near major atmospheric lows were measured from aircraft at 60°N over Canada in 1 977.39 During project Gametag, Danielsen40 found that the ozone concentration increased from 40 p.p.b. u p to 1 0 0 p.p.b. at 5.4 km on a flight from San Francisco to Hawaii. This air was traced back by isentropic analysis to the stratosphere, from where it had originated 3 days previously. This rate of transport is over one hundred times faster than motion due to zonal seasonal mean circulations. Singh et aE.14 and Johnson and Viezeelg report on a programme of aircraft measurements t o observe tropopause folding in the Spring and Autumn of 1978. Measurements in the Spring were made over Colorado, Kansas, Oklahoma, Minnesota, and Mississippi, while those in Autumn were made over Minnesota, Iowa, Illinois, and Tennessee. Ten dates are given when ozone concentrations ranged from 177 p.p.b. t o 362 p.p.b. over an altitude range from 5 . 2 km to 7.5 km. As predicted by Danie1sen:O all the ozone intrusions were found on the North side of the jet stream and were associated with upperlevel low pressure troughs. High ground-level concentrations of ozone due to isentropic transport of ozone from the stratosphere have been measured by a few workers. Attmannspacher and Hartmann~gruber~'reported seven such occasions in the winter of 1971 with concentrations between 2 5 0 and 500 p.p.b. lasting 10 min or longer. Each occurrence was during a snow storm accompanying a passing cold front. Five consecutive hours of ozone levels in excess of 80 p.p.b. with a maximum hourly average concentration of 2 3 0 p.p.b., during the early hours of a cold night in November at Santa Rosa, California, are reported by Lamb.42He concluded the stratospheric ozone was transported to the ground in precipitation-driven downdrafts. Derwent et aZ.35observed ozone levels in excess of 1 0 0 p.p.b. at two rural sites in Britain in Spring 1976 and 1 9 7 7 . In both cases, frontal troughs crossed the area a day before the elevated levels were measured and cross-sections of the atmosphere showed that a stratospheric intrusion in the trailing edge of the jet stream occurred. A maximum concentration of 223 p.p.b. was reported by Haagenson et aZ.43 on 4 March 1 9 7 8 in Denver, Colorado. Isentropic analysis indicated that the air had originated from a stratospheric intrusion 3 days earlier. This air was also subject to enhanced photochemistry due to local pollution. Since there is a positive correlation between radioisotope concentrations and ozone in the region of the tropopause36 it is possible t o identify stratospheric ozone at ground-level by measuring radioactive debris. Husain et a1.l8 used 7Be to indicate the presence of stratospheric air on the summit of Whiteface Mountain. They found two days in July 1975 with peaks in 7Be concentrations which corresponded with ozone peaks. They deduced an upper limit of 37 p.p.b. was due to E. F. Danielsen and R. S. Hipskind, J. Geophys. Res., 1980, 8 5 , 393. E . F. Danielsen, J . Geophys. Res., 1980, 85,401. 4 1 W. Attmannspacher and R. Hartmannsgruber, Pageoph, 1973,106-108, 1091. 4 2 R. G . Lamb, J. Appl. Meteorol., 1977,16, 780. 43 P. L. Haagenson, M. A. Shapiro, P. Middleton, and A. R. Laird, J. Geophys. Res., 1981, 86, 5231.

39 40

Tropospheric Ozone

7

stratospheric ozone. Simultaneous measurements of ‘Be and ozone in the lower stratosphere have shown that their ratio is nearly constant at ca. l l f Ci m-3 ~ . p . b . - ’ . Hence ~~ ground-level 7Be can be used to estimate the associated stratospheric ozone. Dutkiewicz et aZ.45 used this method and found that the greatest impact of stratospheric ozone at Whiteface Mountain occurs during late Spring and early Summer. They estimated that 24 h average ground-level ozone was increased by 12 p.p.b. due to tropopause folding events. Reiter46 correlated wSr with ozone-sonde measurements made between December 1962 and December 1965. He found that stratospheric intrusions giving rise to ozone concentrations over 80 p.p.b. occur on 0.2% of the days of the year. Other instances of high ozone concentrations at ground-level due to stratospheric intrusions have been reported by Shapiro4’ and Singh et aZ.13 Singh and his co-workers measured an ozone concentration of 196 p.p.b. at Zugspitze, W. Germany, in January 1976. The ’Be concentration showed a 600% increase during this episode. The overall contribution of stratospheric ozone to the tropospheric ozone budget will be discussed further later.

3 Photochemistry of the Clean Troposphere I t was originally thought that ozone in the troposphere was inert; it was transported from the stratosphere and destroyed at the ground. However, recently it has been suggested that ozone is not inert, even in the unpolluted troposphere, and that photochemical processes are important in the overal ozone budget. The photochemistry of the troposphere has been studied and modelled by many workers lo, 25,48 in attempts to calculate the relative importance of downward transport from the stratosphere and gas-phase photochemical production and destruction of ozone. The hydroxyl radical plays a dominant role in the chemistry of the troposphere, The primary source of tropospheric OH results from the reaction in which ozone is photolysed to yield O(’D) by ultraviolet radiation in the 300320 nm range, reaction (3). The hydroxyl radicals are then formed from the reaction of O(’D) with water

+

O( ‘D) H2O + 2 0 H

(5)

net cycle 0 3

H2O 4- hv + 2 0 H 3 . 0 2

(6)

I t is in this way that the OH production results in a net cycle of ozone destruction and that ozone acts as the precursor to most unpolluted troposphere

44 45

46

47 48

V. A. Dutkiewicz and L. Husain, Geophys. Res. Lett., 1979, 6 , 171. V. A. Dutkiewicz, L. Husain, and A. Rusheed, Proc. Internat. Ozone Symp., Boulder, Colorado, 1980. E. R. Reiter, Internat. Conf. on Photochemical Oxidant Pollution and its Control, EPA600/3-77-00la, 1977. M. A. Shapiro, Mon. Weather Rev., 1978, 106, 1100. S. C. Liu, D. Kley, M. McFarland, J . D. Mahlman, and H. Levy, J. Geophys. Res., 1980, 85,7546.

Environmental Chemistry

8

photochemistry. The OH formed by reactions ( 3 ) and (5) may react with methane and carbon monoxide t o initiate catalytic cycles which produce ozone in the troposphere. CH4 4-OH + CH3 4- H 2 0 CH3+

0 2

4- M + CH302 4-

(7)

M

(8)

and CO

OH+ C02+ H

H 4-0

2

M j . 0 2 4- M

If NO exists in sufficient amounts then C H 3 0 2 3- NO + CH30 i- NO2 CH30

+ O2

+

CH20 4- H 0 2

If formaldehyde is photolysed, then CH2O 4- hu CHO 4- 0

-+

CHO 4- H

2 + HO2

4- CO

HO2 4-NO + NO2

OH

NO2 4-hu

-+

NO 4- O(3P)

( A < 370 nm)

(A

< 420 nm)

and ozone is produced via reaction (2). The net cycle for methane oxidation is then CH4 4- 8 0 2 + H2O 4- CO 4- 403 4- 2 0 H

(1 7)

For CO, oxidation reactions ( 9 ) and (10) can be followed by reactions (15), (16), and (2) resulting in the net cycle

co + 2 0 2 + c02 + 0

3

(18)

At low concentrations of NO ozone may be destroyed by HO2 + 0 3 + OH

+ 202

(19)

competing with (15 ) . Ozone is also destroyed by its reaction with nitric oxide NO

+0 3

+

NO2 + O2

(20)

Thus the operation of a tropospheric photochemical source for ozone depends upon the background levels of NO,. The amount of ozone which can be formed in the troposphere by this mechanism is limited by the concentrations of CO and CH4. I t is possible that methane oxidation could yield 3.5 molecules of ozone per molecule of methane.'O Methane is released from the earth's surface to the atmosphere at an average rate of 1.5 x 10l1molecules cm-2 s-1.49 Carbon monoxide is produced in the troposphere from the formaldehyde formed during methane 49

D. H. Ehhalt and U. Schmidt, Pageoph, 1978, 116,452.

9

Tropospheric Ozone

oxidation. Other sources of carbon monoxide provide 2.5 x lou molecules CO cm-2 s-l, Hence, the possible yield of ozone from CH4 and CO oxidation could be 8 x 10l1 molecules cm-2 s-l if NO existed in sufficient quantities.1° The equilibrium distribution of 03,in sunlight, is governed by reactions (16), (2), and (20), so in a photostationary stateSo k16 " 0 2 1

[031

=k2o "01

.

Ozone is destroyed via reactions ( 3 ) , (5), ( 9 ) , (lo), and (19) and the net ozone destruction reaction is CO

+ H2O

203

+ hv

+

202

C02

+ 20H

(21)

A schematic representation of the odd oxygen photochemistry in the troposphere is shown in Figure 3 .

NO

O(l D)

O(1

D) H,O +

3

2 OH

U1D)+H, +H+OH LOc D) CH, -+ CH3+OH +

I

Figure 3 Main chemical reactions of the odd oxygen photochemistry in the troposphere

I t is possible, by using numerical models, to calculate the effect of the various reactions in the troposphere and compare the results with in situ measurements. The results of such calculations lead to the proposition that significant amounts of ozone are produced in the troposphere from CH&O/NO, photochemistry.22>51 Fishman and C r ~ t z e nbased ~ ~ their argument on the fact that the hemispheric asymmetry in the tropospheric ozone distribution could not be explained satisfactorily by differences in the stratosphere-troposphere exchange rates in

'"P. A. Leighton, 'Photochemistry of Air Pollution', Academic Press, New York, 1961.

'' W. L. Chameidesand J . C. G. Walker, J. Geophys. Res., 1973, 78, 8751.

Environmental Chemistry

10

the two hemispheres. They postulated that the difference between the fluxes into and out of each hemisphere could be caused by photochemical processes in the troposphere. were criticized for ambiguities in the interpreThe early results of models tation of experimental data and for not including vertical mixing of the lower t r o p o ~ p h e r e . Later ~ ~ , ~models ~ recognized the importance of both transport and photochemistry in establishing the ozone distribution, but these were still dependent upon knowledge of the reaction rates in the troposphere. These models are dependent also on the assumed NO, concentrations, as higher concentrations promote the CH&O oxidation mechanisms. Crutzen 2o estimated that the NO mixing ratio must be greater than 10 p.p.t. for reaction (15) to be more rapid than reaction (1 9 ) . This value is exceeded over most parts of the earth, especially near the strong industrial sources of the Northern hemisphere mid-latitudes. The greatest concentrations are found near the surface and this causes most of the predicted ozone production to be in the lowest 3 km. Liu e t aL4* have suggested that the effects of NO, intrusions from the stratosphere provide an upper tropospheric ozone source which may be larger than the direct injection of ozone from the stratosphere. Vertical profiles of O3 and CO have been measured simultaneously in the free t r ~ p o s p h e r e .The ~ ~ ,data ~ ~ show a positive correlation of CO and O 3 on a short time scale and on a synoptic spatial scale. If there was no chemical input of 0 3 in the troposphere, regions of high 0 3 would be concurrent with low CO mixing ratios. Therefore the results indicate that some in situ photochemical source of O3 exists in the free troposphere. Logan et ul.'* found that the magnitude of the photochemical source was proportional t o NO but less sensitive to CO. I t is not known which oxidation scheme dominates under normal tropospheric conditions, since it depends on the amount of nitric oxide present. Carbon monoxide, NO, and NO2 are influenced by physical and chemical processes in the atmosphere and by anthropogenic sources at the ground. A global set of observations of the minor constituents of the troposphere would be very helpful. In an unpolluted troposphere, stratospheric ozone is the probable primary source of ozone, whereas in an atmosphere polluted with NO,, oxidation of CH4 and CO produce ozone. Generally the local source of ozone in the troposphere is a combination of photochemistry and atmospheric transport. 219

24*25954

4 Ozone Distribution in the Troposphere There is now a considerable volume of observational data on the tropospheric distribution of ozone, though nowhere near the extent of measurements of stratospheric ozone. Most of the information on the vertical distribution of ozone in the troposphere has been obtained from measurements intended primarily to investigate the stratosphere. Regular direct soundings of the vertical distribution have been limited to the North American Ozonesonde Network 52

53 54

P. Fabian, J. Geophys. Res., 1 9 7 4 , 7 9 , 4 1 2 4 . R . Chatfield and H. Harrison, J. Geophys. Res., 1 9 7 6 , 8 1 , 4 2 1 . R . W. Stewart, S. Hameed, and J. P. Pinto, J. Geophys. R e s . , 1977, 8 2 , 3134.

Tropospheric Oz one

11

between 1963 and 1969,55,56a European network which started in 1966,56and those made over Aspendale, Australia, since 1965.57Hering and Borden give results of vertical ozone profiles over the altitude range 0-30 km. These measurements were part of the North American Ozonesonde Network and were made using chemiluminescent dyes on silica gel. Chatfield and H a r r i ~ o n ' ~ , ~ ~ examined these data to derive tropospheric ozone profiles. They found that measurements by chemiluminescent ozonesondes underestimate the concentrations by 50% in comparison with profiles measured by electrochemical ozonesondes. They found that the tropospheric mixing ratios of ozone at all latitudes and seasons in the Northern hemisphere increase with height. Wilcox and Belmont62 and Fishman and C r ~ t z e have n ~ ~both presented vertical ozone profiles in the troposphere by extracting data from stratospheric ozonesonde measurements, Seiler and Fishman 27 and Fishman et aZ.26 obtained two-dimensional distributions of carbon monoxide and ozone in the free troposphere during July and August, 1974. Vertical profiles of ozone were made by a microcoulomb ozone sensor from a series of flights between 67"N and 57"s. The results of some of their profiles are shown in Figure 4. Tropospheric ozone profiles over two stations in Europe have been measured by Dutsch and Ling63and Attmanspacher and Hartmann~gruber.6~ The six years of data were obtained by the electrochemical method. In the Southern hemisphere, measurements of ozone are sparse. Pittock5' measured ozone profiles over Aspendale, Australia (3 8"s)using an electrochemical technique. The ozone concentrations below 2 km are lower in the summer Southern hemisphere than in the Northern hemisphere; also little seasonal variation was found. In the Southern tropics Fishman e t aZ.25 have analysed profiles from Canton (2's) and La Paz (16"s). They found that the ozone levels at 2 km were 35% less than those at a Northern tropical station. Other profiles, in both hemispheres, have been obtained during aircraft flights to measure stratospheric intrusions (e.g., ref. 19). Pelon and Mkgie65,66used the differential absorption laser technique (DIAL) to measure the ozone vertical distribution from the ground up to 25 km. This is the first time that tropospheric 55958-60

"

W. S. Hering and T. R. Borden, Rep. AFCRL-64-30(1), Air Force Cambridge Res. Lab.,

Bedford, Mass., 1964. H. U. Dutsch, W. Zullig, and Ch. Ling, report LAPETH-1, Laboratorium fiir atmospharen physiks ETH, Zurich, Switzerland, 1970. 57 A. B. Pittock, Quart. J. R. Meteorol. Soc., 1977, 103, 575. W. S. Herring and T. R. Borden, Rep. AFCRL-64-30(11), Air Force Cambridge Res. Lab., Bedford, Mass., 1965. '' W. S. Herring and T. R. Borden, R w . AFCRL-64-30(111), Air Force Cambridge Res. Lab., Bedford, Mass.,1965. 6 o W. S. Herring and T. R. Borden, Rep. AFCRL-64-30(IV), Air Force Cambridge Res Lab., Bedford, Mass., 1967. 6 1 R. Charfield and H. Harrison, J. Geophys. Res., 1977, 82, 5965. 6 2 R. W. Wilcox and A. D. Belmont, Rep. FAA-AEQ-77-13, U.S. Dept. of Transport., Washington, D.C., 1977. 63 H. U. Dutsch and Ch. Ling,Pageoph, 1973, 106-108, 1151. 64 W.Attrnanspacher and R. Hartmannsgruber, Beitr.Phys. Atmos., 1976, 49, 18. 6 5 J. Pelon and G. MCgie, J. Geophys. Res., 1982, 87,4947. 6 6 J. Pelon and G. Migie, Nature (London),1982, 299, 137. 56

12

-

Environmental Chemistry Jacksonville (3OoN)

-

m u -

Punta Arenas (53's)

Churchill (SQON)

n

n

E

Y

W

a

3

v) v)

W

a a.

20

10

60

OZONE MIXING RATIO (ppbv) Figure 4

Vertical distribution of ozone mixing ratio with latitude (data from ref. 27)

ozone monitoring has been performed using a ground-based remote sensing technique. All the reported measurements of the vertical distribution of tropospheric ozone show an increase in concentration with height. In the upper troposphere they generally show a large gradient, indicative of its stratospheric source and downward transport. Ozone concentrations in the atmospheric boundary layer are influenced by the local microclimate. There have been few measurements of tropospheric ozone in clean air, far removed from major anthropogenic sources of ozone-

Tropospheric Ozone

13

producing or ozone-destroying contamination. Measurements at 2 m above the ground were carried out at Barrow, Alaska, from January 1965 to September 1967.67Highest concentrations occurred in the Spring, and daily maxima varied between 1 and 70 p.p.b. over the whole year. Stallard et a1.68 obtained a continuous section of surface ozone concentrations in the eastern equatorial and Southern tropical Atlantic. Their measurements were made from a ship on a journey between Dakar (13"N) and Walvis Bay (22"s). Between 1O"N and 1"N maximum concentrations of 3 0 to 48 p.p.b. were recorded. Uniform concentrations of 13 to 15 p.p.b. were obtained over the remainder of the journey. Ozone measurements in clean air have been made as part of the project Tropospharisches Ozon (TROZ) run by the Max Planck Institute fur Aeronomie.16, 69-72 Project TROZ started in 1969 with a meridional chain of 16 stations between Tromso, Norway, and Hermanus, South Africa, monitoring ozone concentrations. When the project finished in 1979, eight stations were still operating. The results of project TROZ are analysed in the next section. Latitudinal Distribution of Ozone. - As was the case with vertical profiles, many workers have extracted data from measurements whose primary concern was stratospheric ozone to obtain latitudinal distributions of tropospheric ozone. Chatfield and Harrison1' anal sed 703 ozonesonde launches between 1966 and B 1969 at six stations near 75 W between 9" and 53"N, and F i ~ h m a n chose ~~,~~ ozonesonde data at Boulder (40°N)55 and the combined data from Bedford (42"N) and Wallops Island ( 3 8"N) and Aspendale (38"s) for their comparisons. They found annual variations in the concentration at 200 mb, with a maximum in late winter and early spring. At 800 mb, the seasonal variation is evident only in the Northern hemisphere and the concentrations in this region were higher than those in the Southern hemisphere. As part of the Global Atmospheric Sampling Program (GASP), ozone was measured by ultraviolet spectrophotometers placed aboard commercial airliners during 1975-7.n-77 The GASP data are confined to the altitude range 8.5 to 13.5 km. At 45"N, east-west variations of ozone have a wavelength of about 2400 km, whereas temperature and wind speed have a wavelength of 3300 km. This difference may be due t o a sampling bias. Ozone amounts in the tropical upper troposphere were fairly uniform and the seasonal mean GASP ozone concentration between 15'N and 15"s showed an annual maximum in or around October. They also found a high level of equatorial symmetry of ozone between 0" and 40" at the altitude range studied. 67

J. J . Kelly,Pugeoph, 1973,106-108,1106,

F. Stallard,J . M. Edmond, and R. E. Newell, Geophys. Res. Lett., 1975, 2 , 289. P. Fabian and P. G. Pruchniewicz,Pageoph, 1973, 106-108,1027. 70 H. K. Tiefenau, P. G. Pruchniewicz, and P. Fabian, Pugeoph, 1973,106-108,1036. 7 1 P. G. Pruchniewicz, Pugeoph, 1973,106-108,1074. 72 M. Schmidt, Proc. Internat. Ozone Symp., Boulder, Colorado, 1980. 73 P, Falconer and J. Holdeman, Geophys. Res. Lett., 1976, 3, 101. 74 R. Pratt and P. Falconer, J. Geophys. Res., 1979, 84, 7876. 7s G. D. Nasaom, J. Appl. Meteorol., 1977, 16, 740. 76 G. D. Nastrom, J. Geophys. Res., 1979, 84, 3683. 77 J. D. Holdemann, T. J. Dudzinski, and M. W. Tiefermann, NASA-TM-81462, 1980.

" R. 69

14

En viro n m en ta1 C hemis try

As part of the Gametag field experiments ozone was measured during 1977 and 1978.78The measurements were made from aircraft between 58"s and 70'N and 48 'S and 7 0 ON over the altitude ranges of 5-6.5 km and 0.3-1 km, respectively. Their measurements were made in the months of April, May, August, and September. They found the lowest ozone levels in the free troposphere between 20's to 12'N and higher average concentrations in the Northern hemisphere than in the Southern hemisphere. The horizontal variability was also greater in the Northern hemisphere than in the Southern hemisphere. Measurements in the boundary layer at remote sites in the Northern hemisphere gave lower concentrations of ozone when compared with free tropospheric values by a factor of approximately 1.5. This factor is only 1.1 for latitudes South of 15"s. Besides ground-based monitoring stations, measurements were also made of ozone aboard airliner^.^^,^ The 34 meridional cross-sections were carried o u t with a Brewer type electrochemical cell between 69.5"N and 34's. Their results indicated enhanced stratosphere-troposphere injection at 30°, 45",and 6S0N, tropospheric ozone concentrations north of 25'N exhibited strong variations with both season and latitude, and between 25"s and 25"N there was no systematic variation of ozone concentration with latitude. Highest values in this region occur between May and June, while the lowest values are found from October to December. In higher Northern latitudes, the annual maximum occurs in April and May. The results from the ground stations indicated that the average meridional distribution of ozone varied by a factor of approximately 2 between the high values in the sub-tropics and Northern mid-latitudes and the lowest levels in the tropics; the amplitudes of the annual variation increased towards Northern mid-latitudes and the Southern tropics and the phase followed that in the free troposphere; and that the phase in the tropics follows the Hadley cell circulation, with maximum concentrations in regions of downward motion. Similarly, Stallard et a1.68 explained their observations of high ozone values in the Inter-Tropical Convergence Zone in terms of strong mesoscale downdrafts within the Hadley cell. Finally, the results of the series of flights during July and August 1974 ' results between 67'N and 57's are discussed by Seiler and F i ~ h m a n . ~Their confirm the asymmetric distribution of tropospheric ozone and the low ozone mixing ratios in the low tropical troposphere which they report are consistent with the findings of Routhier et al.@' In the upper troposphere they found the regions south of 45"s and north of 48'N to be areas where stratospheric air was possibly present, while the region between 15"N and 40'N, was an area of photochemical production. Figure S shows the global regions over which tropospheric clean air ozone measurements have been made. Generally GASP data were obtained on flights between Europe and America, America and Hawaii, and on several flights into D. D. Davis, J. Geophys. Res., 1980,85,7285. W. Bischof and P. Fabian, Pugeoph, 1973,106-108,1041. " F. Routhier, R. Dennett, D. D. Davis, A. Wartburg, P. Haagenson, and A. C. Delany, J. Geophys. Res., 1980,85, 7307. " 79

Figure 5

120

1

I

60

0

LONG I T U D E

,

60

E

Global regions over which tropospheric ozone measurements have been reported

I

li(

120

1

a

0

. 0

0

0

16

Environmental Chemistry C

0

b W -

30.

@ -

ref, 6 1 ref, 27 rd, 80

D-----J

. .. ...**

..'*O

q..''o O''*.O,.,O...*

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South

Figure 6 Latitudinal profiles of ozone mixing ratio at various heights in the troposphere: (a) boundary layer, ( 6 ) middle troposphere 5.5 km, ( c ) upper troposphere

-

rhe Southern hemisphere. I t is evident from Figure 5 that global coverage of tropospheric ozone measurements is a long way from being complete especially for the marine environment. Figures 6a, b, and c show the latitudinal distribution of ozone in the boundary layer, the middle troposphere and the high troposphere,

Tropospheric Ozone

17

as reported by several workers. The main feature of Figure 6a is the asymmetry of the profiles. The greater concentrations of ozone in the Northern hemisphere are possibly due to higher anthropogenic emissions of precursor pollutants. In the Northern hemisphere the annual mean concentration of ozone in the lower troposphere is about 30 p.p.b. and mean concentrations of 50,40, and 30 p.p.b. are expected in the Spring, Summer, and Autumn.14 Figures 6 b and c also exhibit a NortherdSouthern hemisphere asymmetry, with the integrated ozone burden approximately 1-3 times greater in the Northern hemisphere than in the Southern hemisphere.80 Minimum ozone concentrations are found around the equator with maximum levels at mid- or high latitudes. The Spring maximum in mid-latitudes occurs first in the upper troposphere and then propagates down to the boundary layer. The minimum ozone levels in the latitude range 10°N to 10's coincide with the ascending branch of the Hadley cell. The asymmetry in the concentrations is mainly due to the stratospheric ozone flux. This flux is large and downward at about 35'N and 70°N in the Northern hemisphere winter. However, in the Southern hemisphere winter the main downward flux is at 35'S, with a weak upward flux poleward of this. Within the tropics, the ozone concentration follows the configuration of the Hadley cell. When comparing many measurements of the same parameter one must remember that all the measurements are different. This may be due to the large variety of instruments and techniques used, systematic errors, or natural variations. Some of the differences between the profiles in Figure 6 may be due t o these reasons. 5 Sinks of Ozone in the Unpolluted Troposphere

In addition to the chemical sinks for ozone in the clean troposphere, ozone is destroyed by contact with the earth's surface. Ozone, like several other gaseous pollutants, exhibits diurnal variations in concentration. The observed nocturnal depletion is thought to be due to the destruction of the pollutant a t the ground. When a nocturnal radiation inversion forms, due t o cooling of the earth's surface, the ozone removed cannot be readily replenished by that above since the inversion acts as an effective barrier between the convectively stable layer near the ground and the air aloft, thus resulting in a nocturnal depletion of ozone when the influence of the ground is greatest, and a concentration maximum around midday when convective mixing is strongest, This diurnal variation of ozone levels is not observed at many maritime and mountain sites due t o the land-sea breezes1 and the katabatic winds,82 respectively, and, like the other sites,when the meteorological conditions are unfavourable for a radiation inversion to form. The direct absorption/destruction of a gas at the ground surface is called dry deposition. This distinguishes it from removal by precipitation, although water surfaces as well as vegetation and soil may participate. The idea of a deposition , ~a~means of velocity, vg, was first proposed by Chamberlain and C h a d ~ i c k as

''

C. L. Parsons and M. E. Williams, J. Geophys. Res., 1979,84, 7863.

83

B. Broder, Pageoph, 1981, 119,978. A. C. Chamberlain and R. C. Chadwick, Nucleonics, 1953, 11, 22.

18

Environmental Chemistry

explaining the loss of material from the atmosphere t o the underlying surface when the material was too small in size t o be described adequately by Stoke's law sedimentation. In studies of dry deposition of ozone and other pollutants from the atmosphere t o a surface the rate of transfer, or deposition velocity is often expressed by:

where F is the downward flux of the pollutant (pgcm-2s-') and C is the concentration of the pollutant (pg ~ m - ~at ) height z, giving the deposition velocity in units of cm s-l. The deposition velocity varies over different types of terrain and vegetation and because of its dependence on concentration its value will depend on the height at which the concentration is measured. Regener 84 described the conditions which governed the destruction of ozone at the earth's surface. Except when the air is heavily laden with aerosol, he suggested that all the ozone destruction takes place at the surface. The first direct measurements of ozone deposition velocity were made by A l d a ~ who ,~~ found values of 0.02 cm s-' over snow, 0.04 cm s-l over water, and 0.6 cm s-l over land. Since these first measurements, the deposition velocity has been determined by many workers over several types of Table 1 shows the values of deposition velocities as found by various workers. The place, height, and type of surface over which the measurements were made are included. The results are difficult to summarize because of the many factors affecting the estimates : experimental method, reference height, time of day, type of surface, and atmospheric conditions, The best estimates of deposition velocity appear to be in the range of 0.02 to 0.1 cm s-l over snow and water surfaces and 0.3 to 1.7 cm s-' over grass. McMahon and Denkongl report a mean deposition velocity of 0.6 cm s-l for diabatic conditions based on the average of six independent determinations and a value of 1.1 cm s-l for neutral conditions. The diurnal variation in deposition velocity is probably due t o one or a combination of the following: variations of soil moisture at night, changes in soil surface temperature, and the closure of plant stomata at night. During the daytime the leaf stomata of the vegetation open t o permit exchange of water vapour, carbon dioxide, and oxygen between the atmosphere and the leaves. Ozone can then be destroyed at inner leaf surfaces by rapid reactions but at night its pathway t o the inner surfaces is c l 0 s e d . ~ 9 ~ ~ 84

V . H . Regener, J. Geophys. Res., 1957,62,221. L. Aldaz, J. Geophys. Res., 1969,74,6943. I. E. Galbally, Quart. J. R . Meteorol. SOC., 1971,97, 18. J. A. Garland and R. G. Derwent, Quarr. J. R . Meteorol. SOC., 1979,105,169. J. A. Garland, A. W. Elzerman, and S. A. Penkett, J. Geophys. Res., 1980,85, 7488. M. L. Wesely, D. R. Cook, and R. M. Williams, Boundary-Layer Meteorol., 1981, 20,

459. E . Robinson, B. Lamb, and M. P. Chockalingham, EPA/600/3-82-042, 1982. '' T.A. McMahon and P. J. Denison, Amos. Environ., 1979,13,571. 9 2 M. L. Wesely, J . A. Eastman, D. R. Cook, and B. B. Hicks, Boundary-Layer Meteorol., 1978,15,361. 93 J . A. Eastman, M. L. Wesely, and D. H. Stedman, Proc. Internat. Ozone Symp., Boulder, Colorado, 1980.

90

Tropospheric Ozone

19

Table 1 Deposition velocity A uthor

Location

Neightjsurface

Deposition vebcityjcm s-’

Robinson e t al. 90

Illinois

1 mabove soyabean 1 m above grain/grass 1 m above forest snow baresoil lake water 2 m above grass water snow 1 5 m above grass 1 m above

0.6 day 0.17 night 0.6 day 0.1 7 night 1.o

Washington Pennsylvania Wesely e t akE4

Illinois

Galbally and Royb

Australia

Lenschow e t al.loS

Colorado

Garland and Derwent 7 9

Harwell, U.K.

grass

Wesely et al. 9 2 Van DO^ e t al.a

Illinois Indiana Cabauw, Netherlands

maize maize > 5 m, grass

0.03 0.1 0.01 0.6 day 0.24 night 0.05 0.06 0.12

0.58 day 0.29 night

0.2-0.7 0.2-0.6

0.13

‘H. Van Dop, R. Guichert, and R. W.Lanting, Atrnos. Environ., 1977,11,65;bI.E.Galbally and C. R. Roy, Proc. Int. Ozone Symp., Boulder, Colorado, 1980.

The deposition velocities over ‘wet’ surfaces are much smaller than those over dry surfaces. The main factor governing this difference is the chemical nature of the surface. The presence of water at the surface strongly retards ozone removal, possibly due to the small solubility and reactivity of ozone in clean water. In seawater, the presence of iodide makes a substantial contribution to the deposition of ozone. However, an unidentified additional reaction is required to explain fully the deposition rate.88

6 Tropospheric Ozone Budget The main problem in calculating the tropospheric ozone budget is estimating the magnitude of the flux from the stratosphere into the troposphere and the overall rate of destruction at the earth’s surface. The stratosphere-troposphere flux, photochemical production or destruction and mean residence time and transport within the troposphere should be, in total, equal t o the ozone deposition flux in the unpolluted troposphere. A number of estimates of the annual flux of stratospheric ozone into the troposphere are available. Using the stratospheric inventory of 90Sr, Danielsen 30 and Reitera estimate that 70 to 80% of the Northern hemisphere stratospheric

20

Environmental Chemistry

air mass is exchanged annually with the Northern hemisphere troposphere. Reiter assumes that 20% of the mass exchanged is due to tropopause folding, 43%to mean meridional circulation, and 16% to stratospheric exchange between the hemispheres. On the other hand, Danielsen assumes that all the exchange is due to tropopause folding events. Singh et al. l4 estimate that tropopause folding accounts for 60-80% and mean meridional circulation 20-40% of the mass exchanged. Their argument is based on the fact that mean meridional circulation processes are concentrated in the tropics where stratospheric ozone concentrations are small. Danielsen and M ~ h n e and n ~ ~Mohnen and Reiterg4 obtained average ozone fluxes of 0.74 x lou and 0.88 X 10" molecules cm-2 s-l, respectively. They analysed the number of tropopause folding events and estimated the mass of stratospheric air entering the troposphere for each event in the Northern hemisphere per year by assuming an average mixing ratio. Nastrom75 calculated an average ozone flux of 0.78 x lo1' molecules cm-2 s-l by using a mean vertical velocity of 0.5 cm s-l and the average ozone mixing ratio as measured by aircraft flights, Fabian and Pruchniewicz16 divided the annual mean of tropospheric ozone by mean residence time t o compute mean injection fluxes for various latitude belts. The average fluxes varied between 3 X lo1' molecules cm-2 s-l at the equator and 10 x 1 O l o molecules cm-2 s-' at the main injection latitudes (30'N, 45'N, and 65'N). An annual flux of 5 to 8 x 10'O molecules cm-2 s-l was estimated by Singh et based on the uncertainties in the assumption of Danielsen= and Reiter.& Gidel and S h a p i r ~estimated ~~ the vertical flux of ozone in the lower troposphere to be 0.49 x 10l1 and 0.25 x 10'l molecules cm-2 sL1 for the Northern and Southern hemispheres, respectively. These fluxes were obtained from a general circulation model. General circulation models were also used by Cunnold et a1.96 and Mahlman ef aLg7 to calculate the vertical flux of ozone. Cunnold obtained a total global flux of 6.6 X 1O'O molecules cm-2 s-l, while Mahlman found fluxes of 4.8 x lolo, 2.6 X lo'', and 3.7 X 1O1O molecules cm-2 s-l for Northern hemisphere, Southern hemisphere, and the global total respectively. Prior to these estimates, Dutsch in 1946 estimated the amount of ozone leavin the lower stratosphere t o be 7.1, 0.26, and 0.04 x 1O1O molecules cm-2 s-l at 0 , 45', and 80' latitude; Paetzold9* estimated the flux to be between 4 and 8 x 1O'O molecules cm-* s-l, and Jungeg9 obtained the value of 7.5 x 1O1O molecules cmP2s-1. The ozone fluxes presented here are listed in Table 2, All these estimates are of the same order of magnitude and the annual flux of ozone from the stratosphere to the troposphere s e e m to be in the range of 3-10 x 1O'O molecules cm-2 s-l. The vertical flux of ozone to the earth's surface has been calculated by many workers and their estimates are listed in Table 3. Many of the measure-

V. A. Mohnen and E. R . Reiter, Int. Conf. on Oxidants - Analysis of Evidence and Viewpoints, 111, EPS-600/3-77-115, U.S. EPA, Washington D.C., 1977. 9 5 L. T. Gidel and M. A. Shapiro, J. Geophys Res., 1 9 8 0 , 8 5 , 4 0 4 9 . '' D. Cunnold, F. Alyea, N. Phillips, and R . Prinn, J. Atmos. Sci., 1975, 32, 170. 9 7 J. D. Mahlman, H. Levy, and W. J . M o x h , J. Atmos. Sci., 1980, 37,655. 9 8 H. K. Paetzold, J. Atmos. Ten. Phys., 1955, 7 , 128. 99 C. E. Junge, Tellus, 1962, 14, 363. 94

21

Tropospheric Ozone

Table 2 E stirnated stratosphere -trop osp here flux A uthor

Flux (X 10” molecules cm-2 s-’)

Danielsen and Mohnen3’ Mohnen and Reiter94 Nastrom” Fabian and Pruchniewicz16

0.74 0.88 0.78 0.3 (equator) 1 .O (30°,45”, 65’N) 0.5-0.8 0.49 (Northern hemisphere) 0.25 (Southern hemisphere) 0.66 0.48 (Northern hemisphere) 0.26 (Southern hemisphere)

Singh et al. l 4 Gidel and Shapiro” Cunnold et al. 9 6 Mahlman et al. 9 7

ments84,86,100-102used the profile method to determine the ozone flux, which equates the flux with the product of the ozone concentration gradient and the associated eddy diffusivity, while others have measured the ozone flux for of an open-bottomed different surfaces by the box m e t h ~ d . ~lo4~ This , ~ ~consists , box covered -inside with Mylar film. Ozone is injected into the box and then the rate of decay of the partial density of ozone is measured. The rate of decrease of ozone within the system is equal to its destruction rate at the surface being studied. Measurements by this method are useful, but do not represent conditions that actually occur in nature. Since the development of sensitive fast response ozone detectors, direct measurements of the ozone flux by eddy correlation techniques are now possible. The vertical flux, F , is determined by computing the covariance between the vertical wind speed, w ,and the ozone concentration, c, such that F = - c v , where the primes indicate deviations from the average value and the bar represents a temporal average. This method has been employed by Wesely et aZ. 89,92 for ground-based measurements and by Lenschow et aZ.105-107 for measurements from an aircraft. The values for the deposition fluxes in Table 3 show a wide variation, but it must be remembered that the measurements were made using different techniques, over various types of surfaces, different times of the year and places on theearth. The vast majority of the measurements were made in the Northern hemisphere but this has not prevented various estimates of the global surface deposition of ozone from being derived. These are given in Table 4, together with the hemispheric ratio of ozone destruction at the earth’s surface. The differences are J. J. Kellyand J . D. McTaggart-Cowan, J. Geophys. Res., 1968, 73, 3328. I. E. Galbally, Nature, 1968,218,456. lo2 I. E. Galbally and I. Allison, J. Geophys. Res., 1972, 77, 3946. l o 3 V. H. Regener and L. Aldaz, J. Geophys. Res., 1969, 74,6935. I. E. Galbally and C. R. Roy, Quart. J. R. Meteorol. SOC.,1980,106, 599. D. H. Lenschow, A. C. Delany, B. B. Stankov, and D. H. Stedman, Boundary-Layer Meteorol., 1980,19, 249. D. H. Lenschow, R. Pearson, and B. B. Stankov, J. Geophys. Res., 1981, 86, 7291. D. H . Lenschow, R. Pearson, and B. B. Stankov, J. Geophys. Res., 1982, 87, 8833. loo

E n viro n m en tal Chemistry

22

Table 3 Verticalflux of ozone t o the earth’s surface A uthor

Location

Lettau‘ Paetzold” Regenera4 b Kroening and Ney Kelly and McTaggartCowan A1daza5

estimate estimate Nebraska Minnesota Alaska New Mexico

Tiwari and Sreedharand Turner e t aLe

Atlantic Ocean New Mexico estimate Hay, Australia Edithvale, Australia Mt. Baller, Australia Mawson, Antarctica Poona, India Connecticut

Van ~ o etpa1.f Eastman and S t e d m a g

Cabauw, Netherlands Illinois

Wesely et al. 9 2 Leuning e t al. Eastman e t al. 93

Illinois Ontario Illinois Pennsylvania Switzerland

Galbally and Allison lo’

Lenschow e t al. Io6 Lenschow e t al. I o 7 Wesely e t al. 8 9

grass tundra

Flux (X10” molecules c m - 2s - ’ )

0.04 0.4 0.87-2.5 6.0 -8.7-7.3

loo

Regener and Aldazlo3 Fabian and JungeC Galballya6,”*

Broder et aLi

Surface

Colorado Gulf of Mexico Texas Illinois Lake Michigan

sand or dry grass fresh water salt water bare soil dry soil and short grass snow soil maize bare soil grass

grass maize maize tobacco maize soyabean mountainous terrain farmland salt water forest snow bare soil water

3 .O 0.3 0.2 1.7-3.8 0.45-2.0 0.1-5.0 0.5-6.3 - 2.0-1 0.0 4.02-4.83 6-34 8-1 8 1.6-3.7 5 1-40 0.5-14 2.5-14.7 2.5-10 up t o 19 u p to 40 14 k 2 2-5 0.6-1.2 20 0.28-1.13 0.56-3.72 0.03 0.3 9

-

a H. Lettau, Compendium of Meteorology, 3 20-340, American Meteorological Society, Boston, Mass., 1951; bJ. L. Kroening and E. P. Ney, J. Ge0ph.w. Res., 1962, 67, 1867; ‘P. Fabian and C. E. Junge, Archiv. Met. Geoph. Bioklim., Ser. A., 1970, 19, 161; dV. S . Tiwari and C. R. Sreedharan,Pageoph, 1973, 106-108, 1124; eN. C. Turner, P. E. Waggoner, and S. Rich, Nature, 1974, 250,486;fH. Van Dop, R. Guichert, and R. W. Lanting, Atrnos. Environ., 1977, 11, 6 5 ; g J . A. Eastman and D. H. Stedman, ibid., 1977, 1 1 , 1 2 0 9 ; h R . Leuning, M. H. Unsworth, H. N. Neumann, and K. M. King, ibid., 1979,13,115 5 ; B. Broder, H. U. Dutsch, and W. Graber, ibid., 1981, 15,1195.

Tropospheric Ozone

23

Table 4 Estimates of global deposition fluxes Flux (Xl0"molecules cm:'s-') c

A u thor

Global

J ~ n g e ~ ~ 0.96-1.1 5 Aldaz ' 2.1-3.3 Fabian and Jungea 0.69-1.1 5 Fabian and Pruchniewiczb 1.1 Fishman and C r ~ t z e n ~ ~ 1.64 Galbally and RoyIo4 0.77-2.3

Northern hemisphere

Southern hemisphere

Interhemispheric ratio

1.56-2.19 0.44-0.76 0.73 1.22 1 .o

0.57-1.20 0.25-0.38 0.3 7 0.42 0.67

1:2 1 :2 1 :1.5 1 :2.9 1: 1.5

aP. Fabian and C. E. Junge, Archiv. Met. Geoph. Bioklirn., Ser. A., 1970, 19,161;bP. Fabian and P. G. Pruchniewicz, Report MPAE-W-100-76-21, Max-Planck Institiit fur Aeron., Katlenburg-Lindau, W. Germany, 1976.

mainly due to the fact that the authors have used different deposition velocities and in their characterization of global surfaces. Because of the asymmetry in land mass and because ozone destruction is greater over land surfaces than over oceans the ozone destruction in the Northern hemisphere is greater than that in the Southern hemisphere. The ratio of ozone destruction at the surface between the two hemispheres is given in Table 4. Again the main differences can be explained by the use of different deposition velocities. Only the calculation of Galbally and Roy104takes account of the diurnal decrease in turbulent transfer and ozone concentration in the boundary layer as well as any change in the surface resistance at night. From the global values of the fluxes in Tables 2 and 4 it is clear that there is little room in the ozone budget for a large net source or sink from tropospheric photochemistry. However, when the fluxes in each hemisphere are compared it is evident that the stratosphere-troposphere flux is much larger in the North than in the South, but both are smaller than the deposition fluxes in the respective hemispheres. This tends to support the theory of photochemical production in the troposphere, with a possible substantial source of ozone in the Northern hemisphere troposphere. The mean residence time of ozone in the troposphere is in the range of 1 to 3 months, Junge108 found a residence time of 1.1 to 2 months while Fabian and Pruchniewicz16 derive tropospheric residence times for 30° latitude intervals. These range from 42 days for 6O0-90'N to 88 days for 30°-600S. If the latitude interva1.s are weighted according to area, residence times of 62 f 14 daysand 8 3 2 18 days are obtained for the Northern and Southern hemispheres, respectively, the differences being due to the asymmetry in land mass distribution, deposition over land is larger than that over ocean. Singh et al. l4 give a best estimate of the mean residence time of 25 to 3 5 days, lo'

C. E. Junge, 'Air Chemistry and Radioactivity', Academic Press, New York, 1963.

Environmental Chemistry The combination of destruction rate, tropospheric content and tropospheric life-time should be consistent. However, there are large uncertainties in the values of the fluxes due to stratosphere-troposphere exchange mechanisms and deposition at the earth’s surface, especially in the hemispheric distribution of them, as well as uncertainties in the mean residence time of ozone in the troposphere. Until further research is carried out, it is impossible to give exact values to the amount of tropospheric ozone of stratospheric origin and that formed by tropospheric photochemistry. 24

7 Ozone Formation and Destruction in Polluted Air

In the early 1950’s it became clear that, in the polluted air of a number of large cities under specific meteorological conditions, ozone was produced by a complex reaction system involving various trace chemical species. Since the first photochemical smogs occurred in Los Angeles in the 1940’s and caused irritation of the eyes and respiratory system of the inhabitants, this form of pollution has been observed in many large cities in different parts of the world.109-112 In addition to the formation of ozone, air pollution in the form of oxides of nitrogen and hydrocarbons gives rise to the formation of other secondary air pollutants such as aldehydes, peroxyacetyl nitrate (PAN),113 and visibilityreducing aerosol^.^^^^^^^ I t has been shown that under the influence of ultraviolet sunlight low concentrations of nitrogen dioxide and certain organic compounds mixed in air produces ozone116s117and that a similar situation occurs when emissions of automobile exhaust are irradiated.l18 Intensified research since the 1950’s has led to the elucidation of the kinetics of reactions and mechanistic pathways in photochemical air pollution formation. Ozone is formed in polluted air as a result of photolysis of nitrogen dioxide, and its concentration is determined by the three rapid reactions (2), (16),and (20). As shown earlier in a photostationary state the following relationship holds:50 ki6 “ 0 2 1 [031

=

k2o “01

Hence the ozone concentration is dependent on the ratio of nitrogen dioxide t o nitric oxide concentration and the value of k16, highest when the insolation is at its peak value. This relationship has been tested using both ambient air datallg D. J . Ball, Nature, 1976,263,580. J. Schjoldager,J. Air Pollut. Control Assoc., 1981,31, 1187. ‘11 A. Cecinato, M.Possanzini, A. Liberti, and D. Brocco, Sci. Total Environ., 1979,13, 1. 112 J . S. Jacobson and G. D. Salottolo, Atmos. Environ., 1975,9,321. 113 H. Nieboer and J . Van Ham, Atmus. Environ., 1976,10, 115. 1 1 4 J. E Lovelock,Atmos. Enviton., 1972,6,917. ‘ I s D. H.F. Atkins, R . A. Cox, and A. E. J . Eggleton, Nature, 1972,235,372. ‘16 A. J. Haagen-Smit,Ind. Eng. Chem., 1952,44,1352. 11’ A. J. Haagen-Smit, C. E. Bradley, and M. M. Fox, Ind. Eng. Chem., 1953,45,2086. ‘ I 8 A. J. Haagen-Smit and M. M. Fox, Ind. Eng. Chem., 1956,48, 1485. ‘I9 J. G.Calvert, Environ. Sci. Technol., 1976,10,256. ‘09

‘lo

Tropospheric Ozone

25

and smog-chamber data120 which support reactions (2) and (16) as being the only significant sources of ozone in the ambient air. These two reactions cannot alone explain the build-up of ozone, since any ozone formed is destroyed by the rapid oxidation of nitric oxide by ozone, reaction (20). I t is now recognized that N O must be oxidized t o NO2 by a mechanism other than reaction with ozone and that peroxy radicals, formed in air masses polluted with hydrocarbons and CO, can fulfil this role: HO2 4- NO

-+

NO2 4-OH

(14)

RO2 4- NO + NO2 4-RO

(22)

R C ( 0 ) 0 2 4- NO

(23)

-+

NO2 4-RC02

Dermerjian et aZ.121 showed that peroxy radicals are formed by OH attack on methane, n-butane, ethylene, and propylene while Calvert119 demonstrated that hydrocarbon degradation is driven by OH radicals. The concentrations of the HO2 and R 0 2 radicals are dependent upon solar intensity, resulting in peak concentrations around midday within an urban area. 122 An important source of the hydroperoxy radical is the reaction of an alkyloxy radical with molecular oxygen C H 3 0 4- 0

2 + H02

4- CH2O

(12)

The subsequent NO 4- H 0 2 reaction produces another molecule of ozone. Hydroperoxy and alkylperoxy radicals are also formed as a result of attack by oxidizing species on hydrocarbons. The hydroxyl radical produced in reaction (15 ) reacts rapidly with certain hydrocarbons, especially alkenes, to produce, after reaction with molecular oxygen, alkylperoxy radicals RH 4- OH + H2O 4-R

R 4- 0

2

--f

(24)

RO2

For this reason the rate constants for hydroxyl radical reaction with hydrocarbons have been used as an indication of the photochemical smog formation The basis of photochemical ozone potential of specific hydrocarbons. formation is the interaction of free radicals generated in polluted air masses with the NO-N02-03 cycle. Apart from undergoing photolysis to nitric oxide and atomic oxygen [reaction (16)], nitrogen dioxide may also be oxidized to compounds including PAN, alkyl nitrates, nitric acid, and particulate nitrate salts. Gas-phase reactions are the most important sinks for ozone in the urban atmosphere because it is likely that they are more rapid than destruction at the 1239124

120

123

124

.

R J. O’Brien, Environ. Sci. Technol., 1974,8, 579. K. L. Dermerjian, J. A. Kerr, and J. G. Calvert, d d v . Environ. Sci. Technol., 1974, 4, 1. T. E. Graedel, L. A. Farrow, and T. A. Weber, Atmos. Environ., 1976, 10,1095. K. R. Darnall, A. C. Lloyd, A. M. Winer, and J. N. Pitts, Environ. Sci. Technol., 1976,

10,692. H. B. Singh, J. R. Martinez, D. G. Hendry, R. J. Jaffee, and W. B. Johnson, Environ. Sci. Technol., 1981, 15,113.

26

E nviro nmental Chemistry

ground by dry deposition. The major sink mechanism for ozone in the urban atmosphere is its rapid oxidation of nitric oxide [reaction ( 2 0 ) ] . Part of the nitrogen dioxide formed by this reaction undergoes further reactions to give nitric and nitrous acid. Precipitation removes the nitric acid,,while nitrous acid can be photodissociated to give nitric oxide. In certain conditions hydrocarbons can also act as sinks for ozone, although the rate of these reactions are significantly slower than the reaction with nitric oxide. The required conditions for these reactions to become important are high ozone concentrations and low nitric oxide concentrations such as may exist during the afternoon in urban

Table 5 Reactivity scale for atmospheric hydrocarbons based on reaction with hydroxyl radicals123 Compound

Rate constant p.p.m.-' min-'

Methane Ethyne Ethane Benzene Propane n-Butane Isopentane Butan-2-one 2-Methylpentane Toluene n-Pro p ylben zene isoPropyl benzene Ethene n-Hexane 3-Methylpentane Ethylbenzene p-Xylene p-Ethyltoluene o-Ethyltoluene o-Xylene 4-methyl pentan-2-one rn -Ethyl toluene m-Xylene 1,2,3-Trhethylbenzene Propene 1,2,4-Trhethylbenzene 1,3,5-Trhethylbenzene cis-2-Butene 6-Pinene 1,3-Butadiene 2-Methyl-2-butene 2,3-Dimethyl-2-butene d-Limonene

1.2 x 10 2.0 x l o 2 2.7 X lo2 3.9 x lo= 3.2 x 1 0 3 4.4 x lo3 4.9 x l o 3 5.1 x 1 0 3 7.8 x lo3 8.8 X lo3 9.1 x i o 3 9.1 x lo3 9.3 x103 9.3 x 103 1.1 x104 1.2 x lo4 1.8 x 1 0 4 1.9 x lo4 2.0 x lo4 2.1 x lo4 2.3 x lo4 2.6 x lo4

3.5 x104 3.7 x lo4 3.7 x 1 0 4 4.9 x lo4 7.3 x104 7.9 x 10'' 1.0 x 1 0 5

1.1 x i o 5

1.2 x l o 5 1.6 x 1 0 5 2.2 x lo5

Reactivity relarive to methane

1 23 33 180 270 375 420 440 6 70 750 770 7 70 790 790 900 1000

1530 1625 1710 1750 1920 2420 2920 3100 31 5 0 41 70 6190 6730 8750 9670 10000 14 000 18 800

Proposed class (see Table 6 ) I

I1 I1 I11 I11 111 111 111

I11 I11 I11 111

I11 111

111

111-IV IV IV IV IV IV IV IV IV IV IV IV IV

IV IV-v V V V

Tropospheric Ozone

27

Table 6 Reactivity scale for hydrocarbons based on rate of disappearance of hydrocarbon due to reaction with hydroxyl radicals (t% = 0.693/ko~OH)

Reactivity relative to Methane (= 1)

> 9.9 d

< 10

24 h-9.9 d 2.4-24 h 0.24-2.4 h < 0.24 h

10-100 100-1000 1000-10 000 > loo00

Half-life

Class

I I1 111

IV

V

areas. The rate constants for hydroxyl radical reaction with hydrocarbons have been used as an indication of the photochemical smog formation potential of specific hydrocarbons (see Tables 5 and 6).123 The processes of photochemical production and destruction which represent the overall behaviour of the polluted atmosphere can be simply represented by consecutive reactions: 125 Hydrocarbon

ti

secondary

OH + pollutants

A

sink processes

The time scale for secondary pollutant formation is represented by t 1 while the time scale for the loss processes is represented by t 2 . When the OH radical concentrations increase, tl is reduced so that it is shorter than t 2 . This occurs under certain favourable meteorological conditions which results in a build-up of secondary pollutants. Normally the time scale for production and destruction are comparable due to lower OH concentrations and this results in the secondary pollutant levels being lower. The chemistry of photochemical air pollution is clearly extremely complex. The mixture of organic compounds present in the polluted atmosphere is itself complex and these compounds react at different rates t o produce various amounts of secondary pollutants as a function of overall hydrocarbon composition and NO, concentration. Smog-chamber Studies. - Modern day computer techniques have made it possible t o model the photochemistry of smog formation, The modelling of chemical processes in the atmosphere is based on a set of photochemical reactions which reflects current knowledge. The main approach has been t o measure temporal concentration profiles under laboratory conditions in large reaction vessels known as smog chambers and then develop kinetic mechanisms t o simulate the results. Smog chambers have been constructed by several workers; early chambers were constructed indoors and operated at constant temperature, light intensity, and relative humidity. The chambers were used t o explore the photochemical smog phenomenon, to elucidate some of the mechanisms involved in smog 12’

R. G. Derwent and 0. HOV,AERE Report R9434, HMSO, London, 1979.

28

Environmental Chemistry

reactions, and t o derive relationships between photochemical oxidant and its precursors, hydrocarbons and nitrogen oxides."', 126-128 Smog chambers have to be constructed of a material that can transmit light of wavelength down to the lower solar spectral irradiance limit of 290 nm. Pyrex, Tedlar, Mylar, and FEP Teflon are materials with this property.129 Not only is FEP Teflon the most inert of these materials,130 it has also a high transmission for the U.V. and only a few absorption bands in the i.r. This allows photochemical reactions to proceed unhindered and also reduces any greenhouse effects inside the chamber. Early chamber results showed that a decrease in initial hydrocarbon concentration resulted in a proportional decrease in the rate of NOz formation from NO.13J Dimitriades128 found that the maximum NO2 concentrations were independent of non-methane hydrocarbons (NMHC). Indoor chambers are not necessarily representative of those in ambient air since constant light intensity, unrealistic spectral distribution, constant temperature, and relative humidity make it difficult to extrapolate results from the chambers to ambient air. In an attempt to overcome these difficulties Jeffries et al. constructed an outdoor chamber, situated at the University of North Carolina, U.S.A. The chamber consisted of Teflon film supported by a wooden 'A' frame with various mixing fans and manifolds entering the chamber through the floor. The chamber was separated into two halves of equal volume (156 m3) by a heat-sealed Teflon panel. Initial results showed that heterogeneous reactions were less significant than in smaller metal and glass chambers, that diurnal light intensity effects might be significant for oxidant control strategies and that under outdoor conditions at constant initial NO, concentration, lower initial NMHC concentrations resulted in lower maximum NOz concentrations and NO2 dosage during daylight hours.133 Kamens e t aZ.134 used the same chamber t o study a simulated hydrocarbon and NO, mix over 22 h. They found that reductions in hydrocarbons of 50-85% from an initial average concentration of 2.9 p.p.m.C. results in high night-time NO*; similarly low night-time NO2 was correlated to high afternoon oxidant. They concluded that low initial hydrocarbons, poor solar radiation, or a combination of both could result in high night-time NO2. The effect of dilute day-old smog on fresh smog was studied by Kamens et aZ.135in T. A. Hecht, J . H . Seinfeld, and M. C. Dodge, Environ. Sci. Technol., 1974,8, 327. B. Dimitriades, J. Air Pollut. Control Assoc., 1967, 17, 460. 1 2 8 B. Dimitriades, Environ. S ci. Technol., 1977, 11, 80. B. Dimitriades, Smog Chamber Conf. Proc., Report, EPA-600/3-76-029, U.S. EPA, Research Triangle Park, North Carolina, 1976. I3O R. J . Jaffe, Smog Chamber Conf. Proc., Report EPA-600/3-76-029, U.S. EPA, Research Triangle Park, North Carolina, 1976. 1 3 ' M. W. Korth, A. H. Rose, and R. C. Stahman, J. Air Pollut. Control ASSOC., 1964, 14, 168. 132 H . E. Jeffries, D. L. Fox,and R. M. Kamens, Environ. S c i Technol., 1976, 10,1006. 133 H . E. Jeffries, D. L. Fox, and R. M. Kamens, J. Air Pollut. Control Assoc., 1976, 26, 480. 1 3 4 R . M. Kamens, H. E. JeffGes, D. L. Fox, and L. Alexander, Ahnos. Environ., 1977, 11, 225. R. M. Kamens, H. E. Jeffries, I(. G . Sexton, and R. W. Wiener, Amos. Environ., 1982, 16,1027. 126

Tropospheric Ozone 29 the chamber at North Carolina. They hypothesized that 1 5 2 5 % of the increase in ozone, when fresh smog precursors plus dilute residuals from the previous day were compared with fresh systems, was due to residual first day aldehydes. The remaining increase resulted from an increased morning percentage of NO2 and the residual alkanes. Sickles et aZ.ls studied the generation of ozone in four outdoor smog chambers. The chambers consisted of FEP Teflon on aluminium frames, each with a volume of 27 m3. These chambers were at the Research Triangle Institute in North Carolina. Initial concentrations of 1-10 p.p.m.C. of non-methane hydrocarbon and 0.1-1 p.p.m. of oxides of nitrogen resulted in ozone levels in excess of 80 p.p.b. on the second and third day of the experiment. Matched experiments were conducted between the chambers of the University of North Carolina and the Research Triangle 1n~titute.l~' The sites of the chambers were 32 km apart so it was assumed that they experienced similar light intensity and temperature profiles. They found that both sets of chambers displayed similar chemical behaviour and overall agreement between the results was good. The National Institute for Environmental Studies, Japan, have constructed a reaction chamber of Teflon film, 4.28 m3 in volume, irradiated by fluorescent lamps. Studies showed that the maximum ozone formed from l-alkene-nitrogen oxide mixtures is in general proportional to the square root of both the initial concentration of NO, and the primary photodissociation rate of NO2 and thus is proportional to the photostationary state parameter [O3] ps. From this, it was proposed t o represent the ozone formation potential of each hydrocarbon by the ratio of maximum ozone to [O3] ps.138-141 Recently they proposed the use of the 'ozone formation rate' which represents the photochemical reactivity of hydrocarbon mixtures on the basis of ozone formation rate.142 The ozone formation rate for C3H6 in a NO,-dry air mixture was a linear function of the maximum [OH] and initial [ C ~ H S ] . Several experiments in a 17.3 m3 chamber have been conducted t o study nitrogen oxide reaction^.^^^*^‘'‘' The chamber was lined with Teflon, with fluorescent lamps for irradiation. The results of the study showed that the NO, transformation rate was dependent on hydrocarbon composition, relative humidity, and initial NMHC/NO, ratio. Also for an air parcel leaving a typical city with a NMHC/NO, ratio of 7.5, approximately 40% of the original NO, will be in the form of reaction products, and the PANhorganic nitrate ratio will be 0.3 by midafternoon. 136

J . E. Sickles, L. A. Ripperton, and W. C. Eaton, Internat. Conf. on Photochemical

Oxidant Pollution and its Control, EPA-600/3-77-001a, 1977. R. S. Wright, R. M. Kamens, J . E. Sickles, H. E. Jeffries,and W. C. Eaton, J. dirPoNut. Control ASSOC.,1978,20,248. 138 H . Akimoto, F. Sakamaki, M. Hoshino, G. Inoue, and M. Okuda, Enuiron. Sci Technol., 1979, 13,53. 139 F. Sakamaki, H. Akimoto, and M. Okuda, Environ. Sci. Technol., 1980,14,985. 140 F. Sakamaki, H. Akimoto, and M. Okuda, Enuiron. ScL Technol., 1981,15,665. K. Shibuya, T. Nagashima, S. Imai, and H. Akimoto, Enuiron. Scf. Technol., 1981, 15, 662. H. Akimoto and F. Sakamaki, Environ. Sci. Technol., 1983, 17,94. 143 C. W. Spicer, G. M. Sverdrup, and M. R. Kuhlman, d t m o s . Environ., 1981,15,2353. 144 C . W. Spicer, Environ. Sci. Technol., 1983,17,112. 13'

30

Environmental Chemistry

Although outdoor chambers were designed t o be more representative of the atmosphere than indoor chambers, extrapolation of results from any chamber t o ambient atmospheric conditions must be carried out with extreme caution. I t has been postulated that contamination of the chamber from the walls may affect the results. I t has been suggested that nitric acid forms on the walls of smog chamber^'^^,^^ and that the release of NO, species from the walls and their photolytic reaction gives reactive species.147Dimitriades et al. 148 recognized the full significance of residual contamination problems. Teflon film has been found to liberate high concentrations of contaminants such as fluorocarbons, solvents, antioxidants, and manufacturing residuals.149 These contaminants could interfere with both hydrocarbon analyses and reactivity studies. By heat treating the Teflon film at 190°C for 24 h Lonneman et were able to reduce the outgassing contamination. Despite this problem, smog-chamber studies have played an important role in the understanding of the mechanisms controlling photochemical ozone formation. Modelling of Photochemical Pollution. - Early research on photochemical smog showed that a great number of individual organic species and their intermediate radicals and products were involved. As our knowledge of the chemical kinetics of the atmosphere has improved, photochemical models have become more complex with in excess of 100 elementary reactions being involved for a single h y d r o c a r b ~ n . ' ~ Although ~-~~ modern day computer techniques have made it practical to solve complex systems of ordinary differential equations the full chemistry of photochemical pollutant formation is far too extensive to be incorporated into models. This problem is overcome by lumping several reactions together. Some models are based on specific surrogate hydrocarbon ~ h e m i s t r i e s , ' ~ ~while ~'~~ in, ~ others, ~ ~ the general features of the hydrocarbon chemistry are r e p r e ~ e n t e d . ' ~ ~The * ~model ~ ~ - ~of ~ ~McRae et aZ.160 provides a B. W. Gay and J. J. Bufalini, Environ, Sci. Technol., 1971, 5,422. C. W. Spicer and D. F. Miller, J. Air Pollut. Control Assoc., 1976, 26,45. 147 J . J. Bufalini, S. L. Kopczynski, and M. C. Dodge, Environ. Lett., 1972, 3 , 101. 14' B. Dimitriades, M. C. Dodge, J . J. Bufalini, K. L. Dermerjian, and A. P. Altshuller, Environ. Sci. Technol., 1976, 10, 934. 14' J.J. Bufalini, T. A. Walter, and M. M. Bufalini Environ. Sci. Technol., 1977, 11, 1181. I 5 0 W. A. Lonneman, J. J. Bufalini, R. L. Kuntz, and S. A. Meeks, Environ, Sci. Technol., 1981, 15,99. l S 1 D. G. Hendry, A. C. Baldwin, J . R. Barker, and D. M. Golden, Report EPA-600/3-78059,1978. A. H. Falls and J. H. Seinfeld, Environ. Sci. Technol., 1978, 12, 1398. l S 3 R. Atkinson, W. P. L. Carter, K. R. Darnall, A. M. Winer, and J. N. Pitts,Int. J , Chem. Kinet., 1980,12, 779. R. Atkinson, A. C. Lloyd, and L. Winges, Atmos. Environ., 1982, 16, 1341. "'A. C. Baldwin, J . R. Barker, D. M. Golden, and D. G. Hendry, J. Phys. Chem., 1977, 81,2483. 156 W. P. Carter, A. C. Lloyd, J . L. Sprung, and J. N. Pitts, Int. J. Chem. Kinet., 1979, 11, 45. l S 7 R. J. Gelinas and P. D. Skewes-Cox, J. Phys. Chem., 1977, 81, 2468. A. H. Falls, G. J. McRae,and J. H. Seinfeld, Int. J. Chem. Kinet., 1979,11, 1137. l S 9 N. T. Wakelyn and G. L. Gregory, Environ. Sci. Technol., 1980,14,1006. 160 G. J . McRae, W. R. Goodin, and J. H. Seinfeld, Atmos. Environ., 1982,16,679. 14' 14'

Tropospheric Ozone 31 system for modelling urban air pollution, including three-dimensional, grid-based as well as Lagrangian trajectory models, Derwent and Hovl% developed a photochemical model to study the formation of photochemical air pollution in the United Kingdom. They used a simple box model of horizontal dimensions 32 x 50 km and initially a fixed mixing height of 1 km, then with a variable vertical dimension up to the mean inversion height, Altogether, 240 chemical reactions and 41 photochemical reactions were employed to describe the formation of ozone, PAN, and the aerosol species, The results from the ‘complete urban model’, which allowed for vertical mixing and advection of upwind air, showed good agreement with observations in central London during summer periods. It also showed that oxidant* generation in the U.K. is not restricted to urban areas. An urban plume model, whereby an air parcel moves westwards over London with a mean wind speed of 2.4 m s-l, gave similar concentrations of ozone and PAN as those in photochemical episodes. Derwent and Hov161 assume a mixing height of 1300 m for the box model t o calculate the impact of vehicle exhaust emission controls on photochemical air pollution formation in the U.K. Again, the model gave realistic levels of ozone, PAN, peroxypropionyl nitrate (PPN), and aerosol during photochemical episodes. I t was found that with increasing reductions of hydrocarbon emissions secondary pollutant concentrations declined, whereas with increasing reductions of NO, emissions the concentrations of the secondary pollutants began to increase. Thus hydrocarbon control appears to provide the best method of reducing secondary pollution formation in London. Hov and his co-workers in Norway have developed a model to study the chemical evolution and long-range transport of tropospheric 0 z 0 n e . ~ Their ~~-~~~ results showed that ozone stays at high concentrations for several days, allowing for transport over long distances (around 1000 km, depending on wind speed). One of the situations investigated was similar to that when elevated ozone levels occur in Britain. Here, an air mass is exposed to continuous emissions of hydrocarbons and NO, for 4 days at rates typical of continental sources. The air mass then moves over the sea for several days and is subsequently exposed t o a 3 h injection of hydrocarbons and NO, at a rate typical of urban areas. The full effect of these pollutants appears the following day. The predicted values agreed well with an episode reported by Cox.166 All model results are dependent on the initial conditions. They also contain aspects of uncertainty in unknown rate constants, in the importance of competing *The term ‘oxidant’ arises from the use of oxidation of potassium iodide as an analytical method for atmospheric ozone. Oxidant includes not only ozone, but also peroxyacylnitrates which quantitatively oxidize KI, and NO, to which KI has an approximately 10% stoicheiometric response. R. G. Derwent and 0. HOV,Enuiron. Sci. Techno!., 1980,14,1360. E. Hesstvedt, 0. HOV, and I. S. A. Isaksen, Geophys. Norvegicu, 1977,31, 27. E. Hesstvedt, 0. Hov, and I. S. A. Isaksen, Int. J. Chem. Kine?., 1978,10,971. 164 I. S. A. Isaksen, K. H. Midtbo, J . Sunde, and P. J. CrutZen, Geophys. Noruegicu, 1977, 31, 11. 16’ 0 . Hov, E. Hesstvedt, and I. S. A. Isaksen, Nature, 1978,273, 341. 166 R . A. Cox, Tellus, 1977,29,356. 16’

162

32

Environmental Chemistry

Figure 7 The typical diurnal variation of ozone (m), nitrogen dioxide (*) in an urban area

nitric acid (A), and

(Reproduced by permission from Adv. Environ. Sci. Technol., 1976, 7 , 7 5 )

reaction paths and in the methods of lumping reactions together. Hence considerable uncertainties still associate with the results of such models, although great progress has been made in modelling in recent years. Urban Ozone. - As noted before, anthropogenic sources of ozone dominate in urban areas. Emissions of nitric oxide and hydrocarbons are high in the early morning, as a result of the morning rush hour. These emissions will destroy oxidant present, whether it be of natural origin or left from the previous day, during this time. This is followed by an increase in nitrogen dioxide concentrations, initially slow since a t this level of concentration the reaction of NO with molecular oxygen is extremely slow. The oxidation of nitric oxide becomes rapid once sufficient quantities of ozone have been formed, Photochemical activity will be most intense around midday giving a maximum ozone concentration. Concentrations fall as the solar intensity diminishes and fresh emissions of nitric oxide rapidly deplete the ozone in the air, leading to the diurnal variaton of ozone as shown in Figure 7 . Often ozone produced during the day is trapped aloft by a nocturnal radiation inversion formed during night hours. If vertical mixing occurs this ozone can be mixed to ground level. This generally occurs due to solar warming in the early morning but may occasionally, for other reasons, occur during the night t o give elevated ozone concentrations. Not all high urban ozone levels are the result of local photochemical processes. In order to estimate the natural ozone component in urban air, measurements

Tropospheric Ozone 33 are made in rural areas and from these the natural ozone concentrations are estimated. For a typical natural rural concentration of 40 p.p.b. in the U.S.A. it has been estimated that urban concentrations will have 20 p.p.b. of natural 0 2 0 n e . l ~Coffey ~ and Stasiuk168found evidence of high ozone concentrations in rural areas of New York State being transported into urban areas through advection and vertical mixing. Such transport was suggested by Frankhauser 169 as a possible source of elevated ozone in some cities in the U.S.A. Further evidence of long distance transport of ozone into urban areas was provided by Chan et aZ.170 who measured ozone aloft and at the surface upwind of Philadelphia. By relating ozone aloft t o surface measurements1" they found the ozone transmeasured surface port ranged from a few p.p.b. t o 123 p.p.b. Freas et concentrations of ozone in excess of 80 p.p.b. upwind of Philadelphia, Washington, Pittsburgh, and Cleveland. The measurements, made between 1100 to 1300 LDT, included hydrocarbons and oxides of nitrogen. The concentration of the hydrocarbons ranged from 0.05-0.3p.p.m.C. and those of nitrogen oxide from
34

Environmental Chemistry

air is continuously advected from an urban area and ozone formation may continue in the resultant urban plume which still contains oxides of nitrogen and hydrocarbons. Dimitriades et al. have shown that the hydrocarbon-NOx ratio increases downwind of an urban area, while lsaksen et al.i74 showed that the higher this ratio the more effective the ozone production becomes with 5-9 ozone molecules produced for each NO, molecule released during the first day of transport. This is due, initially, to the large amounts of nitrogen oxides which suppress the build-up of peroxyradicals, and the consequent reduction in the number of NO t o NO2 conversions that can take place before the mixture is chemically deactivated. When all the ozone precursors have been consumed, the ozone levels will remain elevated as the chemical sinks have been removed until a substantial amount of dilution occurs through mixing with other air masses. Once chemical deactivation has occurred, the ozone-rich air may be trapped aloft at nigh t-time between an anticyclonic subsidence inversion and a surfacebased radiation inversion. if these inversions form, then this ozone reservoir above the radiation inversion is effectively cut off from all sinks, although Harrison et ~ 1 . have l ~ ~ shown that the concentration of the reservoir may be slightly reduced from the daytime peak. The ozone in the convectively stable shallow surface layer is depleted by dry deposition. Ozone levels at the surface will rise the following morning as the inversion breaks up. This increase is generally observed prior to approximately 1000 GMT and is due to atmospheric mixing bringing down ozone-rich air from above. Figure 8 shows a typical diurnal variation of ozone at a rural site; the nocturnal depletion and rapid increase of ozone in the morning is clearly shown. I t has been suggested by Chameides and Stedman176 that ozone levels in excess of 80 p.p.b. may be reached through the reaction of nitric oxide emitted by anthropogenic sources and methane produced naturally. This result has been widely criticized. Jeffries and Saeger 177 have shown that the steady-state assumption model of Chameides and Stedman is unreliable for solving atmospheric hydrocarbon and NO2 kinetic systems. By using the Gear method for solving kinetic equations, Dimitriades et al.148predicted an ozone yield of only 12 p.p.b. and 16 p.p.b. for the two sets of initial concentrations used by Chameides and Stedman. Their model also ignores the organic pollutants that accompany urban NO,. The model of Isaksen et al.'" showed that tropospheric air with methane as the only hydrocarbon, together with anthropogenic NO,, was unable to produce ozone above 80 p.p.b. When both mixing with background air and ground removal of ozone were included, the concentration was always below 60 p.p.b. Although global natural emissions of NMHC and other organic emissions are several times larger than man-made emissions, they are spread over large areas so the ambient hydrocarbon concentrations are low. Freas et ~ 1 . measured l ~ ~ NMHC concentrations of 0.1 p.p.m.C. in rural areas not dominated by urban 174

17'

17'

I. S . A . Isaksen, 0. Hov, and E. Hesstvedt, Environ. Sci. Technol., 1978, 12,1279. R . ,M. Harrison, C. D. Holman, H. A. McCartney, and J . F. R. McIlveen, A m o s . Environ., 1978,12,2021, W. L. Chameides and D. H . Stedman, Environ. Sci. Technol., 1976, 10, 150. H. E. Jeffries and H . Saeger, Environ. Sci. Technol., 1976, 10, 936.

Tropospheric Ozone

and NO, (-) Figure 8 The diurnal variation of ozone (-) near Heysham, Lancs on 29 May 19 78

35

at a rural site

(Reproduced by permission from Atmos. Environ., 1979,13, 1 5 3 5 )

plumes. Vegetation emits some hydrocarbons such as isoprene and various terpenes and for low NO, levels they can act as an ozone sink with concomitant formation of aerosol. Hence, elevated levels of ozone in rural areas are caused primarily by longrange transport of ozone and/or ozone precursors from urban areas. The urban plume results from the emission of hydrocarbons and oxides of nitrogen and as the plume is transported downwind it becomes nearly uniformly distributed in the vertical plane because of strong daytime mixing. There is, however, negligible lateral mixing over middle distances, 8 Elevated Ozone Levels

As discussed earlier, photochemical air pollution has been observed in many regions of the world. There now follows a brief survey of reported elevated ozone levels as a result of photochemical oxidants and their precursors. It is only relatively recently that monitoring of tropospheric ozone concentrations has been carried out systematically in Britain. However, ozone concentrations which are elevated well above the natural background levels have been reported on numerous occasions. In June/July 1972 an anticyclone over the North Sea and Scandinavia resulted in light easterly winds over southern England. Ozone concentrations in excess of 100 p.p.b. were recorded at Harwell and a continental

Environmental Chemistry

36

precursor source area was sugge~ted."~Cox et al.'% in their study based on measurements of ozone and halocarbons a t three sites (Sibton, Suffolk; Harwell, Oxon; Adrigole, Country Cork, Eire) in August 1973 concluded that ozone and/or its precursors in suitable meteorological conditions can be transported over distances in the order of 100-1000 km, and that on occasions, continental emissions provide a major contribution to photochemical pollution in Britain. Hence, during ozone episodes photochemical pollution is essentially a regional rather than local phenomenon. Maximum ozone concentrations of 100-1 50 p.p.b. were reached at all the sites and from air trajectories, transport times of 1-2 days were e ~ t i m a t e d . " Cox166 ~ reports on measurements of NO, NO2, and ozone at the Adrigole site during July-November 1974. Again, long-range transport was evident. During the summer of 1976 the meteorological conditions were particularly favourable for the formation of photochemical ozone over Britain. Apling et ~ 1 .measured l ~ ~ ozone at sites in central London, the suburbs of London, and rural areas of Southern England during this period. In urban areas levels exceeded 200 p.p.b., and in rural areas 250 p.p.b. They pointed out that the primary pollutant plume requires several hours of solar radiation before photochemical ozone can be detected. In this time the plume has travelled 50 km or more downwind of the primary source, so identification of the source is difficult in densely populated and highly industrialized regions. In central London, Derwent and Stewart180found maximum ozone concentrations, up t o 11 1 p.p.b., occurred in light or calm wind and little cloud, while the results of Stewart e t a l . 181 showed that maximum hourly ozone concentrations occur early in the morning or between 1500-1600 local time, due to diffusion of stratospheric ozone and photochemistry, respectively. Non-local production of ozone was suggested as the source for maximum ozone between 1900 and 2100 local time. The results of ozone measurements in Greater London during the summers of 1975 and 1976 are described by Ba11lW and Ball and Bernard.la2 They suggest that there are two possible sources for elevated levels (150 p.p.b. in 1975, 180 p.p.b. in 1976) in and around London. The first is long-range transport from the European continent and the second is from photochemical reactions involving locally emitted precursor pollutants. These precursor pollutants only appear in central London on calm days, but under the meteorological conditions of the 1976 summer they produced an additional maximum downwind ozone concentration of 80 p.p.b.lS2 More recently, Harrison and Holman 183 and Harrison and McCartneylM have discussed measurements at a rural site near Heysham, Lancashire, made in 1977 R. A. C o x , A. E. J. Eggleton, R. G. Derwent, J. E. Lovelock, and D. H . Pack, Nature, 1975,255,118. 179 A J. Apling, E. J . Sullivan, M. L. Williams, D. J. Ball, R. E. Bernard, R. G . Derwent, A. E. J . Eggleton, L. Hampton, and R. E. Waller, Nature, 1977,269,569. l a * R. G.Derwent and H. N. M. Stewart, Nature, 1973.241, 342. l a ' H.N. M. Stewart, E. J . Sullivan, and M. L. Williams, Nature, 1976,263,582. l S 2 D.J. Ball and R. E. Bernard, Atmos. EnM'ron., 1978,12,1391. 1 8 3 R. M,Harrison and C. D. Holman, A m o s . Enuiron., 1979,13, 1535. l a 4 R. M. Harrison and H . A. McCartney, Atmos. Enuiron., 1980,14,233.

.

Tropospheric Ozone 37 and 1978. During the episodes in May and June 1978, two distinct types of polluted air masses were identified: one from the North Sea and Northern England, and the other from the continent and the South-East and Midlands of England. The former air mass represents long-range continental sources and the latter represents both long-range and middle distance (British) sources. This study has shown that at this site 50-80 p.p.b. of ozone can be attributed to longrange transport of ozone from continental sources, while middle-distance sources within Britain can contribute an additional 20-50 p.p.b. Finally, Martin and BarberlS5 found 10 days in 1978 and 25 days in 1979 when the hourly ozone concentration exceeded 80 p.p.b. at a rural site at Bottesford, Leicestershire, in central England. These results can be compared with the results of Holman,186 who during the summers 1977-9 observed 28 days at Heysham of ozone levels in excess of 80 p.p.b. These summers have been noted for the poor weather, thus even in bad summers, levels in excess of accepted ambient air quality standards have been recorded. Table 7 shows the maximum hourly ozone levels observed each year at various sites in England, and illustrates the considerably higher levels of 1976.

Table 7 Highest hourly average ozone concentrations observed at various sites in England Ozone concentrationip.p. b. * A

/

L oca tion Central London (urban) London suburbs Harwell (rural) Lancaster (rural)

1971

101

\

1972

1973

1974

1975

1976

1977

1978

112

136

164

150 130

210 210

122a

1 59a

177

258

121b

1 14b

ll0C

120c

128

141

120

1979

122=

Results from ref. 179 except: “ref. 240;bref. 125;‘ref. 186.

In south-eastern Norway ambient ozone measurements have been made since 1975.110,187,188 Due to the variable climate the total number of ozone episodes can vary considerably from one year to another. The episodes may be caused by long-range transport from the continent or Britain, by local and mesoscale production, or by a combination of both. Concentrations of up to 200 p.p.b. have been recorded. A. Martin and F. R . Barber, Atmos. Environ.,

1981,15, 567.

C.D.Holman, Ph.D. Thesis, University of Lancaster, 1980.

’” J . Schjoldager, B. Sivertsen, and J. E. Hanssen, Atmos. Environ., 1978,12, 2461. la’

J. Schjoldager, Atmos. Environ., 1979,13, 1689.

38

E nviro n men tal Chemistry

In Sweden, GrennfeltlS9has measured a maximum hourly ozone concentration of 210 p.p.b. at a rural coastal site and levels of 100-200 p.p.b. in the Gothenburg area. These ozone episodes were the result of long-range transport and mesoscale production. A series of studies in the Cologne/Bonn area of Germany from 1975 have been reported by Becker et aZ.lg0 Their conclusions as t o the source of elevated ozone levels were in agreement with previous work, namely that the ozone levels resulted from local precursor emissions, in addition to long-range transport. Systematic measurements of ozone have been carried out in the Netherlands since 1968191-193and it has been established that hourly ozone concentrations may reach 250 p.p.b. Guicherit and Van Doplg3 studied the occurrence of high ozone levels in Western Europe during 1969-75. When a stagnating anticyclone was situated over Western Europe, ozone concentrations in excess of 100 p.p.b. could occur, both in rural and urban environments, over distances of more than a thousand miles. No discrete source areas could be inferred from trajectory analyses. They concluded that, due t o transport, precursors are widely distributed over Europe so that under the appropriate meteorological conditions photochemical air pollution can occur simultaneously over large areas, A similar study was conducted by Schjoldager et a!.'* who examined photochemical oxidants in North-Western Europe during 1976-9. They concluded that in some of the episodes long-range transport played the dominant role, whereas in others local and mesoscale ozone formation appeared to be more important. Cecinato et al."' monitored ozone during the summer of 1977 at two sites in Rome. At an urban site the maximum hourly concentration exceeded 100 p.p.b. on 9% of the days, while at a suburban site it was exceeded on 10%of the days. Levels in excess of 100 p.p.b. were exceeded on 74% of the days studied by Nasralla and Shakour in Cairo during 1979. A concentration of 500 p.p.b. was exceeded on two of the days. Ozone concentrations have been routinely measured in several urban locations in Israel since 1975.19591% During the spring and autumn ozone levels between 100 and 240 p.p.b. were observed. This ozone was either formed locally or transported from Europe or North Africa.

P. Greenfelt, Internat. Conf. on Photochemical Oxidant Pollution and its Control, EPA-600/3 -7 7-001 a, 19 7 7. K. H . Becker, J . Lobel, U. Schurath, H. Deimel, H . W. Georgii, and W. Fricke, Proc. 4th Internat. Clean Air Congress, Japanese Union of Air Pollut. Prevention ASSOC., Tokyo,1977. 191 J . A. Wisse and C. A. Velds, Atmos. Environ., 1 9 7 0 , 4 , 79. 1 9 2 R. Guichert, R. Jeltes, and F. Lindquist, Environ. Pollut., 1972, 3, 91. '93 R . Guichert and H. Van Dop, Atmos. Environ., 1977, 11, 145. 1 9 4 J. Schjoldager, H. Dovland, P. Greenfelt, and J . Saltbones, Photochemical Oxidants in North-Western Europe 1976-79, A Pilot Project, Norwegian Institute for Air Research, Lillestrom, Norway. E. Ganor, Y . Beck, and A. Donagi, Atmos. Environ., 1978, 12,1081. 196 E. Ganor and A. Donagi, Proc. Internat. Ozone Symp., Boulder, Colorado, 1980. 1*9

Tropospheric Ozone

39

In Australia, ozone concentrations have been monitored as part of the Sydney Oxidant Study.1g7s1g8Levels above 210 p.p.b. can result from transport and recirculation of photochemical smog across the Sydney Basin. Ozone precursors were carried offshore where they react to form ozone. In the afternoon the sea breeze advects back air containing these high ozone concentrations. Elevated levels of ozone in North America are very well documented. In Canada, the most frequent and highest values of ozone were found to occur in south-western 0 n t a r i 0 . l ~ Concentrations ~ of up to 150 p.p.b. were observed with south-south-westerly wind flows suggesting that pollution of anthropogenic ozone, from the U.S.A., was brought into the region.200 In August, 1974, an hourly average of 8 0 p.p.b, was exceeded 49% of the time in Toronto.201 Their observations pointed to transport processes rather than local photochemical production leading to these elevated ozone concentrations. In the U.S.A. both individual plumes from cities and regional pollution have been reported. Clearly defined ozone plumes are frequently observed downwind of major centres of population and industry on photochemically active days. The St Louis urban plume was found to cause ozone concentrations in excess of 8 0 p.p,b. 160 km or more downwind of the city.202 Spicer et aLm3 monitored a concentration of 130 p.p.b. upwind of St. Louis and found that emissions from Springfield, Illinois (population 100 000) lead to a definable ozone plume. Aerial measurements of the plume from the Chicago-Gary area recorded a peak enhancement of 70-80 p.p.b. across the 50 km wide plume.204Evidence of urban plumes in the north-eastern area of the United States has been presented by several a ~ t h o r ~whose ~ ~ results ~ , ~indicated ~ ~ - significant ~ ~ ~ ozone generation in urban plumes.

G . S. Hawke, A. C. Heggie, R. Hyde, A. Mitchell, and R. Rothwell, Proc. Internat. Clean Air Conf., Brisbane, Australia, 1978. l g 8 R. Hyde, G. S. Hawke, and A. C. Heggie, Proc. Internat. Clean Air Conf. Brisbane, Australia, 1978. 199 Y . S. Chung, Proc. Internat. Clean Air Conf., Brisbane, Australia, 1978. Y . S. Chung, J. Appl. Meteorol., 1977,16, 1127. * O 1 K. G. Anlauf, M. A. Lusis, H. A. Wiebe, and R. D. S. Stevens, Atrnos. Environ., 1975, 197

'O'

'03

'04

205

'06

'O'

9 , 1137. W. H. White, J . A. Anderson, D. L. Blumenthal, R. B. Husar, N. V. Gillani, J . D. Husar, and W. E. Wilson, Science, 1976, 194, 187. C. W. Spicer, D. W. Joseph, and P. R. Sticksel, J. Air Pollut. Control ASSOC.,1982, 32, 278. K . Sexton and H . Westberg, J. Air Pollut. Control ASSOC., 1980, 30,911. G. T. Wolff, P. J . Lioy, G. D. Wright, and R. E. Pasceri, J. Air Pollut. Control ASSOC., 1977, 27,460. W. S . Cleveland, B. Kleiner, J . E. McRae, R. E. Pasceri, and J. L. Warner, paper 75-526, Annual Meeting of the Air Pollut. Control ASSOC., Boston, Mass., 1975. W. S . Cleveland, B. Kleiner, J. E. McRae, and J . L. Warner, Science, 1976, 191,179.

40

E nuironmental Chemistry

On the regional scale, ozone sources and transport in the north-eastern United States are well d o c ~ m e n t e d . ~ They ~ - ~ ' observed ~ a fairly uniform ozone distribution which indicated that transport of ozone and its precursors over several hundreds of miles in the north-eastern United States plays a large part in determining the observed concentration distribution, in addition to natural ozone and ozone generated in urban plumes. To obtain information on air masses which approach the eastern U.S. from the west and north-west, Kelly et ~ 1 . ~measured ' ~ various parameters over South Dakota and concluded that the stratosphere was the dominant source of ozone in the air masses which affected the north-eastern U.S. Wolff et aL2'0 estimated that over 100 p.p.b. of ozone are contained in air parcels transported from the midwest to the northeastern U.S., and above the nocturnal inversion the mean half life of ozone is 46.8 h. These studies, representative of many on this topic, serve t o give some indication as to the extent of photochemical air pollution and transport in both urban and rural areas. Long-range transport phenomena are now well established in both the European and North American context. Meteorological Conditions Associated with Ozone Pollution. - The occurrence of tropospheric photochemical ozone formation is highly dependent on meteorological conditions. High ozone concentrations are normally produced on warm, anticyclonic days when the solar radiation is high, wind speed light, and pollutants are confined by a subsidence inversion. In Europe, elevated ozone levels generally occur with stagnating anticyclones over central Europe, Scandinavia, or Finland. In Britain an episode might develop as follows: an anticyclone situated over the North Sea, North Germany, or Scandinavia results in light easterly winds over Britain. Cloud cover breaks up and warm sunny spells persist for several days. Only on the first day of such a situation is there any guarantee that precursor emissions from the U.K. affect ozone levels in the U.K. On subsequent days, long-range transport provides an additional major source of ozone. Elevated levels of ozone will persist as long as the anticyclone prevails. Such a situation occurred a t the end of May 1978 when elevated ozone levels were measured on eight consecutive days at Heysham. Figure 9 shows the 1200 GMT synoptic chart for 30/5/78when the maximum hourly ozone concentration reached 98 p.p.b.

'08

'09

'lo 211

212 213

214

215

G . T. Wolff, P, J . Lioy, G. D. Wright, R. E. Meyers, and R. T. Cederwall, Armos. Environ., 1977, 11, 797. G . T . Wolff, P. J . Lioy, R. E. Meyers, R. T. Cederwall, G . D. Wright, R. E. Pasceri, and R . S. Taylor, Environ. Sci. Technol., 1977, 11, 506. G . T. Wolff, P. J . Lioy, and G . D. Wright,J. Environ. Sci. HeaIrh, 1980, A15, 183. F . L. Ludwig and E. Shelar, Report EPA 901/9-76-005, 1977. F . L. Ludwig and K. Maughan, Report EPA 901/9-78-001, 1978. C . W . Spicer, J . L. Gemma, and P. R. Sticksel, Ozone: transport considerations and predictive models for southern New England, Battelle-Columbus Laboratories Report, Columbus, Ohio. C. W. Spicer, D. W. Joseph, P. R. Sticksel, and G . F. Ward, Environ. Sci. Technol., 1979, 13,975. N. A. Kelly, G . T. Wolff, and M. A. Ferman, Armos. Environ., 1982,16,1077.

Tropospheric Ozone

41

Figure 9 Synoptic situation at 1200 G M T on 30 May 1978

In North America, high ozone levels occurred most often when a high pressure system was near the reporting station. During these periods of air stagnation, ozone may be trapped in the lowest atmosphere layer, resulting in a build-up of large concentrations. Vukovich et al. 216,217 have found that high ozone occurred most often on the backside of the high pressure system in air that had large residence times in the system. Also high ozone occurred most often when the high pressure was in the eastern U.S.A. showing that as the high pressure moved from west to east, the air became more polluted as it moved east. Several meteorological parameters have been investigated for correlations with ozone. From multiple regression analysis, a correlation coefficient of 0.69 has been found between high ozone values and daily average wind speed, daily maximum temperature, and solar radiation.Ig3 In the St Louis area, Karl and De Marrais218 concluded that the day-to-day variance in solar radiation was highly F. M. Vukovich, W. D. Bach, B. W. Crissman, and W. J . King, dtmos. Environ., 1977, . l l , 967. F . M. Vukovich, Atmos. Environ., 1 9 7 9 , 1 3 , 2 5 5 . T. R . Karl and G. De Marrais, Internat. Conf. on Photochemical Oxidant Pollution and its Control, EPA-600/3-77-001a, 1977.

216

'I8

42

Environmental Chemistry

correlated with maximum temperature, the height of the 500 mb surface, and the number of days since rain occurred and that these variables can influence ozone levels. Measurements of pollutant and meteorological data from Chicago were analysed by Wadden et aZ.,219 who found a trend of higher ozone with increased temperatures (r = 0.57). N o well defined trends were shown between maximum ozone and the previous day’s ozone maximum, solar radiation, hours of sunlight, and height of the 500 mb surface. From Japanese data, Wadden et al.220 found a high correlation (r = 0.66) between maximum ozone and temperature. By analysing the synoptic weather patterns they showed that the maximum value of hourly ozone occurred 4 days after the passage of a front. This compares well with the value of 3-5 days found by Wadden et aL219 for Chicago. Mukammal et al. 221 examined the influence of various meteorological parameters on maximum ozone concentrations in Ontario. They derived a curvilinear relationship between ozone concentration and wet bulb potential temperature (WBPT). A relationship between ozone concentration and WBPT is not unexpected because WBPT is a conservative parameter, characteristic of air masses and previous workers have associated high ozone in eastern North America with the presence of maritime tropical air m a s ~ e s . ~ ~ ~ ~ ~ ~ Attempts have been made t o forecast ozone levels by empirical models. Wolff and Lioy222 developed a model based on the upwind ozone maximum on the previous day, maximum temperature, the previous day’s upwind maximum temperature, and the mean wind speed from the surface to 1000 m. The model results gave predictions within f 20% of the observed values for 85% of the cases. The model developed by R e ~ l e t was t ~ ~used ~ to forecast ozone levels at Newport, Kentucky. The most significant variables were nitrogen dioxide concentration, temperature, and wind speed. Weekday-Weekend Variations. - Since the traffic patterns in cities show a marked decrease in volume at weekends, it is expected that precursor emissions are reduced and hence ozone levels increase, Anthropogenic emissions, especially NO, can decrease or increase ozone concentrations. Since the immediate effect of NO is to destroy ozone then it is expected that ozone concentrations, in urban areas, should be lower on weekdays than on weekends. The same emissions that critically reduce ozone concentrations will result in increased concentrations somewhere downwind. In areas without important precursor emissions, a reverse effect is expected if they were appreciably affected by ozone produced from imported anthropogenic emissions. Bruntz et aZ.ln have reported that ozone diurnal patterns in New Jersey and New York show only minor differences on weekdays and Sundays, whereas Cleveland et aL2% found slightly higher levels on Sundays in the same area.

220

’11 222

lZ3 224

R . A. Wadden, E . D. Ross, and J . E. Quon, J. Environ. Eng. Div. ASCE, 1979, 105,621. R. A. Wadden, Y . Ogawa, M. Okuda, and K. Murano, J. Environ. Eng. Div. ASCE, 1982, 108,680. E . I. Mukammal, H . H . Newmann, and T. J. Gillespie, Atrnos. Environ., 1982, 16, 2 0 9 5 . G. T. Wolff and P . J . Lioy, J. A i r Pollut. Control ASSOC.,1978, 28, 1034. G . H . Revlett, J. Air Pollut. Control Assoc., 1978, 28, 338. W . S . Cleveland, T. E. Graedel, B. Kleiner, and J . L. Warner, Science, 1974, 186, 1037.

Tropospheric Ozone

43

Cleveland and McRae225 found reductions of 10-25% of ozone concentrations at weekends downwind of New York while upwind sites showed little change. Data from Los Angeles between June and September 1972 and 1973 showed that differences between weekday and weekend concentrations were insignificant in the central region, but weekday levels were greater in the north and less in the south than weekend levels.226 Again in Los Angeles, Elkus and Wilson227 found lower ozone concentrations during the summer on Sundays for some inland stations and the opposite effect for stations located ,closer to the coast, In St Louis the difference in the concentrations of photochemical pollutants NO, NO2, NMHC, and 0 3 from weekends to weekdays depended to a large extent on the distance of the measurement from the urban area.228 Ozone concentrations decrease from weekends to weekdays inside the city, in contrast t o the results outside the city. The results of Ludwig and Maughan212 show weekend increases of ozone in urban areas and decreases in rural areas. At the sites in New England, higher weekend ozone concentrations were found in Manchester and Burlington while at Nashua weekday levels were higher than weekend levels. In summary, there are conflicting results regarding the behaviour of ozone on weekdays versus weekends; i. e., areas having relatively large local precursor emissions are expected to experience higher ozone concentrations on weekends than weekdays, but this is not always observed.

9 Biological Effects of Ozone People, animals, plants, ecological systems, and human artefacts are all potential targets for pollution. Ozone is known t o have an adverse effect on human health and vegetation, in particular, agricultural crops. There is a scarcity of quantitative data available on the potential damage t o human health from ozone, although rather more is known about its effect on vegetation. Effects on Humans. - Photochemical smogs are known to produce symptoms such as sore throats, running noses, eye irritation, headaches as well as more serious effects such as bronchitis and other respiratory diseases. The eye irritation is now known to be caused by photochemical pollutants other than ozone,229 but it is still not clear what part ozone plays in producing the other symptoms. Most experimental studies have been carried out on animals, but there is no reliable method of extrapolating the animal dose-response data t o humans. Clinical and epidemiological studies have shown that asthmatic and other persons with reactive airways appear most sensitive to changes in ozone concentration and that exercise effectively increases the ozone dose delivered to the target tissues in the respiratory tract. Of the studies made, the general conclusion is

225 226 227 228 229

W. S. Cleveland and J. E. McRae, Environ. Sci. Technol., 1978,12,5 5 8 . S. B. Levitt and D. P. Chock,J. Air Pollut. Control ASSOC.,1976,26,1091. B. Elkus and K. R. Wilson,Amos. Environ., 1977,11,5 0 9 . T.R . Karl, Atmos. Environ., 1978,12, 1657. A. P.Altshuller, J. Air Pollut. Control ASSOC., 1978,28, 594.

Environmental Chemistry

44 Table 8 Ozone effects on man Author

Reported effects

Schoettlin and Landau'

When daily maximum hourly 0, exceeded 0.25 p.p.m. the average number of asthma patients having attacks was statistically significantly elevated When daily maximum hourly 0, exceeded 0.3 p.p.m. increased rates of chest discomfort and headaches were observed in student nurses Subjects undergoing light intermittent exercise showed signs of discomfort and significant changes in lung function when exposed'to ozone levels of 0.37 p.p.m. for 2 h Reduction in pulmonary function and symptoms of discomfort in subjects undergoing vigorous exercise and exposed t o ozone levels of 0.3 p.p.m. for 1 h No significant changes in respiratory function of asthmatic patients when exposed to 0, levels of 0.2-0.25 p.p.m. for 2 h and undergoing light intermittent exercise, small but significant blood biochemical changes observed

Hammer e t al.

Silverman e t ai.c Folinsbee et al.d

De Lucia and Adamse

Lin e t a1.f

'C. E. Schoettlin and E. Landau, Public Health Rep., 1961, 76, 545; bD. I. Hammer, V. Hasselblad, B. Portnoy, and P. F. Wehrle, Arch. Environ. Health, 1974, 28, 255; 'F. Silverman, L. J. Folinsbee, J. Barnard, and R. J. Shephard, J. Appl. Physiol., 1976, 41, 859; dL. J. Folinsbee, F. Silverman, and R. J. Shephard, J. AppL Physiol., 1975, 38, 996;eA. J. De Lucia and W. C. Adams, J. Appl. Physiol., 1977, 43, 75;fW. S. Linn, R. D. Buckley, C. E. Spicer, R. L. Blessey, M. P. Jones, D. A. Fisher, and J. D. Hackney,Arn. Rev. Respir. Dis., 1978,117,835.

that ozone has adverse health effects but the concentrations at which these effects become important are the subject of debate, The studies do indicate that the intensity and significance of effects increases as the pollutant level increases. Table 8 shows some of the concentrations and exposure times used in studies when adverse health effects have been reported, Recent work suggests that the effects of short-term exposure to ozone are first observed in the 150250 p.p.b. concentration range,230 while evidence from controlled human and community health studies has shown that the first adverse effects in man appear when the ozone concentration is in the range 100-250 ~ . p . b . ~ ~ l These concentrations give little room for manoeuvre above the natural background levels, especially as the effects reported in some studies may occur a t 230

National Academy of Science, Medical and Biological Effects of Environmental Pollutants, Ozone and other Photochemical Oxidants; Washington, 1977. World Health Organisation, Environmental Health Criteria ( 7 ) Photochemical Oxidants, WHO, Geneva, 1978.

Tropospheric Ozone

45

lower concentrations when ozone is present in combination with other urban pollutants. Effects on Vegetation. -Plants are more sensitive t o ozone than are humans; damage can be observed at concentrations as low as 40 p.p.b. Ozone is extremely phytotoxic at concentration levels found in polluted atmospheres and plants can be used to indicate air pollution. The visible effects are identifiable pigmented chlorotic and necrotic foliar patterns resulting from major physiological disturbances. These may be measured on the basis of growth and long-term biochemical changes. By observing the visible damage it is possible to estimate concentration levels of the pollutant causing the injury, Leaf injury can be correlated with average ozone only in areas where environmental conditions and daily ozone fluxes Nicotiana tabacum cultivars Bel-W3 and Bel-C have are particularly been employed to indicate ozone concentrations. The variety Bel-W3 is very sensitive t o ozone exposure, the threshold for injury being 40p.p.b., and has been used in several s t ~ d i e s . ~ ~Kelleher ~ - = ~ and Feder2” used both a commercial ozone monitor and Bel-W3 on Nantucket Island, Massachusetts, and found high concentrations and high amounts of leaf injury only when the wind was from the New York/Washington area, which further supports the evidence described earlier of long-range transport. As a result of studying the leaf injury of Bel-W3 at 60 sites in the British Isles during 1977, Ashmore et aZ.238concluded that in the absence of anticyclones, local sources with populations greater than 75 000 are as important as larger more distant cities for ozone and leaf injury. Ashmore e t aLm used cultivars of Pisum satiuum and Raphanus satiuus as indicators. The Pisum sativum plants were grown in filtered and unfiltered air; leaf injury was only found in the plants in the unfiltered air. Also the cotyledons of Raphanus sativus showed an accelerated rate of senescence. The maximum ozone concentration during the experimental period was 1 38 p.p.b., and levels exceeded 80 p.p.b. each day. Injury to vegetation does not always manifest itself by visible damage to leaves. I t can occur as reduced growth, reduced capacity for flowering, and reduced yield, Although roots are not exposed to air pollution, they may be more affected than shoots which are exposed. The root/shoot ratio has been

2. Naveh, S. Chaim, and E. H. Steinberger, Environ. Pollut., 1978,16, 249. W.W.Heck and A. S. Heagle, J. Air PolZut. Control ASSOC.,1970,20, 97. 234 J. S. Jacobson and W. A. Feder, Bull. Mass. Agric. Exp. Stn., 1974,604. 2 3 5 J. N. B. Bell and R. A. Cox, Environ. PolZut., 1975,8,163.

232 233

A. C. Posthumus, Proc. of the Kuopio meeting on Plant Damage Caused by Air Pollution, Kuopio, Finland, 1976. 2 3 7 T. J. Kelleher and W. A. Feder, Environ. Pollut., 1978,17,187. 238 M. R. Ashmore, J. N. B. Bell, and C. L. Reily, Environ. Polluf., Ser. B , 1980,1, 195. 2 3 9 D.C. Horsman,Environ. Pollut., Ser. B , 1981,2,69. 240 M. R. Ashmore, J . N. B. Bell, C. Dalpra, and V. C. Runeckles, Environ. Pollut., 1980,

236

21,209.

46

Environmental Chemistry

seen to decrease in many species when exposed to ozone.2c11-244If the root is a crop, the yield is reduced, as in radishw5 or onion.246 Although the exposure time is important, it has been found that concentration has the dominant role. Exposure over a period of weeks t o concentrations in the range 50-100 p.p,b. has produced reduced growth in radish,245 sweet ~ o r nand , ~soyabean.a8 ~ Onions (spartan era) exposed to an ozone concentration of 20 p.p.b. for 24 h show no weight reduction but when exposed t o a concentration of 100 p.p.b. for 1 and 4 h showed weight reductions of 21 and 48%.249 Long-term growth reduction can result from exposure to ozone concentrations found in typical photochemical episodes. Since ozone damage to vegetation may manifest itself only in the form of a reduction of yield, it is impossible t o assess reliably these consequences of photochemical pollution in economic terms. Environmental Health Guidelines. - Various ambient air quality standards for to~120 ' ~ . p . b . ~have ~ O been ozone ranging from an hourly average of 5 0 ~ . p . b . ~ set and are listed in Table 9 . Until recently the United States EPA had a much more stringent standard of 80 p.p.b. (hourly average, not t o be exceeded more than one hour each year). However, as a result of clinical and epidemiological

Table 9 Air quality standards for ozone Urganisation

Air quality standard

World Health Organi~ation~~'

50-100 p.p.b. (1 h average) as guideline 6 0 p.p.b. (1 h average) best single value 80 p.p.b. (1 h average) 120 p.p.b. (1 h average)

Greater London CouncilZS2 US. Environmental Protection Agencyzso

studies a two tier system was proposed251 with a primary standard .(human health effects) of 100 p.p.b. and a secondary standard of 80 p.p.b. (welfare effects). After a comment period the EPA, in February 1979, raised both standards t o 120 p.p.b- and changed the definition of the point a t which the standard is attained t o 'when the expected number of days per calendar year with maximum hourly average concentrations above 120 p.p.b. is equal to or less than These new standards leave a lower margin of safety than the previous standards. 24 1 24 2

243

G. Ogato and E. V. Maas, J. Environ. Qual., 1973, 2, 518. J . P. Bennett and R. J . Oshima, J. Am. SOC. Hort. Sci., 1976, 101,638. R. J. Oshima, J . P. Bennett, and P. K . Braegelmann, J. A m . SOC. Hort. Sci., 1978, 103,

348. 244

G . Hofstra, A. Ali, R. T. Wukasch, and R. A. Fletcher, A t m o s . Environ., 1981, 15,483. D.Tingey, W.W. Heck, and R. A. Reinert, J. A m . SOC.Hort. Sci., 1971, 96, 369. 246 R . T. Wukasch and G . Hofstra, J. A m . SOC.Hort. Sci., 1977, 102, 543. 2 4 7 A. S. Heagle, D. E. Body, and E. K. Pounds, Phytopathology, 1 9 7 2 , 6 2 , 6 8 3 . 248 A. S. Heagle, D. E. Body, and G. E. Neely, Phytopathology, 1974, 64,132. 249 W. W. Heck and C. S . Brandt, in Air Pollution, Vol. 11, ed. A. C. Stern, Academic Press, New York. 2 s 0 U.S. Environmental Protection Agency, Fed. Reg., 1979,44, 8202. 2 51 U S . Environmental Protection Agency, Fed. Reg., 1978, 43,26 962. 245

Tropospheric Ozone 47 In Britain, no ambient air quality standard exists, although the Greater London Council252 has produced a guideline of a maximum hourly average of 80 p.p.b. 10 Analytical Techniques

Early measurements of ambient ozone concentrations were determined by measurement of the capacity of an air sample to oxidize potassium iodide.253 This method is sensitive not only t o ozone but other oxidizing substances in air such as PAN, hydrogen peroxide, and to a limited extent nitrogen dioxide. The potassium iodide method has two main variants. In the more common method 1 or 2% neutral buffered KI is used as an absorbing reagent and the iodine released is determined spectrophotometrically.254The other technique uses 2% unbuffered KI as the reagent and a visual end-point titration is used for calibration.255 The overall reaction is: 0 3 +2 ~ + +21-+ I2

+ KI

+ H 2 0 + o2

+ K13

A stoicheiometry of 1 :1 is assumed; however, Boyd and c o - w o r k e r ~compared ~~~ the KI method with an independent measure of ozone concentration and found a stoicheiometry for 12/03 of 1.5. They also found that the stoicheiometry was pH dependent and a complex mechanism was proposed. Flamm257found that ozone analysis with 1% neutral buffered KI gave results 1 5 3 0 % larger than those obtained by U.V. absorption, if an 12/03 stoicheiometry of 1.0 was assumed, A second product of the 03-KI reaction which slowly liberates additional iodine was tentatively identified as hydrogen peroxide and it was suggested that previous disagreements between investigators may be explained by variations in ( H 2 0 2 ) / ( 0 3 ) caused by differences in flow rate, collection time, interval before spectrophotometric analyses and by trace contaminants in some KI sources. Following the report by Negerbradt et al. 258 that the chemiluminescent ozone-ethylene reaction could be used for continuous monitoring of atmospheric ozone numerous commercial analysers were produced based on this principle. Ambient air and ethylene from a cylinder enter a reaction chamber at atmospheric pressure through concentric tubes facing a photomultiplier tube. Emission is centred on 435 nm and hence no interference from the reaction of ozone with nitrogen oxide occurs. This method is sensitive t o ozone concentrations in the range 0.001-100 p.p.m., over which the response is linear. The light Greater London Council, Joint Report of Planning Committee and Public Services Committee, Council Minutes, 1975. 2 5 3 B. E. Saltman and N. Gilbert, Anal. Chem., 1959, 31,1914. 2 5 4 APHA Intersociety Committee, ‘Methods of Air Sampling and Analysis’, 2nd Edn., American Public Health ASSOC., Washington D.C., 1977. 2 5 5 J. N. Pits, J. M. McAfee, W. D. Long and A. M. Winer, Environ. Sci. Technol., 1976, 10, 787. 2 5 6 A. W. Boyd, C. Willis, and R. Cyr, Anal. Chern., 1970’42,670, 2 5 3 D. L. Flamm, Environ. Sci. Technol., 1977, 11,978. l S 8 G . W. Negerbradt, A. VanderHorst, and J. Van Diujn, Nature, 1965, 206, 87. 252

Environmental Chemistry

48

emission is due principally to formation of electronically excited formaldehyde, HCHO ( 'A2) and a-dicarbonyl compounds.259 C2H4

+0 3

CHzO*

+

+ Ozonide + CH20*

CH2O 4- h~

The main problem with chemiluminescent analysers has been that of calibration. By using a Pen-Ray U.V. lamp with a stabilized power supply, steady concentrations of ozone may be generated ;however, there is no simple means of determining the concentration of the standard atmosphere. Because of the problems with KI methods, specific ozone calibration techniques have been developed including U.V. and i.r. photometry and gas-phase titration. In a comparison of calibration techniques, Paur260reported that gas-phase titration and U.V. photometry agree within 2.5% in the 150-700 p.p.b. 0 3 range, and deviate by only 3% from the borate-KI procedure. Good agreement between gas-phase titration and U.V. photometry was also found by De More and Patapoff,%' who recommended the latter method for routine use as the former is more susceptible to experimental error. The U.V.photometric technique also forms the basis of commercially available ambient air ozone analysers. These analysers measure the absorbance of ozone at 254 nm and may determine concentrations down t o 0.001 p.p.m. of ozone, Interferences due t o other U.V. absorbing atmospheric components (e.g., aromatic hydrocarbons, mercury vapour) are eliminated by alternately drawing air through the cell direct, and through a catalytic converter which decomposes ozone without causing loss of other atmospheric constituents. The difference in the absorbance readings in the two operational modes is due only to ozone. This instrument has the advantage of being a primary standard, not requiring supplemen tary Calibrat ion equipment.

259 260

16'

W. A. Kummer, J . N. Pitts, and H. Akimoto, Chem. Phys. L e t t . , 1972,12,495. R.J . Paur, Natl. Bur. Stand. (U.S.) Spec. Publ., 1977,464,15. W. B. DeMore and M. Patapoff, Environ. Sci. Technol., 1976,10,897.

2 The Environmental Chemistry of Organotin Compounds BY S. J. B L U N D E N , L. A. HOBBS,AND P. J. S M I T H

1 Introduction

The worldwide production of organotin chemicals has, over the last 25 years, risen from under 5000 tons in 1955 t o at least 30 000 tons at the present time (Figure l),representing approximately 5% of the current tin consumption. This increase is primarily due to the wide range of industrial applications discovered for the organotin compounds, and, in all probability, tin has a larger number of its organometallic derivatives in commercial use than any other element (Table 1 and Figures 2, 3 , 4 , and 5). On a tonnage basis, the biocidal uses192of the triorganotin compounds, R3SnX (-8000 tons), are exceeded by the non-toxic application^',^ of the diand mono-organotins, RzSnXz and RSnX3 (- 22 000 tons), The tetraorganotins, RqSn, have no large volume industrial outlets at the present time,5being primarily used as intermediates in the manufacture of the other types of organotin camp ou nds , R, SnX 4-n. In view of the diversity of organotins used industrially, a knowledge of their environmental chemistry is of fundamental importance. Certain areas of organotin environmental chemistry have been reviewed and, recently, a

’ C. J. Evans, Speciality Chemicals, 1981, 1, 26. C. J. Evans, Tin Its Uses, 1974, 100, 3. C. J . Evans, Speciality Chemicals, 1981, 1, 12. C. J. Evans, TinIts Uses, 1974, 101, 12. W. Wehner and H. 0. Wirth, in ‘22 Deutscher Zinntag: Organozinnchemie’, ed. W. P. Neumann and M. Gielen, Rev. Si, Ge, Sn and Ptt Compds., Special Issue, Freund Publish-

ing House, Tel Aviv, 1980, p. 7. Bokranz and H. Plum, ‘Industrial Manufacture and Use of Organotin Compounds’, Schering A.G., Bergkamen, 1975. Anon., ‘Environmental Health Criteria, 1 5 : Tin and Organotin Compounds - A Preliminary Review’, World Health Organisation, Geneva, 1980. P . J. Craig, in ‘Comprehensive Organometallic Chemistry’, ed. G. Wilkinson, Pergamon Press, Oxford, 1982, Vol. 2, p. 979. H. Plum, In$ Chim., 1981,220, 135. l o J. J. Zuckerman, R. P. Reisdorf, H. V. Ellis, and R. R. Wilkinson, in ‘Organometals and Organometalloids: Occurrence and Fate in the Environment’, ed. F. E. Brinckman and J. M. Bellama, ACSSymp. Ser., 1978, 8 2 , 388. ” F. E. Brinckman, J. Organornet. Chem. Library, 1981, 12, 343. P. A. Jones and M. F. Millson, ‘Organotins in the Canadian Environment: A Synopsis’, Environ. Can., Econ. Tech. Rev, Rep. EPS 3-EC-82-1, Environ. Impact Control Directorate, Quebec, 1982.

‘ A.

Environment a1 Chemistry

50 3oooa

25000

I

2000a

TOnS

isooa

lOOO(1

5000

1965

1967

1969

1971

1975 1973

1979

1977

1981

Figure 1 Estimated world production of organotin chemicals. (I.T.R.I. photograph)

Table 1 Industrial applications of organotin compounds Compound

Application R,SnX

Agriculture (Fungicides) (Antifeedants - Figure 3) (Acaricides)

Ph,SnX (X = OH, OAc) F'h,SnX (X = OH, OAc) (cyclo-C,H ,,),SnX (X = OH, -N-C ((PhMe,CCH 2) ,Sn) 20

Antifouling paint biocides (Figure 4)

I

= N-C = N)

I

H H Ph,SnX (X = OH, OAc, F, Cl,S CS NMe,, 0 . C 0 . C H 2 C l , O*CO*C,H,N-3) Ph,SnO - CO - CH2CBr, * CO OSnPh, Bu,SnX (X = F, Cl,OAc)

.

-

51

The Environmental Chemistry of Organotin Compounds Table 1-contd. Compound

Application R,SnX

(Bu3Sn)20 Bu,SnO CO * CH,CBr, CO OSnBu, Bu,SnO CO (CH,),. CO OSnBu, (-CH,CMe(CO OSnBuJ-1, Wood preservative fungicides (Figure 5 )

-

-

-

-

-

(Bu3Sn)20 Bu ,Sn(naphthenate) m-l,Sn),PO,

Stone preservation

(Bu,Sn)20

Disinfectants

Bu,SnO CO Ph (Bu3Sn)20

Molluscicides (field trials)

Bu ,SnF (Bu3Sn)20

-

RZSnX, Heat and light stabilisers for rigid PVC

-

R,Sn(SCH,. CO OOct'), (R = Me, Bu, Oct, BuO CO CH,CH,) (R,SnO. CO CH :CH CO O), (R = Bu, Oct) R,Sn(O * CO CH :CH CO OR'), (R = Bu; R' = Oct) Bu,Sn(O CO C,,H,,), Bu,Sn(SC ,,H,,),

.

Homogeneous catalysts for RW silicones, polyurethane foams and transesterification reactions

Precursor for forming SnO, f on glass3

~

Anthelmintics for poultry

-

.

-

-

- - -

Bu,Sn(O CO Me), Bu,Sn(O. CO - Oct'), Bu,Sn(O CO C, ,H2,), Bu,Sn(O - CO C,,H,,), (Bu,SnO),

s

Me ,SnCl,

-

Bu,Sn(O - CO C, ,H,,),

Heat stabilisers for rigid PVC

RSn(SCH2. CO OOct'), * (R = Me,Bu, Oct, BuO CO CH,CH,) (BuSnS1.5)4

Homogeneous catalysts for transesterification reactions

(BuSn(O)OH), BuSn(OH),Cl

Precursor for SnO, films on glass

BuSnC1, MeSnC1, (20%; i- 80% Me,SnCf,)

-

* These compounds are used in combination with the corresponding R,SnX,

derivatives.

52

Environmental Chemistry

h

c a 2 b13 0 0

4 4

c a Y

d‘

N

The Environmental Chemistry of Organo tin Com p oun ds

53

54

Environmental Chemistry

Figure 4 Seawater immersion tests are often used in assessing the efficiency of antifouling paints in preventing the attachment of marine organisms. The panels which have not been protected with an organotin based paint are heavily encrusted with marine growth. (Photograph by courtesy of International Marine Coatings, Gateshead)

The Environmental Chemistry of Organotin Compounds

55

56

Environmental Chemistry

specialized survey of the triphenyltin compounds has been published. 13' This review will, therefore, concentrate primarily on those aspects not already covered in depth. However, before these are discussed, a brief description of the toxicological characteristics of, and trace analytical techniques for, organotins will be given. 2 Toxicological Pattern of Organotins

Toxicology and Mode of Action. - The toxicological properties of organotins follow a very complex pattern,'3b but, in general, progressive introduction of organic groups a t the tin atom in any member of an R,SnX+, series produces a maximum biological activity when n = 3 , i.e., for the triorganotin compounds, R3SnX,I4 Within any R3SnX series, species specificity is markedly dependent upon the nature of the organic groups (Table 2), the higher tri-n-alkyltin compounds being essentially non-toxic to all organisms. Additionally, the inorganic radical, X, has generally very little effect on the biological activity. l4

Table 2 Species specificity of triorganotin compounds, RSSnX Species Insects Mammals Gram-negative bacteria Gram-positive bacteria, fish, fungi, molluscs, plants Fish, fun@,molluscs Fish, mites

R in most active R,SnX compound Me Et Pr

Bu

Ph cyclo-C,H,, PhMe,CCH,

Of particular importance to the environment is the high toxicity of tributyl-, triphenyl-, and tricyclohexyl-tin derivatives t o fish (Table 3 ) and other aquatic life. The symptoms of organotin toxicity can be manifested in a variety of ways, and, although their biological activities are not fully understood, di- and triorganotins have been shown t o have a number of effects on metabolic processes and enzymes. The tetraorganotins, R4Sn, show delayed toxic action, and studies I3'R. Bock, in 'Residue Reviews', ed. F. A. Gunther, Springer Verlag, New York, 1981, VOl. 79. 13b0 Wada, . S. Manabe, H. Iwai, and Y . Arakawa, J p n J. Ind. Health, 1982, 24, 24. l 4 G . J . M. van der Kerk and J. G. A. Luijten, J. A p p l . Chem., 1954, 4, 314. '' M. Polster and K. HalaEka, Tag. Ber. A k a d . Landwertsch.-Wiss., DDR Berlin, 1974, 126, 117. E. Linden, B, E. Bengtsson, 0. Svanberg, and G. Sundstrom, Chemosphere, 1979, 8, 834. l 7 T. E. Tooby, P. A. Hursey and J . J. Alabaster, Chem. Ind., 1975, 523. Anon., 'Plictran Miticide', Tech. Inf. Bull., Dow Chemical C o . , Midland, Michigan, 1979. l 9 I. Hammann, K. L. Biichel, K. Bungarz, and L. Born, Pflanzenschultz-Nuchr. Bayer (Engl. Ed.), 1978, 31, 61,

* LC,,

H

I

=N-C

H

1

=N

0.05 0.026 0.06-0.08 0.07 10.0 1.o

Leuciscus idus melanotus (Golden orfe) Lebistes reticulatus (Guppy) Albumus alburnus (Bleak) Leuciscus idus melanotus (Golden orfe) Leuciscus idus melanotus (Golden orfe) Leuciscus idus melanotus (Golden orfe) Leuciscus idus melanotus (Golden orfe) Salmo gairdneri (Rainbow trout fry) Lebistes reticulatus (Guppy) Alburnus alburnus (Bleak) Microterus salmonoitles (Large mouth bass) Carassius auratus (Goldfish) 0.015 0.034 0.40 0.06 0.0 1-0.1

>45

LC,,*/p.p .m.

Fish species

is the initial concentration of a compound killing 50% of the test organisms during the time denoted in p a r e n t h e s e ~ . ' ~

(Bu ,Sn),O Bu,SnOAc Bu,SnF Bu ,Sn(naphthenate) Bu,Sn Bu,SnCl, BuSnC1, Ph,SnOH Ph ,SnO Ac Ph,SnF (cyclo- C,H ,),SnOH (cyclo- C,H ,),Sn-k-C

Compound

Table 3 T o x i c i t y of organotin compounds t o fish

16 16 18 19

17

16 9 9 9 9

15

9

ReJ:

s-

r/,

R

x

2 2x 5

$

0

%ij

0

%

5

5

0

58

Environmental Chemistry

in mammals have shown them to be converted in uivo and in vitro t o the triorganotin species, R3SnX, particularly in the liver.20y2’ The underlying cause of the broad spectrum of acute toxicity shown by the triorganotin compounds is thought to be due to derangement of mitochondrial functions which can be brought (i) by interaction with mitochondrial membranes t o cause swelling and

disruption ; ( i i ) by secondary effects derived from their ability as ionophores to derange mitochondrial function through mediation of Cl-/OH- exchange across the lipid membrane; (iii) by their ability t o inhibit the fundamental energy conservation processes involved in the synthesis of ATP, of which living organisms share many common features, e.g., triorganotins inhibit mitochondrial oxidative phosphorylation and also photosynthetic phosphorylation in chloroplasts. On a macromolecular scale, work has been carried out by a number of groups to identify the possible binding sites for triorganotins, and they have been found to bind to a number of proteins, e.g., yeast mitochondrial membrane.25 Recent work with cat haemoglobin has found that cysteine residues and histidine are i n v ~ l v e d . ~ In , ~ ’general, the toxic effects of triorganotins have been found t o be reversible, although it has recently been observed that trimethyltin compounds can cause selective and irreversible neuronal destruction in the brain.28, The toxic effects of diorganotins have also been found t o be generally reversible. As in the case of triorganotin derivatives, the diorganotin species are inhibitors of oxygen uptake in mitochondria, but the mechanism of their toxic action is fundamentally different. Diorganotins have been shown to inhibit a-ketoacid ~ x i d a t i o n , probably ~ ~ , ~ ~ by combining with enzymes or coenzymes possessing vicinal dithiol groups, One particular effect of certain dialkyltins, e.g., OctzSnC12, noted by Seinen,30*31is their toxicity towards lymphocytes and 29a929b

J . E. Cremer, Biochem. J., 1958, 68, 685. E. C. Kimmel, R. H. Fish, and J . E. Casida, J. Agric. Food Chem., 1977, 25, 1. 2 2 W . N. Aldridge, in ‘Organotin Compounds: New Chemistry and Applications’, ed. J . J . Zuckerman, ACS Adv. Chem. Ser., 1976,157, 186. 23 W . N. Aldridge, in ‘Proc. 2nd Int. Conf. Si, Ge, Sn and Pb Compds.’, eds. M. Gielen and P. G . Harrison, Rev. Si, Ge, Sn and Pb Compds., Special Issue, Freund Publishing House, Tel Aviv, 1978, p. 9 . 24 M. J . Selwyn, i n ‘Organotin Compounds: New Chemistry and Applications’, ed. J. J. Zuckerman, ACS Adv. Chem. Ser., 1976, 157, 204. 2s K. Cain and D. E. Griffiths, Biochem. J., 1977, 162, 575. 26 B. M. Elliott, W . N. Aldridge, and J . W . Bridges, Biochem. J . , 1979, 177,461. F. Taketa, K. Siebenlist, J. Kasten-Jolly, and N. Palosaari, Arch. Biochem. Biophys., 1980,203,466. 28 A. W . Brown, W . N. Aldridge, and B. W. Street, Neuropath. Appl. Neurobiol., 1979, 5 , 83. 29aW.N. Aldridge, A. W . Brown, J . B. Brierley, R. D. Verschoyle, and B. W . Street, Lancet, 1981,692. 29bW.D. Ross, Am. J. Psychiatry, 1981,8,1092. 30 W. Seinen, Vet. Sci Commun., 1979/80, 3 , 279. 31 W. Seinen, Immunol. Consid. Toxicol., 1981, 1, 103. 2o 21

’’

59 The Environmental Chemistry of Organotin Compounds the suppression of thymus dependent immunity in rats, This appears t o be more marked for derivatives of increasing chain length, in contrast to the general pattern of acute toxicities, as expressed by LD50 values.32 The mono-organotin compounds, RSnX3, do not appear t o have any important toxic action in mammals and the inorganic tin derivatives are essentially nonMetabolism. - A knowledge of the metabolic fate of organotin compounds in mammals is obviously of considerable importance. It was first demonstrated by Cremer20 that tetraethyltin is metabolized in vivo in rats to a triethyltin derivative. It has also been shown that diethyltin species are broken down in vivo in rats to monoethyltin salts, which are rapidly eliminated from the body.% The metabolism in vivo in rabbits of a series of tetraalkyltin compounds has been and it was found that the extent studied by Wada and his of conversion of R&n+ R3SnX decreased as the length of the alkyl chain increased.35 Freitag and Bock have examined the metabolism of Ph3SnC1 in rats, using a tin-113 labelled compound, and, by analysis of the degradation products in urine, found that di- and mono-phenyltin compounds were formed, along with inorganic tin.38 Additionally, Fish and his co-workers21 have studied the breakdown of tin-113 labelled Ph3SnOAc in rats, and found a similar breakdown pattern of the triaryltin compound. In vivo studies in rats and mice using tric y c l ~ h e x y l - ,t~r i~e t h ~ l - , ~and ' t r i b ~ t y l - t i n ~ compounds ~ $ ~ - ~ ~ have indicated that they are excreted essentially intact from the animals and there appears to be no problem of long term accumulation. However, Blair reported43 that tricyclohexyltin hydroxide is metabolized in vivo in rats t o di- and monorecently demonstrated, in organotin and inorganic tin species. Wada e t al. rats, that tributyltin fluoride, once transported to the liver, is dealkylated, and Ward et al. 44b found that bis(tributy1tin) oxide was metabolized in sheepshead minnows, Cyprinodon variegatus, t o form dibutyl-, monobutyl-, and inorganic tin products. P. J. Smith, 'Toxicological Data on Organotin Compounds', I. T.R.I. Publication No. 538, 1978. 3 3 J . M. Barnes and L. Magos, Organornet. Chem. Rev., 1963, 3 , 137. 34 J . W. Bridges, D. S. Davies, and R. T. Williams, Biochem. J., 1967, 105, 1261. 35 Y . Arakawa, 0. Wada, and T. H. Yu, Toxicol. Appl. Pharmacol., 1981, 60, 1. 36 H. Iwai and 0. Wada, Indust. Health, 1981, 60, 1. 37 H. Iwai, 0. Wada, Y. Arakawa,and T. Ono, J. Toxicol. Environ. Health, 1982, 9 , 4 1 . 38 K. D. Freitag and R. Bock, Pestic. Sci., 1974, 5 , 731. 39 E. C. Kimmel, J . E. Casida, and R. H. Fish, J. Agric. Food Chem., 1980, 2 8 , 117. 40 R. A. Brown, C. M. Nazario, R. S. de Tirado, J. Castrilton, and E. T. Agard, Environ. Res., 1977, 13, 56. 41 W. H. Evans, N. F. Cardarelli, and D. J. Smith, J. Toxicol. Environ. Health, 1979, 5 , 871. 4 2 J . P. Anger, PhD. Thesis, Universitd Rene Descartes de Paris, 1975. 43 E. H. Blair, Environ. Qual. Safety Suppl., 1975, 3 , 406. 44aH. Iwai, 0. Wada, and Y. Arakawa, J. Anal. Toxicol., 1982, 5 , 300. 44bG. S. Ward, G. C. Cram, P. R. Parrish, H. Trachman, and A. Slesinger, Aquat. Toxicol. Haz. Asses.: 4th Con$, ASTM STP 737, ed. D. R. Branson and K. L. Dickson, Am. SOC. Testing and Materials, 1981, p. 183. 32

60

Environmental Chemistry

Although in vitro animal studies are not directly related t o the environment, it has been demonstrated, using rat liver microsomal mono-oxygenase enzyme systems, that the primary metabolic reaction for butyltin compounds is not Sn-C bond cleavage, but carbon hydroxylation of the butyl groups, where the a- and 0-carbon-hydrogen bonds are found to be more susceptible to hydroxylation.21 Similarly, results indicating hydroxylation of the organic group have been obtained with tricyclohexyltin hydroxide,39 but this process does not appear to occur with triphenyltin ~ p e c i e s . ~ ~ , ~ ~ In the majority of studies carried out t o date on the metabolism of organotin compounds, there is very little information on the dealkylation or dearylation products derived from the organic moieties. Recent work by Prough et al.* on the NADPH- and oxygen- dependent microsomal metabolism of the ethyltin series, Et,SnX4-n ( n = 2-4), has demonstrated that the major organic metabolite is ethylene and the minor product, ethane, Similarly, Williams et al. found that ’ Fish and benzene was formed in the metabolism of triphenyltin a ~ e t a t e , ~and his co-workers21 detected but-1-ene from the butyltin compounds, Bu4Sn and BuJSnX (where X = C1, OAc, and OSnBu3).

3 Analysis of Organotins a t Environmental Levels As World Governments and Authorities become increasingly concerned with environmental pollution, the need for new analytical techniques with increasingly lower limits of detection becomes more important. This is borne out in the organotin field by the upsurge of interest in analytical procedures for these compounds, at environmental levels, in recent years. Methods for the analysis of organotin compounds up to 1976 have been comprehensively reviewed by L ~ i j t e and n ~ ~by Price and Smith,49but, in general, these early techniques have not been able easily t o determine the species of the organotins present, or have not had sufficient sensitivity for trace analysis. Recent advances in instrumental methodology have been aimed a t overcoming both of these problems by combining a separation step with a suitable means of quantitative determination. A recently developed separation technique is the conversion of the organotins to their volatile hydrides, R,SnH4-,, which are then separated on the basis of their differing boiling points. This speciation method, coupled t o a hydrogen rich, hydrogen-air flame emission detector, has been used by Braman and Tompkinsso to determine methyltin species, Me,SnX4-n, in the environment.

45

4b

47 48

49

R. H. Fish, E. C. Kimmel, and J . E. Casida, in ‘Organotin Compounds: New Chemistry and Applications’, ed. J . J. Zuckerman, ACS Adv. Chem. Ser., 1976, 157, 197. R . A, Prough, M. A. Stalmach, P. Wiebkin, and J. W. Bridges, Biochem. J., 1981, 196, 763. B. Williams, L. G . Dring, and R. T. Williams, Toxicol. AppL Phamacol., 1978, 46, 567. J . G. A. Luijten, ’A Bibliography of Organotin Analysis’, I.T.R.I. Publication No. 41 7, 1970. J. W. Price and R. Smith, ‘Handbuch der Analytischen Chemie, Part 111: Tin’, Springer Verlag, Berlin, 1978. R. S. Braman and M. A. Tompkins, Anal. Chem., 1979, 51, 12.

The Environmental Chemistry of Organotin Compounds

61

Hodge et al.51 employed a similar separation procedure, but with detection by atomic absorption spectrometry, for the determination of mono- and di-methyltin and mono- and di-butyltin species at trace levels, and Soderquist and Crosbys2 determined phenyltin compounds, Phn SnX4-n, in aqueous solution by electroncapture gas-liquid chromatography, after conversion to their hydrides. Organotin speciation has also been achieved by chromatographic methods. For example, gas chromatography ( G C ) interfaced to a graphite furnace atomic absorption (GFAA) spectrometer has been used for the determination of methyltin compounds, MenSnX4-n, at environmental levels,53,54 and, later a system developed by Brinckman and his ~ o - w o r k e r s , based ~ ~ , ~ on ~ ion-exchange highpressure liquid chromatography/GFAA, for the direct speciation of di- and triorganotins in the environment. Arakawa et aLS7have used GC, with an electroncapture detector, for the determination of trialkyltins in animal tissue, and, with a hydrogen flame ionization detector, for the determination of tetraalkyltinsS8 in animal tissue. GC with a flame photometric detector has been used, for the determination of methyltin 59 and butyltin60 compounds in water. The high sensitivity and good specificity of spectrofluorescence should give it potential as an analytical technique for individual organotin derivatives in environmental samples, However, at present this method has only been applied t o triphenyltin6'-@ and trimethyltin@" compounds, A survey of analytical methods for the determination of organotin compounds in water has recently been p ~ b l i s h e db~ .~~ 4 Modes of Entry into the Environment

Since organotin compounds find such a wide variety of industrial applications, there are, consequently, a large number of pathways which can be envisaged for their entry into the environment (Table 4).

V. H. Hodge, S. L. Siedel, and E. D. Goldberg, Anal. Chem., 1979,51, 1256. C. J. Soderquist and D. G. Crosby, Anal. Chem., 1978,50,1435. 53 G.E. Parris, W. R. Blair, and F. E. Brinckman, Anal. Chem., 1977,49,378. " Y. K.Chau, P. T. S. Wong, and G . A. Bengert, Anal. Chem., 1982,54,246. s 5 F. E. Brinckman, W. R. Blair, K. L. Jewett, and W. P. Iverson, J. Chromatogr. Sci., 1977,

51

52

15,493. 57

s9

K. L. Jewett and F. E. Brinckman, J. Chrornatogr. Sci., 1981,19,583. Y.Arakawa, 0. Wada, and T. H. Yu, J. Chromatogr., 1981,216,209. Y.Arakawa, 0.Wada and H. Iwai, J. Chrornutogr., 1981,207,237. J. A. Jackson, W. R. Blair, F. E. Brinckman, and W. P. Iverson, Environ. Sci

Technol.,

1982,16,110. 6o R. J. Maguire and H. Huneault, J. Chromutogr., 1981,209,458. F. Vernon, Anal. Chim. Actu, 1974,71,192. '' S.J. Blunden and A. H. Chapman, Analyst, 1978,103,1266. 63 P. G. Baker, D. S. Farrington, and R. A. Hoodless, Analyst, 1980,105,282. 64aW. N. Aldridge and B. W. Street, Analyst, 1981,106,60. 64bA.H. Chapman, Proc. Anal. Chem., 1983,20,210. 64cS. J. Blunden and A. H. Chapman, 'A Review of the Degradation of Organoth Compounds in the Environment and their Determination in Water', I.T.R.I. Publication No. 626,1983.

Environmental Chemistry

62

Table 4 Possible direct modes of entry of organotins into the environment Medium Gir

soil

Water

Source

Species R ,SnX

Agricultural spraying. Volatilization from biocidal treatments. Antifouling paint sprays.

R,SnX, R,SnX,, and RSnX,

Incineration of organotin treated or stabilized waste materials.

R,SnX, and RSnX,

Glass coating operations - spraying of organotins on to glass at high temperatures to give SnO, films.

R,SnX

Agricultural applications.

R,SnX, R,SnX,, and RSnX,

Burial of organotin containing waste materials.

R ,SnX

Antifouling coatings. Molluscicides. Overspray from agricultural applications. Land run-off from agricultural usage. Industrial processes, e.g., slimicides in paper manufacture.

R,SnX, and RSnX,

Leaching from organotin stabilized PVC.

The principal commercial use of organotins, on a tonnage basis, is as heat and light stabilizers in rigid PVC, and this accounts for some 66% (an estimated 20 000 tons) of all organotins produced worldwide. Organotin stabilized PVC finds many applications, including potable water piping, drainage piping, food contact grade PVC (mainly beverage bottles and food packaging), roofing and glazing materials, and, more recently, window frames. Hence, the possible routes by which these stabilizers (which are exclusively the less toxic mono- and dialkyltin derivatives) may enter the environment are: ( a ) leaching/weathering, (b) land burial, and (c) incineration of waste material. Of these, atmospheric as they are release of organotins by incineration is probably not likely t o undergo thermal decomposition to inorganic tin compounds at the combustion temperatures. This method of disposal is also much less common than land filling. Leaching the organotin stabilizers from PVC has been studied in some depth, due to their use in food contact applications, for which they have been approved in many countries; this subject has been reviewed.65b The levels of stabilizers employed are typically 0.5-2.0% m/m66 and studies on the leaching rates from 65aEPA-OTS,Draft Report, ‘Assessment of the Need for, the Character of, and the Impact Resulting from, Limitations on Selected Organotins’. Phase I - Assessment of the Need for Limitations on Organotins, July, 1977, quoted in ref. 10. 6sbG. A. Senich, Polymer, 1982, 23, 1385. 66 L. R. Brecket, Pure AppL Chern., 1981, 53, 577.

The Envirolamental Chemistry of Organotin Compounds

63

potable water p i ~ i n g and ~ ~ food ~ ~ contact have shown that these are generally very low. The actual rates of loss depend upon the nature of the leachant (pH and aqueous or organic character) and the alkyl chain length of the organotin species ~ r e s e n t I .t is ~ of ~ interest ~ ~ ~ ~to note that, although leaching rates are very low, it has been found that organotin stabilized PVC is not suitable for certain medical applications, due to localized adverse tissue reactions towards the o r g a n o t i n ~ . ~ ~ , ~ ~ With regard t o the environmental fate of organotin stabilizers in PVC waste, disposed of by land-fill, little is known about, and no systematic studies have been carried out in, this area. It seems reasonable to assume that leaching by aqueous media continues to occur, but the relationships between chemical environment, soil adsorption, leaching rates, and degradation are unknown, However, it can reasonably be inferred that the waste would be distributed evenly in domestic rubbish and the concentrations involved would be very low in deed, The use of mono- and di-alkyltins as homogeneous catalysts (accounting for some 2000 tons of organotin compounds annually) in the production of polyurethane foams and silicone elastomers, and for esterification processes, results in their incorporation into the finished product at low levels, especially in terms of the final volume. A typical concentration of organotin catalyst, e.g., dibutyltin dilaurate, in a flexible polyurethane foam formulation is of the order of 0.02% r n / m 7 1 Therefore, the methods of release of these compounds will be similar to those encountered with the PVC stabilizers. From the foregoing discussion it can be concluded that, although PVC stabilizers and homogeneous catalysts together comprise the largest consumption of organotin compounds, it is unlikely that these contribute significantly t o the levels found in the environment. Thus, although the biocidal uses of organotin chemicals comprise only approximately 30% of the total world consumption, they probably give rise to the largest proportion of free organotins in the environment, due to their direct introduction into natural media. One example is the use of tri-organotin compounds as non-systemic agrochemicals (& 2000 tons worldwide), where, because of the manner in which they are applied (often aerial spraying), there is the possibility that not only the soil, but also the air and adjacent waterways, could be contaminated by air-borne sprays. Volatilization of triorganotin biocides, after application, is unlikely t o be a problem owing to the general involatility of these compounds, as reflected by their very low vapour pressures (Table 5 ) . However, volatilization by co-distillation from moist surfaces has been observed for tricyclohexyltin hydroxide, l2 67aG.R. Dietz, J. D. Banzer, and E. M. Miller, in ‘Safety and Health with Plastics’, Society of Plastics Engineers, Denver, Colorado, 1977, p. 25. 67bE. A. Boettner, G. L. Ball, Z. Hollingsworth, and R. Aquino, EPA Rep. No. EPA-600/ 1-81-062,U.S. EPA Health Effects Res. Lab., Cincinnati, Ohio, Sept. 1981. 6 8 K. Figge, W. Freitag, and W. D. Beiber, Dtsch. Lebensm.-Rundsch., 1979, 7 5 , 3 3 3 . 69 J. Heller and S. K. Brauman, Nar. Inst. Heulth Rep. NIH/DTB-79/01, No. PB-299744, Bethesda, Md., 1977. ’O W. L. Guess and T. B. Stetson, J. Am. Med. ASSOC., 1968, 204, 580. 71 S. Karpel, Tin Its Uses,1980, 125, 1.

64

E nviro nm en tal Cbe mistry

Table 5 Vapour pressures of organotin compounds Compound Ph,Sn Bu,SnOAc Bu,SnO. CO Ph (Bu,Sn),O

Vapour pressurelmm Hg

-

(cyclo- C,H II),Sn-N-C

I

H

=N-C = N

t

9.2 X lo-" 2.7 x 10-3

(25 "C) (20 "C)

1.5 X 7.5 x 1.2 x 1 0 - ~ 6.4x 1 0 - ~ 1.1 x 10-5 < 5 x 10-5

(20 "C) (20 "C) (20 "C) (20 "C) (25 " C ) (25 " C )

Ref. 72 73 14U

74a 73 74b 75

19

H

which is essentially non-volatile in the dry state, although it is not known t o what extent this occurs under field conditions, A low vapour pressure is particularly important in wood preservation, where the active tributyltin fungicide must remain for many years in the treated timber. Triorganotin compounds have been shown to adsorb strongly on to soil. Barnes et al.76 found that I4C-triphenyltin acetate could not be leached with water from a 25 cm layer of an agricultural loam over a period of 6 weeks, and subsequent analysis of the soil for radioactivity showed that over 75% of the organotin compound had remained in the top 4 cm. The adsorption behaviour of the same radiolabelled triphenyltin derivative in two types of soil has been studied by Suess and Eben,77 and of tin-113 labelled triphenyltin chloride in clay, by Leeuwangh et a1.% Strong adsorption of the triphenyltin compounds to the soil was again found, and, in the latter case,78 it was established that the ratio of Ph3SnC1 in the sediment to that in water was approximately 20 : 1. The adsorption characteristics of bis(tributy1tin) oxide and tributyltin fluoride in several soils (clay, sand, topsoil, and silt) have been examined",80 and it was observed79 that, over a 16-week period, no significant leaching of the tributyltin compounds occurred. Strong affinity of soil particles for other triorganotin

P. G . Laye, University of Leeds, unpublished work, 1982. U. Thust, Tin Its Uses, 1979, 122, 2. ','Anon., 'Specifications and Technical Data: Organotin Compounds', Schering Industrial Chemicals, Bergkamen, 1981. 74bR. J . Maguire and E. J. Hale, Can. Nut. Water Res. Znst. Rep., Sept. 1982. 7 5 Anon., 'Tributyltin Oxide: Notes on Handling and Toxicity', Tech. Service N o t e , Albright and Wilson Ltd., Oldbury, May, 1969. 76 R. D. Barnes, A. T. Bull, and R. C. Poller, Pestic Sci., 1973, 4, 305. 7 7 A. Suess and Ch. Eben, Pflanzenschultz, 1973, 80, 288. 7 8 P. Leeuwangh, W. Nijman, H. Visser, 2. Kolar, and J . J . M. de Goby, Med. Fuc. Landbouww. Rijkuniv. Ghent, 1976,41/2, 1483. 79 L. R. Harris, C. Andrews, D. Burch, D. Hampton, and S. Maegerlein, Report No. DTNSRDC/SME-78/2A, D. W. Taylor Naval Ship R . and D. Centre, Bethesda, Md., Sept., 1979. 8o D. Barug and J . W. Vonk, Pestic. Sci., 1980, 11, 77. 72 73

The Environmental Chemistry of Organotin Compounds

65

derivatives has also been demonstrated.81,82It is therefore likely that, after spray application of triorganotin biocides, most of these will be removed by adsorption on to the soil before they can enter surface waters. Additionally, it has been shown83a that up t o 100 p.p.m. of bis(tributy1tin) oxide has no adverse effect on soil microorganisms or on their ability to maintain soil fertility. The interaction of the methyltin chlorides, MenSnCLpn, with humates, peat, and related materials, using the tin-113 labelled compounds, has recently been studied.83b Direct entry of organotins into the aqueous environment is primarily due to their use in agriculture and in antifouling paint systems. In agricultural applications, contamination could result from run-off water and overspray, as mentioned previously, whereas, in marine antifouling paints M,85 and in antifouling rubber coatings,& which utilize some 3000 tons of tributyl- and triphenyl-tin derivatives annually, the triorganotin compounds are released directly from the paint or rubber matrix into the water in the vicinity of the ship’s hull and subsequently dispersed. In this respect, the release of the triorganotin biocide in a static environment, e.g., harbours and bays, is likely to be more significant than its loss when the vessel is moving in the open sea. Although the fate of the organotin compounds released in this way is not known, it has been proposed by Good e t al. 87 that triorganotin hydroxides are formed. A further related mode of entry of tributylin compounds into the fresh water environment is their probable future use in slow release rubber pellets for the control of the snail vectors of schistosomiasis.86~88~89 Other possible modes of entry of organotins into the environment (Table 4) result from low tonnage uses, e.g. , anthelmintic treatments for poultry, disinfectants, etc., and are therefore not considered to have a significant impact on environmental levels. 5 Aqueous Chemlatry

Studies of the nature of organotin compounds in aqueous solution have mainly been limited to methyltin derivatives, since other organotins do not have a sufficiently high solubility in water to permit spectroscopic investigation. The water solubilities of some triorganotin compounds are given in Table 6 . In general, the aqueous solubility of tri-n-alkyltin species decreases as the alkyl chain length increases. However, exceptions to this are known, e.g.,

A. E. Slesinger, Proc. 17th Ann. Marine Coatings Conf., Biloxi, , Miss., March, 1977. T. Katsumura, JP. 21 360 165/1975. 83aW. B. Bollen and C. M. Tu, Tin Its Uses, 1972, 94, 13. 83bM.Omar and H . J. M. Bowen, J. Radioanal. Chem., 1982, 7 4 , 2 7 3 . 34 C. J. Evans and P. J . Smith, J. Oil Col. Chem. ASSOC., 1975, 58, i60. 8 5 C. J. Evans and R. Hill, Rev. Si, Ge, Sn and Pb Compds., 1983/4, 7, 57. 86 N. F. Cardarelli, ‘Controlled Release Pesticide Formulations’, Chemical Rubber Co., Cleveland, Ohio, 1976. 87 C. P. Monaghan, J . F. Hoffman, E. J . O’Brien, L. M. Frenzel, and M. L. Good, Proc. 4th Int. Control. Rel. Pestic. Symp., Oregon State Univ., Oregon, Aug., 1977. 8 8 N. F. Cardarelli, in ‘Molluscicides in Schistosomiasis Control’, ed. T. C. Cheng, Academic Press, New York, 1974, p. 177. 89 J. Duncan, Pharmacol. Ther., 1980, 10, 407. 81

82

66

Environmental Chemistry

Table 6 Aqueous solubilities of triorganotin compounds at room temperatu re Soh bility */p.p .m.

R,SnX

- 35 000

Et,SnOH Et ,SnOAc Pr ,SnC1 (PI 3 w 2 0 P c SnCl Bu ,SnF Bu ,SnC1

Bu ,SnOAc

(Bu,Sn),S Bu,SnSO ,Me Bu,SnSO,Et Ph,SnF Ph,SnCl

-

-

7500 50 50 25 6 4.5 50 17t 16 5.4 2567 657 50 16 6.4 5 19.5f 1 8-t 8 8-10 3 1.4 <1 3 1OOOf 29 OOOt 1.2 5.2

<1

19

1.27 1.2.f <1

Ph,SnOAc

<3.3t

(cyclo-C,H,,),Sn-N-C

2.9 <1 <1 13007 700T

<1

1

H ((PhMe,CCH,),Sn],O

=N-C = N

I

93

<1

<1

I

90 91 91 91 31 92 93 91 94 94 95 94 91 94 93 95 95 94 94 96 91 95 91 97 97 92 93 91 32 38 91 76 99 91 91 100 100 91 18

Ph,SnOH

(Ph 3Sn) 2 0 Ph,SnSO,Et Ph,SnSO,Ph Oct ,SnC1 (cyclo-C,H, ,),SnOH

Ref

H

<0.005

101

* Solubility values marked t refer to distilled water; unmarked refer to seawater.

The Environmental Chemistry of Organotin Compounds

67

B U ~ S ~ S Oin~ which R , ~ the ~ organic radical is highly hydrophilic. In the case of triphenyltin compounds, the measured solubilities have, in general, been of the same order of magnitude, and Soderquist and Crosby suggested 98 that the solubility of triphenyltin species should be independent of counterion when bonding is primarily ionic (e.g., Cl-, Br-, OAc-). It has been demonstrated that trimethyltin compounds in aqueous solution exist, at pH less than 5 , primarily as the trimethyltin cation, Me3Sn+, and, at higher pH, as Me3SnOH.lm The trimethyltin cation has been shownlW to be hydrated and to have a trigonal bipyramidal tin atom geometry with axial water molecules (1 ; R = Me) and l19Sn n.m.r. studies105aof a 0.05M aqueous solution of Bu3SnS03Et revealed that the organotin species present in solution, at pH 2.5, has structure ( 1 ; R = Bu). In addition, the hydrated tributyltin cation, { B U ~ S ~ ( H ~ Ohas ) ~ )recently +, been demonstrated crystallographicallylM to have structure (1 ; R = Bu). It has been shown93-95that the presence of chloride from

(1)

seawater inhibits the solubility of tributyl- and triphenyl-tin compounds (Table 6), probably by association with the cation to form the covalent organotin chloride.93

P. N. Heron and J. S. G. Sproule, Indian Pulp Paper, 1958, 510. H. P. Vind and H. Hochman, Proc. Am. Wood Pres. Assoc., 1962, 5 8 , 170. 92 C. Beiter, J. E. Engelhart, A. Freiman, and A. W. Sheldon, Proc. ACS Symp. Mar. Freshwaterpestic., Atlantic City, New Jersey, Aug., 1974. 93 M. Ozcan and M. L. Good, Proc. ACS Div. Environ. Chem., Houston, Texas. March, 1P80. 94 A. H. Chapman, I.T.R.I., unpublished work, 1981. 9 5 L. Chromy and K. Uhacz, J. Oil Col. Chem. ASSOC., 1978, 61, 39. 96 H. A. Meinema, T. Berger-Wiersma, G. Versluis-de Haan, and E. Ch. Gevers, Environ. Sci. Technol., 1978, 12, 288. 91 S. J. Blunden, I.T.R.I., unpublished work, 1981. 98 C. J. Soderquist and D. G. Crosby, J. Agn’c. Food Chem., 1980, 28, 111. 99 A. H. Meyling and R. J. Pitchford, Bull. World Health Org., 1966, 34, 141. 90 91

p . J. Smith, ‘Structure/Activity Relationships for Di- and Tri-Organotin Compounds’, I.T.R.I. Publication No. 569, 1980. lo’ Anon., ‘Vendex Miticide’, Tech. Data Bull., Shell Chemical Co., San Ramon, California, 1973. l o 2 S. J. Blunden, A. H. Chapman, A. J. Crowe, and P. J. Smith, Int. Pest Control, 1978, 2 0 (July/Aug.), 5 . l o 3 R. S. Tobias, in ‘Organometals and Organometalloids: Occurrence and Fate in the Environment’, ed. J. M. Bellama and F. E. Brinckman, ACS Symp. Ser., 1978,82, 130. l o 4 F. E. Brinckman, G. E. Parris, W. R. Blair, K. L. Jewett, W. P. Iverson, and J. M. Bellama, Environ. Health Perspect., 1977, 19, 11. J. Blunden, Ph.D. Thesis, University of London, 1981. losbA. G. Davies, J. P. Goddard, M. B. Hursthouse, and N. P. C. Walker, J. Chem. SOC., Chem. Commun., 1983,597. 100

E nviro n men tal Chemistry

68

Table 7 Aqueous solubilities of diorganotin compounds at room temp erature Solubility */p.p.m.

R,SnX,

20 000 92.t 50 6-8

Me,SnCl, Bu,SnCl,

4 6

Bu,Sn(OAc), Bu,Sn(O - CO . nC, IH23)2 Bu,Sn(O .CO - Oct'), (Bu,SnO - CO * CH :CH - CO - 0-ln Oct,SnCl, Ph,SnCl,

* Solubility values marked t

3 6 6
Re$ 91 34 91 96 94 91 91 91 91 91 13

refer t o distilled water; unmarked refer to seawater.

PH

M and 1 0 - 2 M soluFigure 6 Species distribution as a function of p H for tions of Me2Snz+. (Reproduced with permission from ACS Symp. Ser., 1978, 82, 130)

Although there is less data available o n the water solubilities of diorganotin compounds (Table 7), it appears that they follow a similar pattern to the triorgan otins. Speciation of dimethyltin compounds in aqueous solution, with respect to pH, has been investigated,lm and the pattern found to be more complicated

The Environmental Chemistry of Organotin Compounds

69

than exists with trimethyltin derivatives (Figure 6 ) . The dimethyltin cation, present at low pH, has been found to be hydrated and has an octahedral tin atom geometry with trans-methyl groups (2; R = Me).106,107However, at environmental pH (6-8), the main species is seen to be Me2Sn(OH)2(Figure 6 ) . Very few studies have been made of the hydrolysis products of the monoalkyltin derivatives and it has been suggestedlW that these exist in solution only

r

R

2+

as the hydrated oxides, However, recent worklo8 with methyltin trichloride has shown that, at pH 1.4, a concentration dependent equilibrium exists [reaction (l)]. At low concentrations ( 5 ) predominates, is undoubtedly hydrated and, is presumably in equilibrium with other hydroxy derivatives [reaction (2)]. As the pH is reduced, the species to the right are increasingly favoured, and, from Tobias’ worklo3 with tri- and di-methyltin compounds, it might be suggested that, at environmental pH, MeSn(0H)S will predominate. MeSn(OH)C12 - 2H20+MeSn(OH)2C1- nH20 (3 1 (4 1

*

* (MeSn(OH))2+

MeSn(OH)3+M C ~ ~ ( O H MeSn(OH)2+ )~+ =+MeSn3+

(1)

(51

(2)

Finally, it is of interest to describe the nature of inorganic tin species in aqueous solution, since these could be relevant to the transformations of tin compounds in the environment, which are discussed in the next section. Pettine et al, studiedlog the hydrolysis of stannous salts in seawater, from 0.1 to l.OM, at 2 0 ° C , and found that, at pH 8.1, the predominant species is Sn(OH)2. In a similar investigation of stannic nitrate,l1° at a 8 x g ion 1-1 level, Nazarenko et al. concluded that, above pH 3 , the inorganic tin(1V) hydroxo-complex was primarily Sn(OH)4. Hence it may be concluded that, at environmental pH, tin compounds, RnSnX4-n (n = 0-3), and SnX2, will exist in aqueous solution as simple neutral hydroxides.

M. M. McGrady and R. S. Tobias, Inorg. Chem., 1964,3,1157. M. M.McGrady and R. S. Tobias, J. Am. Chem. SOC.,1965,87, 1909. S. J . Blunden, P. J . Smith, and D. G. Gillies, Inorg. Chim. Acta, 1982,60, 105. l o 9 M. Pettine, F. J. Millers, and G. Macchi, Anal. Chem., 1981,53, 1039. ‘ l o V. A. Nazarenko, V. P. Antonovich, and E. M. Nevshaya, RUSS. J, Inorg. Chem., 1971, Io6

lo’

16,980.

Environmental Chemistry

70

6 Transformations in the Environment Since the pioneering work by Challenger on the biomethylation of arsenic, selenium and i r 0 n , l ~ ~ -there ~ ~ 3has ~ been considerable interest in the possible routes for environmental methylation of other metalloids and heavy metals.'14a The advances in analytical techniques for tin, described in Section 3 , have enabled the presence of five independent groups in N. America to report50~51~54~59~114b methyltin species, MenSnX4-,, where n = 1-4,in natural media (e.g., seawater, lake water and sediments) at very low (p.p.b.) levels. In this context, it is of interest to note that, in the U.S.A., there is a large utilization (-3000 tons in 1981)" of di- and mono-methyltin derivaties as stabilizers for PVC potable water pipe (see Table 1). Some of these methyltin species which have been detected could, theref ore, possibly result from leaching of the organotins. The first laboratory studies to be conducted on the methylation of tin in the environment were carried out in 1974 by Brinckman and his ~ o - w o r k e r s , ~ ~ ~ using pure cultures of tin-resistant Pseudomonas bacteria from Chesapeake Bay. Inoculation with SnC14 5 H 2 0 ied to the production of what was believed t o be a dimethyltin species. Later experiments by the same workers59 showed that in vitro biomethylation of inorganic tin (IV) by the same bacterial strain produced methylstannanes, MenSnH4-,, where n = 2-4. The formation of methyltin compounds in sediments has recently been described:116-118both SnClz and SnC14 were found t o be methylated, and, in the study by Hallas,''* the species formed from SnC14-5H20 were the hydrides, Me2SnH2 and Me3SnH (on the basis of gas-chromatographic retention times and mass spectral data). Trimethyltin chloride117 and hydroxide116 are converted t o tetramethyltin in sediments and this process appears to be slow. Since Me4Sn was also produced from Me3SnOH in sterile sediments, the tetraalkyltin derivative could be formed U ~ Qa chemical pathway.l" This may be due t o disproportionation of the initially formed bis( trimethyltin) sulphide119 [reaction ( 3 ) J . However, a 3 Me3SnSSnMe3+= (MezSnS)3-t 3 Me4Sn (3) study of tricylohexyltin hydroxide in soils under aerobic and anaerobic conditions, indicated an absence of methylated products.120

-

F. Challenger and C. Higginbottom, Biochem. J., 1935, 29, 1957. F. Challenger, Quart. Rev. Chem. Soc., 1955, 9, 255. '13=F. Challenger, in 'Organometals and Organometalloids: Occurrence and Fate in the Environment', ed. F. E. Brinckman and J . M. Bellama, ACS Symp. Ser., 1978,82, 1. F. Challenger, 'Aspects of the Organic Chemistry of Sulphur', Butterworths, London,

1959. S. Thayer and F. E. Brinckman, Advan. Organometal, Chem., 1982, 20, 313. '4I' J . T. Byrd and M. 0. Andreae, Science, 1982, 218, 565. 11s C. Huey, F. E. Brinckman, S, Grim, and W. P. Iverson, Proc. Inf. Con$ Transp. Persist. Chem. Aquat. ECOS.,Ottawa, 1974, p. 73. 116 H. E. Guard, A. B. Cobet, and W . M. Coleman, 111, Science, 1981, 213, 770. I * ' Y . K. Chau, P. T. S. Wong, 0. Kramar, and G. A. Bengert, in 'Proc. Internat. Conf. Heavy Metals in the Environment (Amsterdam)', ed. W. H. 0. Ernst, CEP Consultants, Edinburgh, 1981. L. E. Hallas, J . C. Means, and J . J. Cooney, Science, 1982, 215, 1505. P. J. Craig and S. Rapsomanikis, J. Chem. SOC.,Chem. Commun., 1982, 114. Anon., '1973 Evaluations of Some Pesticide Residues in Food, The Monographs', WHO Pesticide Residues Series, No, 3 , WHO, Geneva, 1974, p. 448.

""J.

The Environmental Chemistry of Organotin Compounds

71

There have been a number of laboratory investigations using methylcobalamin,121,122 the methyl coenzyme of cyanocobalamin (vitamin B12), or methyl iodide,l17 which is produced by certain seaweeds and algae,123 as chemical methylating agents for tin( 11) and tin( IV) compounds. Although methylcobalamin does not react with tin(1V) chloride,122 ic has been reported by Wood and his co-worke:rs121,122to be demethylated by SnC12 in aqueous HCl solution, in the presence of an oxidizing agent (Fe3+ or Co3+), to form monomethyltin trichloride. Although the alkyltin derivative was suggested to be MeSnCl3, on the basis of 270 MHz 'H n.m.r., detailed n.m.r. studieslo8of this compound in aqueous solution are not fully in accordance with this assignment, Thayer has recently reported124,125 that methylcobalamin reacts with finely divided Sn02 to form methyltin species, but the reaction appears t o be slow, The same author ~ t u d i e d ' ~ ~the , ' ~reaction of four organotin compounds (Me3SnOAc, Et3SnOAc, Ph3SnOAc, and Me2SnCl2) with methylcobalamin, although the products were not identified. Chau and his co-workers found117 that methyl iodide could methylate inorganic tin(1I) salts in an aqueous medium t o form monomethyltin(1V) species, along with small amounts of di- and trimethyltin derivatives, whereas tin (IV) compounds did not react. In summary, it may be concluded that, whereas both tin(I1) and tin(1V) compounds appear t o be biologically methylated under simulated environmental conditions in sediments, chemical methylation may only be important for tin(I1) species. Thus, with regard to the question of methylation of tin in the environment, there appears t o be circumstantial but no direct unequivocal evidence for this phenomenon.'17, 12' Although it is too early to assess the environmental significance of these findings in detail, Brinckman has described" a simple 'through-put' model for the occurrence and fate of methyltin species (Figure 7). The importance of degradation processes as a mode of removal of organotins, including the methyltin derivatives, is discussed in the next Section.

7 Degradation of Organotin Compounds

Owing t o the widespread industrial uses of organotins, along with their biological activity and the possibility of biomethylation, their fate in the environment is of considerable interest. Consequently, work has been carried out over a number of years t o investigate the possible ways in which organotin compounds may degrade in the environment. L. J . Dizikes, W. P. Ridley, and J. M. Wood, J. Am. Chem. Sac., 1978, 100, 1010. Y. T. Fanchiang and J . M. Wood, J. Am. Chem. Soc., 1981, 103, 5 1 0 . P. J . Craig and S. Rapsomanikis, in 'Proc. DoE/NBS Workshop Environ. Spec. Monitor. Needs', ed. F. E. Brinckman and R. H. Fish, Natl. Bur. Stand. (U.S.) Spec. Publ., No. 618, U.S. Dept. Commerce, Washington D.C., 1981, p. 54. 124 J. S. Thayer, i n 'Organometals and Organometalloids: Occurrence and Fate in the Environment', ed. F. E. Brinckman and J. M. Bellama, ACS Symp. Ser., 1978,82, 188. J. S. Thayer, Atrstr. 10th Internat. Con$ Organomet. Chem., Toronto, Aug., 1981. 1 2 6 J . S. Thayer,Inorg. Chem., 1979, 18, 1171. P. J . Craig, Environ. Tech. Lett., 1980, 1, 2 2 5 .

Environmental Chemistry

72 SEWAGE

DEGRADATION METHYLCOBALAMIN

INDUSTRY

(MICROBIAL, PHOTOLYTIC)

Me,Sn"-"'+

P

(Me2SnX2+ MeSnX,)

7

\=3

MICROORGANISMS

MeHg++ Me2Snz+

BIOGEOCHEMICAL

Figure 7 Environmental throughput of methyltins. (Based on ref. 11) The degradation of an organotin compound may be defined as the progressive removal of the organic groups from the tin atom [reaction (4)]. This stepwise loss of organic groups from the tin atom is attractive to potential users of organotin compounds because it is accompanied by a progressive lowering in biological activity (Section 2), and, as such, the use of these compounds would be unlikely to lead to environmental pollution. R4Sn + R3SnX + R2SnX2+ RSnX3+ SnX4

(4)

Early s t ~ d i e s ' ~ * -of' ~the ~ fate of organotins emphasized the 'disappearance' of the compounds, either after having been applied to crops or on exposure to ultraviolet (u.v.) light, and it is only recently that attempts have been made to establish the mode of degradation and to identify the breakdown products. Degradation involves the breaking of a Sn-C bond and this can occur by a number of different processes. These include: ( a ) U.V. irradiation; ( b ) biological cleavage; (c) chemical cleavage; ( d ) y-irradiation; (e) thermal cleavage. Of these processes, y-irradiation will have little effect on environmental degradation owing to its negligible intensity a t the earth's surface. I t is worth noting, however, that the importance of studying the effects of 7-irradiation on organotin compounds may increase because of its possible use for the sterilization of food, some of which is packaged in organotin stabilized PVC, and Dunn and Oldfield131 have shown that y-radiation will degrade tributyltin compounds. Thermal cleavage is also unlikely to be of environmental significance, because the Sn-C bond is reported to be stable at temperatures up to 200 OC.l0 Therefore, only processes (a), ( b ) ,and ( c ) are considered below, ( a ) U.V. Irradiation. - Within the electromagnetic spectrum, U.V. light contains wavelengths in the range 10-400 nm and is adjacent to visible light, which extends from 400 to 700 nm. The wavelengths of U.V. radiation below 200 nm are, however, absorbed by air and so are only transmitted in a vacuum. The

'" J. Bruggemann, K. Barth, and K. H. Niesar, Zenfralbl. Veterinamzedzin, 1964, 11, 4. 129

130

13'

J. Bruggemann, 0. R. Klimmer, and K. H. Niesar, ZentruZbZ. Veterinamzedzin, 1964, 11, 40. H. J . Hardon, A. F. H. Besemer, and H. Brunik, Dtsch. Lebensm.-Rundsch., 1962, 58, 349. P. Dunn and D. Oldfield, Aust. DeJ S c i Ser., Tech. Note No. 298, 1973.

The Environmental Chemistry of Organotin Compounds

73

light emitted by the sun, reaching the earth's surface, consists mostly of wave lengths above 290 nm, although it has been estimated that the quantum flux density below 290 nm is about 10l6photons cm-2 month-l, and therefore a long term effect of the shorter wavelengths may be 0 b ~ e r v e d . lU.V. ~ ~ light of wavelength 290 nm possesses an energy of approximately 300 kJ mo1-I and the mean bond dissociation energies for some Sn-C bonds are found133to be in the range 190-220 kJ mol-'. Therefore, provided that absorption of the light takes place, Sn-C bond cleavage could occur, and, in fact, the maximum absorption wavelength of organotin compounds is generally within the U.V. region. Many studies have been made of various photochemical reactions of organotins and these have been discussed previously.134-136 However, it is only in the last ten years that the emphasis of some work has been placed upon,investigating degradation processes and this summary is limited t o those studies that are of direct environmental interest. Cenci and Cremonini13' investigated the rate of disappearance of triphenyltin acetate and triphenyltin hydroxide in different types of soil when exposed to U.V. light, without identifying the nature of the breakdown products. Getzendaner and C ~ r b i n studied ' ~ ~ the U.V. degradation of tricyclohexyltin hydroxide, suggesting that dicyclohexyltin, monocyclohexyltin, and inorganic tin species were produced, but no further details were given. Akagi and Sakagami139 irradiated several triphenyl-, dibutyl-, and dioctyl-tin compounds on a watch glass and concluded that they all decomposed to inorganic tin. The breakdown of the triphenyltin compounds was suggested to occur stepwise, i.e., reaction ( 5 ) . Barnes et aE.76 showed that triphenyltin acetate was degraded t o inorganic Ph3SnX + Ph2SnX2-+PhSnX3 +inorg. Sn

(5)

tin by U.V. light, while Massauxlm irradiated triphenyltin chloride on a watch glass with light wavelengths of 254nm and 350nm, showing that decomposition occurred faster at the shorter wavelength. A similar study was made by Chapman and Price141 for triphenyltin acetate. In this case, irradiations were carried out using light of wavelengths greater than 235 nm and greater than 3 5 0 nm, and diphenyl-, monophenyl-, and inorganic tin species were identified and quantitatively determined. Again, degradation was shown to occur at a faster rate with the shorter wavelength light. In a similar with bis(tributyltin) oxide and tri-n-butyltin acetate, the pattern of degradation was found to follow that observed for the triphenyltin compounds. The u.v.-induced D. A. M. Watkins, Chem. Ind., 1974, 185. H. A. Skinner, Adv. Owanomet. Chem., 1964, 2 , 49. 134 R. C. Poller, Rev. Si, Ge, Sn and Pb Compds., 1978, 3 , 243. 1 3 5 A. G . Davies, in 'Organotin Compounds: New Chemistry and Applications', ed. J . J. Zuckerman, ACS Adv. Chem. Ser., 1976, 15 7 , 2 6 . 1 3 6 A. G. Davies, J. Organomet. Chem. Library, 1981, 12, 181. 1 3 7 P. Cenci and B. Cremonini, Ind. Sacc. Ital., 1969, 6 2 , 313. 13' M. E. Getzendaner and H. B. Corbin, J. Agric. Food. Chem., 1972, 2 0 , 881. 1 3 9 H. Akagi and Y . Sakagami, Bull. Inst. Publ. Health Jpn., 1971, 2 0 , 1. 140 F. Massaux, Cafe'Cacao Thk, 1971, 15, 221. 14' A. H. Chapman and J . W. Price, Int. Pest Control, 1972, 14 (JanJFeb.),11. 14' A. H. Chapman and J. W. Price, I.T.R.I., unpublished work, 1973. 13'

133

En vir u n rn en t a1 Che rn istry 74 decomposition of bis(tributyltin1 oxide has also been studied by K l O t ~ e r l ~ ~ under a variety of conditions of temperature, irradiation intensity and on different matrices. The same author found144 that the relative rates of breakdown of tributyltin chloride and bis(tributy1tin) oxide were similar, while dibutyltin dichloride broke down faster than dibutyltin oxide. These studies were, however, only concerned with the decomposition of the starting compounds and did not suggest possible products or follow the reaction through to inorganic tin. The U.V. breakdown of bis(tributy1in) oxide has also been reported by Komora and P 0 p 1 ' ~and ~ Woggon and Jehle.146 The study of the U.V. degradation of organotins in aqueous solution has not received much attention, presumably because their low aqueous solubility makes the analysis particularly difficult. In a paper reporting the rates of hydrolysis of organotins, Mazaev14" noted that irradiation of aqueous solutions of diethyltin dicaprylate and dibutyltin bis(SS'-isooctylthioglycollate) produced an increase in the rate of disappearance of the compounds compared t o non-irradiated solutions. Soderquist and Crosbyg8reported the action of U.V. light on triphenyltin hydroxide in water, showing that, under simulated environmental conditions, a diphenyltin species was produced, which underwent further breakdown, possibly forming a polymeric monophenyltin derivative, (PhSnO, H,,).. A study of the U.V. degradation of the methyltin chlorides, M ~ , S I I C I ~ -in ~ , aqueous solu tion147bhas established the approximate relative rates of breakdown of the methyltin species, and the final product of the reaction was shown to be hydrated tin( IV) oxide.

( b ) Biological Cleavage. - The question of biodegradation of organotins is particularly important in situations where, for example, the compounds are not directly exposed to light, e.g., in the soil or on the sea bed. I t has been shown by Barnes et ai.76 that 14C labelled triphenyltin acetate in soil is broken down to inorganic tin. Since carbon dioxide was evolved, and breakdown did not occur in sterile soil, it was concluded that degradation was due to the ability of certain microorganisms to metabolize the organotin compound. The degradation of 14C labelled triphenyltin acetate in soil has also been studied by Suess and Eben." Barug and VonkSO have shown that bis(tri{l-l4C)butyltin) oxide undergoes breakdown in soil, due to the action of microorganisms, and it is also reported43 that tricyclohexyltin hydroxide will degrade in soil. Sheldon reportediw the breakdown of 14C labelled bis(tributy1tin) oxide, tributyltin fluoride and triphenyltin fluoride in soil, showing that degradation occurred faster under aerobic than anaerobic conditions. Additionally, the biodegradation of bis(tributy1rin) oxide to inorganic tin by biological sewage treatment has been studied by Stein and K U ~ t e r . ' ~ ~ ~ D. Klotzer and U. Thust, Chem. Tech. (Leipzig), 1976, 28, 614. D. Klotzer, Zentral. Ins?. Kernforsch. Rossendorf Dresden zfk., 1977, 340, 84. V. F. Komora and M. Popl, Holzrech., 1978, 19, 145. * 4 6 H. Woggon and D. Jehle, Nahrung, 1975, 19, 271. 147aV.T. Mazaev, P. V. Golovanov, A. S. Igumnov,and V. H. Tsay, Gig. Sanit., 1976, 17. 147bS. J . Blunden, J. Organomet. Chem., 1983, 248, 149. I4"A. W. Sheldon, Proc. 18th Annual Marine Coatings Conf., Monterey, California, March, 1978. 14sbV.T. Stein and K. Kuster, 2. Wasser A bwasser Forsch., 1982, 15, 178. 14'

144 145

The Environmental Chemistry of Organotin Compounds

75 The degradation, in wood, of bis(tributy1tin) oxide149-1soband tributyltin naphthenatelSobhas been examined, and di- and mono-butyltin species have been detected as breakdown products. This process could be due to fungal degradation, since Henshaw et demonstrated that pure cultures of the wood rotting fungi, Coniophora puteana (cerebella) and Coriolus (Polystictus) versicolor, will degrade bis(tributy1tin) oxide, forming di- and mono-butyltin derivatives. Other microorganisms that have been shownlS1 to dealkylate bis(tributyltin) oxide are pure cultures of the fungi, Tramatis versicolor and Chaetomium globosurn, and the Gram-negative bacteria, Pseudomonas aeruginosa and Alcaligenes faecalis. In contrast, certain Gram-negative bacteria have been foundlS1 not to degrade bis(tributy1tin) oxide, and Brinckman e t al. lS2 have recently demonstrated the exclusive uptake of the tributyltin cation, Bu3Sn(H20)i, by a number of Gram-negative tin resistant heterotropes, without breakdown. There is, however, the additional possibility that organotin degradation in wood is due to acid cleavage, because both formic and acetic acids may be present in some types of timber.lS3 ( c ) Chemical Cleavage. -The Sn-C bond is capable of polarization in either direction, (Sn(6 +)-C(6 -) or Sn(6 -)-C(S +)),lS4 and is therefore susceptible to attack by both nucleophilic and electrophilic reagents. Hence, for reactions of the type $Sn-Cf

+ A-B

-+

+Sn-A

+ >C-B

A-B may be one of a wide variety of compounds, e.g., mineral acid, carboxylic acid, alkali, etc. In addition, the trimethyltin cation has been shown t o transmethylate with various hydrated metal cations, e.g., T13+, Pd2+, Au3+, Hg2+, forming a dimethyltin species and the corresponding monomethyl-metallic derivative.'l Free radical processes can cause homolytic Sn-C bond fission, the Sn-C bond being a fairly good radical trap.lS4 Owing to the very wide range of chemical reactions that result in Sn-C bond cleavage, a discussion is outside the scope of this review. However, these reactions are described e l s e ~ h e r e . ~ ~ ~ ~ From the breakdown studies that have been reviewed here and elsel56 it may be concluded that, within a generally consistent pattern of behaviour, organotins will degrade in natural media, and this has been demon, ~ ~ t r i c y ~ l o h e x y l t i n 'compounds. ~~ strated for t r i ~ h e n y l t i n ,lS7 ~ ~t, r i b ~ t y l t i n and A suggested breakdown route for triphenyl- and tributyl-tin compounds, which B. G. Henshaw, R. A. Laidlaw, R. J. Order, J. K. Carey, and J. G. Savory, Record of the 1978 Annual Convention of the B. W.P.A., Cambridge, 1978, p. 19. l s o a H . Plum and H. Landseidel, Holz. Roh. Werkstoff, 1980, 38,461. l s o b M . L. Edlund, W. Hintze, J . Jermer, and S. Ohlsson, Swedish Wood Preservation Institute, Stockholm, Rep. No. 143, 1982. D. Barug, Chemosphere, 1981, 10,1145. l S 2 W. R. Blair, G. J. Olsen, F. E. Brickman, and W. P. Iverson, Microb. Ecol., 1982, 8, 241. L. E. Wise, in 'Wood Chemistry', ed. L. E. Wise and E. C. Jahn, Reinhold, New York, 1952, Vol. I. 1 5 4 W. P . Neumann, 'The Organic Chemistry of Tin', Wiley, New York, 1970. R. C. Poller, 'The Chemistry of Organotin Compounds', Academic, New York, 1970. l S 6 S. J. Blunden and A. H. Chapman, Environ. Tech. Lett., 1982, 3, 267. Is' C. P. Monaghan, V. I. Kulkarni, M. Ozcan, and M. L. Good, U.S. Govt. Report, Office of Naval Research, Tech. Rep. No. 2, 1980, AD-AP87374. 14'

76

U V or microorganisms

organisms

OH

I

(RSnO-1

1

UV or microorganisms

SnO, Figure 8 Errvironmental degradation scheme for tributyl- and triphenyl-tin compounds. (Based on ref. 158) probably applies to other organotin compounds, is shown in Figure 8.158Bis(tributyltin) oxide, formed by hydrolysis of the tributyltin derivative, Bu3SnX, is shown to form bis(tributy1tin) carbonate, since it is known159 that bis(tributyltin) oxide reacts readily with carbon dioxide. The effect of the anionic radical, X, in an RnSnX4-n species has not been considered, because very little is known about how this affects the breakdown. However, at the levels that the organotins will be found in the environment, they will either exist as, or will be rapidly converted to, oxides or hydroxide^,'^ carbonates, lS9 or hydrated cations. lo4, lo37

8 Summary

In general, most commercially used organotins are characterized by relatively low mobilities in environmental media, having low vapour pressures, low aqueous solubilities and high affinities for soil and organic sediments. While it is true to say that some commercial organotins, particularly the biocidal triorganotin species, have acute effects on plant and animal life, the exposures are mainly localized, and result from direct applications or accidental spillage. In addition, although not mentioned in Section 2, no carcinogenic effects have been shown by those compounds tested t o date (Table 8), and it is of interest to note that a similar study for bis(tributy1tin) oxide is under way.165 I t has also been shown that organotins will degrade under environmental conditions (Section 7) and so a serious long term pollution hazard should not occur. '59

A. W . Sheldon, J. Paint Technol., 1975, 47, 54. P. J . Smith, A. J . Crowe, D. W. Allen, J . S. Brooks, and R. Formstone, Chern. Ind., 1977, 8 74.

The Environmental Chemistry of Organotin Compounds Table 8 Carcinogenicity studies on organotin chemicals Compound Ph,SnOAc Ph ,SnOH

Bu ,SnF (cyclo-C,H, ,),SnOH Bu,Sn(OAc),

((PhMe,CCH,),Sn) 2O

Species Mice (male and female) Mice (male and female) Rats (male and female) Mice (male) Rats (male and female) Mice (male and female)

77

Q p e of test

Result

Ref.

18 month feeding study 78 week feeding study 78 week feeding study 6 month dermal study 2 year feeding study 78 week feeding study

Negative

160

Negative

16 1

Negative

16 1

Negative

158

Negative

162

No conclusive 163 evidence fox carcinogenicity Rats 78 week No conclusive 163 (male) feeding study evidence for carcinogenicity Mice ’ 18 month Negative 164 (male and female) feeding study

However, in certain specialized areas, e.g., harbours, due t o continuous release of organotins from antifouling paints, coupled with their low mobility, short term elevated concentrations of organotins could occur. The importance of the possible environmental methylation of tin is still open to discussion, but the levels of methyltins reported, so far, do not justify concern, Although there has been much interest in the environmental chemistry of organotin compounds, it is apparent that there is still scope for further research. In this context, it is of interest to note that, in 1978, an association of world organotin manufacturing companies - The Organotin Environmental Programme (ORTEP) Association - was formed to promote and foster the dissemination of scientific and technical information on the environmental effects of organotin compounds,166 Acknowledgements. - The International Tin Research Council, London, is thanked for permission to publish this review, The authors would like to express their appreciation to Drs. P. J. Craig (Leicester Polytechnic), F. E. Brinckman (U.S. National Bureau of Standards), and the late Professor F. Challenger, for valuable comments on the manuscript. I6O

J. R. M. Innes, B. M. Ulland, M. G. Valerio, L. Petrucelli, L. Fishbein, E. R. Hart, A. J . Palotta, R. R. Bates, H. L. Falk, J . J . Gart, M. Klein, I. Mitchell and J . Peters, J. Natl.

Cancer Inst., 1969,42, 1101. Anon., U.S. Natl. Cancer Inst. Carcinogen. Tech. Rep. Ser., 1979, No. 139. 16’ Anon., ‘1970 Evaluations of Some Pesticide Residues in Food’, FAO/WHO, Rome, 1971, p. 527. 163 Anon., U.S. Natl. CancerInst. Carcinogen. Tech. Rep. Ser., 1979, No. 183. Anon., ‘1977 Evaluations of Some Pesticide Residues in Food’, FAOWHO, Rome, 1977, p. 232. K. E. Mott, WHO, Geneva, personal communication, 1983. Anon., Tin Its Uses, 1978, 116, 14.

3 Determination of Heavy Metals in Sewage Sludge S. A. K A T Z 1 Introduction

The disposal of municipal sewage sludge has become a major environmental problem. Municipal sewage treatment works in the United States generate some 20 t o 2 5 thousand tons of sludge per day, The ultimate fate of this material is land fill burial (40%),land application (20%), or ocean dumping (10%).Some sludge (30%) is incinerated, and its ash is discarded to the land and sea. Regardless of the disposal procedure, the heavy metals in the sludge are a potential hazard. Ground water contamination by land fill leachate, air pollution from the incineration or land application process, toxicity to sea plants and animals following ocean dumping, and entrance into the food chain after land application are possible consequences of metal-laden sludge disposal. In order to minimize these impacts and make best use of scarce disposal sites, the determination of heavy metals in sewage sludge prior t o disposal is necessary. This need is not unique to the United States: it prevails throughout the industrialized world. In addition to the obvious contributions from industrial sources, heavy metals in sewage originate from a wide variety of domestic, commercial, and ambient sources as well as from chemicals used in the sewage treatment process. Klein et al.’ have identified non-industrial sources as responsible for 61% of the cadmium, 48% of the chromium, 81% of the copper, 35% of the nickel, and 80% of the zinc in the influents t o 12 New York City sewage treatment works. Gurnham et al. have made an extensive engineering study on the origins of heavy metals in sludges from the wastewater treatment works serving a residential section of Chicago and from that of a suburban residential community near Chicago. Among the sources of heavy metals they identified were: 273

(1) the ambient mineral content of the water supply and the corrosion

products from its distribution system; (2) the trace metals excreted in urine and faeces; L. A. Klein, M. Lang, N. Nash, and S. L. Kirschner, ‘Sources o f Metals in New York City Wastewater’, Conf. Roc., New York Water Pollution Control ASSOC.,New York, January, 1974. C. F. Gurnham, R. R. Ritchie, A. W. Smith, and B. A. Rose, ‘Source and Control of Heavy Metals in Municipal Sludge’, Peter F. Loftus Corp., Chicago, 1979. C. F. Gurnham, B. A. Rose,H. R. Ritchie, W. T. Fetherston, and A. W. Smith, ‘Control of Heavy Metal Content of Municipal Wastewater Sludge’, Peter F. Loftus Corp., Chicago, 1979.

Determination of Heavy Metals in Sewage Sludge

79 ( 3 ) the trace metals extracted from foods during their preparation and those contained in food waste from garbage grinders; (4) the trace metals in soaps and cosmetics as well as those in laundry detergents, bleach, household cleaners, and drain openers; ( 5 ) the chemicals in sewage wastes from school, hospital, commercial, and government laboratories; (6) the chemicals in the sewage wastes from laundries, car washes, photographic laboratories, and similar commercial establishments; (7) the trace metals in restaurant, slaughter house, and mortuary wastes; (8) the trace metals washed from the atmosphere, roof tops, streets, and gardens by storm water entering the sewage collection system; (9) the lime, iron compounds or aluminium compounds used in some sewage treatment works,

Of the many items evaluated, the following consumer products were found to have high nickel contents: bath soap (100-700 p.p.m.), scouring powder (100800 p.p.m.), powdered dishwasher detergent (600 p,p.m.), powdered laundry detergent (400-700 p.p.m.), dry bleach (800 p.p.m.), and household cleaning powder (300 p.p.m.). The daily per capita contribution of nickel to the sewage system from these sources was calculated t o be approximately 20 mg. Using Farrell’s4 value of 90 g for per capita, daily sludge production on a dry weight basis, it appears that the nickel content of domestic sewage sludges can easily exceed 200 p.p.m. Similarly, it appears that domestic sources make a significant contribution to the levels of the other heavy metals in sewage sludges. Literature values for selected ambient metals, copper and zinc, polluting metals, chromium and nickel, and toxic metals, cadmium and lead, in a dozen different sewage sludges are tabulated in Table 1. 2 Analysis of Sewage Sludge

To assess the potential consequences of discharging sewage sludge to the environment, it is necessary to have some insight into its heavy metal content. The procedures by which this information is collected must be designed and executed with great care, Sampling must assure that the material collected is representative of the sludge. The sample container, transport conditions, holding times and handling procedures must be controlled t o assure that there is no gain or loss of analyte prior to receipt by the laboratory. Similarly, neither contamination nor loss may occur during sample preparation. Finally, the determination of the heavy metals must be made by techniques of adequate selectivity and sensitivity to assure reliable results.

Sample Collection, - Sewage sludges range from liquids containing 5 % solids to solids with moisture contents of 10%.The sludges are found in pipes, pits, ponds, and piles as well as in a variety of tanks, digesters, dewaterers, etc. Hence, a single approach t o sampling is not possible. J . B. Farrel, ‘Overview of Sludge Handling and Disposal’, Conf. Proc., Nat. Conf. Municipal Sludge Management, Pittsburgh, June, 1974.

Environmental Chemistry

80

Table 1 Levels of copper, zinc, chromium, nickel, cadmium and lead in selected sewage sludges (mg kg-’ dry wt.) Works location Canada’ Canada‘ Germany7 South Africa* UK, Oxfordshire’ USA, ATiZona” USA, Colorado” USA, DC12 USA, Indiana13 USA, 10wa14 USA, Michigan” USA, MontanaI6 USA, New Jerseyi7 USA, New York” USA, Ohio” USA, WisconsinZo

cu

Zn

Cr

Ni

Cd

1330

2540 7 20 701 1780 1410 3770 1500 890

369 27 77 155 4 70 692

36 7

19 2

1150 100

52 94 230 125

6 31 19 21 10 24 7 19

349 500 190 780 425 1520 223 264 296 3 220 540 3230 155

6 06 875 95 8 710 4 20 450 363 137 95 8 3400 645 2150 285

5200

1210 264 1310 8540 1250 11 100

45 89 20 200 256 156

215 24 13 57 76 169 654

10 74 9 414

7

Pb

’ J . R. Knechtel and J. Fraser, Anal. L e f t . , 1974, 7 , 497. A. Chattopadhyay, ‘Multielement Instrumental Photon Activation Analysis of Digested Sewage Sludges’, Conf. Proc., Measurement, Detection, and Control of Environmental Pollutants, International Atomic Energy Agency, Vienna, 1976. J. I. Kim, I. Fiedler, H. J. Born, and D. Lux,lnt.J. Environ. Anal. Chem., 1981, 10,135. * R. Smith, ‘Determination of Trace Metals in Sewage Sludge’, NIWR Interlaboratory Domparison Study Report No, 395, Council for Scientific and Industrial Research, Pretoria, 1981. P. H. T. Beckett, Environ. Pollut., 1981, 1, 27. l o J. Artiola-Fortuny and W. H. Fuller, Compost Sci Land Utiliz., 1980, May/June, 30. E. W. Kienholz, G. M. Ward, D. E. Johnson, J. C. Baxter, G. Braude, and G. Stern, J. Animal Sci., 1979, 48, 735. L. J. Sikora, W. D. Burge, and J. E. Jones, J. Environ. Qual., 1972, 11, 321. l 3 A. W. Kirleis, L. E. Sommers, and D. W. Nelson, Cereal Chem., 1981, 58, 5 30. l 4 M. A. Tabatabai and W. T. Frankenberg, ‘Chemical Composition of Sewage Sludges in Iowa’, Research Bulletin 586, Agric. and Home Economics Exper. Station, Iowa State Univ. of Sci. and Technol., Ames, May, 1979. l 5 S. L. Daniels and E. S. Conyers, ‘Land Disposal of Chemically Treated Waters and Sludges’, Conf. Proc., 2nd Nat. Conf. Complete Reuse, Chicago, 1975. B. Ammons, Proc. Montana Acad. Sci., 1980, 39, 117. l 7 S. A. Katz, S. W. Jenniss, T. Mount, R. E. Tout, and A. Chatt, Int. J. Environ. Anal. Chem., 1981,9, 209. I s L. D. Tyler, Jamell-Ash Plasma Newsl., 1980, 2,6. 19 S. C. Bergman, C. J. Ritter. E. F. Zamierowski, and C. R. Cothern, J. Environ. Qual., 1979,8,416. J. J. Delfino and R . E. Enderson, Water Sewage Works,1978, 125,R32.

’

Determination of Heavy Metals in Sewage Sludge

81

Sewage sludge samples are collected into pre-cleaned, glass or plastic containers. The US Environmental Protection Agency (EPA)21 recommends one quart, wide-mouth, glass jars with Teflon disc lid liners, and glass containers are specified by the UK Department of the Environment (DOE).22 The former are prepared as follows:23 (i) (ii) (iii) (iv) (v) (vi) (vii)

scrub thoroughly with detergent and water; rinse with 1 :1 nitric acid; rinse with water; rinse with 1 :1 hydrochloric acid; rinse with water; rinse thoroughly with deionized distilled water; dry at 105 ‘C.

The sampling equipment must also be pre-cleaned t o prevent contamination during collection. Both the US EPA21 and the UK DOEM have described various devices for sampling sewage sludge in its many forms and locations. The composite liquid waste sampler (COLIWASA), the pond sampler, and the weighted bottle sampler are described by the former, and the vacuum sampler, the multiple depth sampler, and the core sampler are described by the latter. The design of the COLIWASA permits representative sampling of multiphase liquid wastes having a wide range of viscosities and solids contents, It consists with of a plastic sample tube (5‘ x 14”) containing a plastic stopper rod (53’ x a neoprene rubber stopper. Use of the COLIWASA is limited to shallow ponds, pits, and lagoons. The COLIWASA is inserted into the liquid waste to a depth of up to 4; feet with the stopper rod depressed. The stopper rod is retracted thereby seating the stopper in the bottom of the sample tube, and the COLIWASA is withdrawn from the liquid waste, The sample is drained from the COLIWASA into the sample container by depressing the stopper rod. Replicate samples or composite samples are obtained by repeating the operation at other locations in the liquid waste. The pond sampler consists of an adjustable clamp attached to two or three sections of telescopic aluminium tubing. The clamp is used to secure the sample container, and the telescopic aluminium tubes allow sampling t o depths of up t o 1 5 feet. Replicate sampling and compositing are easily accomplished by simply replacing the sample container. Problems may arise, however, when trying to get low density sludges to replace the overlying liquid which fills the container upon immersion.

i”)

21

United States Environmental Protection Agency, ‘Interim Method for the Analysis of Elemental Priority Pollutants in Sludge’, EMSL, Cincinnati, December, 1978. 22 United Kingdom Department of the Environment, ‘Mercury in Waters, Effluents, and Sludges by Flameless Atomic Absorption Spectrophotometry, 1978’, HMSO, London, 1978. 23 United States Environmental Protection Agency, ‘Test Methods fox Evaluating Solid Wastes, Physical and Chemical Methods’, dashington, May, 1980. 24 United Kingdom Department of the Environment, ’The Sampling and Initial Preparation of Sewage and Waterworks’, Sludges, Soils, Sediments and Plant Materials Prior to Analysis, 1977’, HMSO, London, 1977.

82

Environmental Chemistry

The weighted bottle sampler is used for collections at depths greater than those accessible to the COLIWASA and for collections of low density sludges that do not displace the overlying liquid which fills the pond sampler. The weighted bottle sampler consists of a weighted, stoppered bottle with a line for removing the stopper and raising the filled bottle, The stoppered bottle is lowered into the sludge tank or pond, the stopper is removed by a sharp tug of the line, and the filled bottle is retrieved. The bottle can serve as the sample container, or its contents can be transferred to a sample container. The vacuum sampler is useful in collecting sludges from sedimentation tanks and digesters. The sampler consists of a shielded ten litre vacuum bottle connected t o a collection probe fabricated from sections of aluminium tubing (6’ x 1”). The sampling line and the vacuum line contain valves. The collection probe should be grounded to the tank, and, if an electric motor drives the vacuum pump, it should be spark proof. The collection probe is inserted into the tank or digester through a roof port, The sample bottle is evacuated before opening the sample line valve for collection of sludge. By adding sections of aluminium tubing to the collection probe, the sludge can be sampled at different depths. The multiple depth sampler can simultaneously sample low-solids content sludges at up to 12 different depths. This is accomplished by inserting a small plastic T-piece at a different level in 11 of the 12 small bore plastic tubes connected to the bottles in a modified commercial 12-bottle sampIer. Eleven of the plastic tubes are plugged at the bottom which is weighted t o keep the sampling lines straight. Depth profiles of solids in final settling tanks have been successfully determined in this way. The core sampler, a metal tube with a stainless-steel nose piece and a Teflon tube insert, is used t o sample dry material. A core is taken through the sludge cake or air-dried sludge lifted from drying beds. The Teflon insert can be closed with Teflon end caps and thereby serve as the sample container, or the core can be extruded into a pre-cleaned container. Because of possible inhomogeneity, multiple cores should be taken and composited by coning and quartering. Sample Preservation. - Samples should be transported t o the laboratory under refrigeration or packed in ice as soon as possible after collection. The US EPA21, 23 prohibits the addition of chemical preservatives. The heavy metal determinations should not be delayed. In the event that speed is not possible, storage under refrigeration is required. If the analytical procedure calls for the destruction of the organic matrix by acid digestion, the digested samples may be stored for at least several weeks at room temperature in nitric acid s o l ~ t i o n . ~ ~ - ~ ~ 25

26

21

28

29

Environment Canada, ‘Analytical Methods Manual’, Inland Water Directorate, Ottawa, August, 1979. Environment Protection Authority (Victoria), ‘A Guide to the Sampling and Analysis of Water and Wastewater’, 95/79, East Melbourne, August, 1979. New Jersey Department of Environmental Protection, ‘Field Procedures Manual for Water Data Acquisition’, Trenton, 1980. United Kingdom Department of the Environment, ‘General Principles of Sampling and Accuracy of Results, 1980’. HMSO, London, 1980. United States Environmental Protection Agency, ‘Manual of Methods for Chemical Analysis of Water and Wastes’, Washington, 1979.

Determination of Heavy Metals in Sewage Sludge

83

Sample Preparation. - The extent of sample preparation prior to measuring the heavy metal content of sewage sludge depends, to a large extent, upon the measurement technique employed. For measurements by activation analysis, sample preparation can be as simple as dehydration and encapsulation. When spectrometric or electroanalytical techniques are used, complete destruction of the organic matrix and complete solubilization of the heavy metals are required. I t is necessary that there be neither contamination nor loss in the course of sample preparation : recovery must be complete. Drying. The results o f heavy metal determinations in sewage sludges are usually expressed on a dry weight basis, mg kg-l. This can be accomplished by determining the solids content on one portion of the sludge and measuring the heavy metal levels in another. Problems in obtaining these sample portions, however, lead to a preference for dried, pulverized and thoroughly mixed laboratory samples. The US EPA2' reports that sludge samples may be dried at 60 OC without loss of mercury. This agency2' also recommends drying at 6OoC followed by pulverizing and thoroughly mixing sludge samples prior to digestion for the atomic absorption spectrometric determination of antimony, arsenic, beryllium, cadmium, chromium, copper, lead, nickel, selenium, silver, thallium, and zinc. The UK DOE22 calls for the determination of moisture content in one portion of the sludge, and the use of another for the determination of its mercury content. Knechtel and Frasers and other^^^,^* have used air drying at room temperature. H ~ r a c k Rethfeld,32 ,~~ and Miller and B0swe11~~ dried their samples at temperatures of 40,65, and 70 "C,respectively.

Dissolution. Dissolution of the sample is necessary prior t o the measurement of its heavy metal levels in most instances, While the regulatory agencies require wet oxidation, both dry ashing and acid digestion procedures have found frequent application in the dissolution of sewage sludge samples. Ritter et al. have compared dry ashing at 550 "C followed by hydrochloric acid extraction of the ash with digestion in nitric-perchloric acid mixture, aqua regia digestion, digestion in hydrofluoric acid, and extraction with nitric acid for preparing replicate sub-samples of two sewage sludges prior to the measurement of cadmium, lead, nickel, copper, and zinc by conventional flame atomic absorption spectrometry, Comparison of the results obtained from samples prepared by these five procedures indicated that digestion in nitricperchloric acid mixture and digestion in hydrofluoric acid were equivalent to the dry ashing procedure in terms of heavy metal recovery. The samples prepared by aqua regia digestion and by nitric acid extraction showed lower 30

3'

32 33 34

A. Chattopadhyay, J. Radioanal. Chem., 1977, 37,785. 0. Horak, 'Schermetallgehalte in Pflanzen auf sechs verschiedenen Boden unter dem Einfluss hoher Klarschlammgaben', Landwirtsch. Forsch. Kongressband, 1980, Sauerlander's Verlag, Frankfurt. H. Rethfeld, 2. Anal. Chem., 1982, 310, 127. J . Miller and F. C. Boswell, Environ. Health Perspect., 1981, 42, 197. C. J . Ritter, S. C. Bergman, C. R. Cothern, and E. E. Zamierowski, At. Absorpt. Newsl., 1978, 17, 7 0 .

Environmental Chemistry

84

heavy metal levels. On the basis of speed, simplicity, and superior precision, they concluded that the dry ashing procedure was best for the preparation of sewage sludge samples prior t o the determination of their heavy metal contents by atomic absorption spectrometry. Delfino and Enderson20 carried out a similar study, but they obtained different results. They compared three acid decomposition procedures (nitric acid digestion, digestion with nitric acid and hydrogen peroxide, and aqua regia digestion), muffle furnace ignition at 5 5 0 OC followed by nitric acid extraction of the ash, and low temperature plasma ashing followed by leaching with nitric acid, The silver, cadmium, chromium, copper, nickel, and lead contents of the sludge samples prepared by these five procedures were measured by furnace atomic absorption spectrometry. Conventional flame techniques were employed for the measurement of their calcium, magnesium, sodium, potassium, manganese, and zinc levels. For the most part, the results obtained by the wet digestion procedure were equivalent. The two dry ashing techniques gave less complete recoveries for some of the metals: iron and nickel recoveries from the ashed samples were particularly poor. Artiola-Fortuny and Fullerfo have also compared wet and dry oxidation procedures for the dissolution of sewage sludges, They evaluated six procedures: repeated additions of hydrogen peroxide with intermittent evaporation at 8OoC to near dryness followed by dissolution in 0.2M nitric acid; digestion overnight at gentle reflux with nitric acid in a covered beaker on a hot plate maintained at 83 OC followed by repeated additions of hydrogen peroxide while evaporating to near dryness ; overnight, room temperature pre-digestion with nitric acid followed by the addition of perchloric acid and digestion at 90 O C ; procedure (2) above with subsequent aqua regia-hydrofluoric acid digestion in a high pressure decomposition vessel; digestion with nitric acid at 80 O C in a platinum crucible followed by muffle furnace ignition at 450 *C and subsequent dissolution of the ash with hydrofluoric acid; low temperature, 450 O C , gas flame ignition followed by high temperature, 9OO0C, furnace or flame ignition with subsequent sodium carbonate fusion, decomposition of the melt cake with hydrochloric acid, and evaporation to dryness, and final dissolution in nitric acid. Atomic absorption spectrometry was used to measure the levels of cadmium, cobalt, chromium, copper, iron, manganese, nickel, lead, and zinc in the final solutions obtained with these six procedures. The data for the nine metals and the six procedures were ranked by statistical means. On this basis, methods 4, 5 , and 6 gave the highest metal recoveries, but the sodium interference from the sodium carbonate fusion step limited the usefulness of method 6 . Corrondo et have made an interesting comparison between the direct injection of homogenized and diluted sewage sludge samples into the electro35

M. J. T. Carrondo,

J. N. Lester, and R. Perry, Talunta, 1979,2 6 , 9 2 9 .

Determination of Heavy Metals in Sewage Sludge

a5

thermal atomizer and wet and dry oxidation methods for preparing these samples prior to conventional flame atomization. Sludge samples were diluted fifty-fold with high purity water, acidified, and homogenized with a commercial apparatus. Three wet oxidation procedures were included in their comparison: nitric-sulphuric acid digestion, nitric acid-hydrogen peroxide digestion, and nitric-perchloric-hydrofluoric acid digestion. The dry oxidation procedure involved charring the sample in a 2OO0C furnace for one hour, gradually increasing the temperature to 45OoC over a two-hour period, and continued ashing at this temperature for 14 hours followed by the addition of nitric acid and ashing for an additional hour at 450OC. The ash was then extracted with dilute nitric-hydrochloric acid mixture. The five sets of results for aluminium, calcium, magnesium, and iron were treated statistically. N o significant differences were found between the five sample preparation methods in the determination of iron, but the nitric-sulphuric acid digestion yielded calcium values significantly lower than those obtained by the other four methods. The highest recoveries of aluminium and magnesium were observed in the samples prepared by nitric-perchloric-hydrofluoric acid digestion. Muffle furnace ignition (2 hours at 450°C), digestion with nitric acid, digestion with nitric acid and hydrogen peroxide, and digestion with nitric acid in high pressure decomposition vessels were compared by Jenniss et aZ.36 for the preparation of sewage sludge samples prior to the determination of cadmium and lead by flame atomic absorption spectrometry. They found superior recoveries from the samples prepared by nitric acid-hydrogen peroxide digestion and digestion with nitric acid in the high pressure decomposition vessels. Katz et a2.l7 have employed the methods of Jenniss et al.36 t o compare recoveries of cadmium, chromium, copper, iron, nickel, lead, and zinc from two sewage sludge samples. Statistical evaluation of the results, which were obtained by conventional flame atomic absorption spectrometry, led them to the conclusion that dry ashing was not an acceptable method for the preparation of sewage sludge samples prior to atomic absorption spectrometry. They found the nitric acid-hydrogen peroxide digestion to be an efficient, convenient, and rapid method for this purpose. Ritter37 has recently compared his dry ashing method34 t o the nitric acid digestion in high pressure decomposition vessels36 in preparing sewage sludge samples for cadmium and lead determinations by atomic absorption spectrometry. He found equivalent cadmium recoveries by the two methods, and superior lead recoveries with the high pressure decomposition vessels. Nonetheless, he concluded, from the standpoint of time and expense, that the dry ashing method is the most practical. The foregoing comparisons are summarized in Table 2. I t appears that there is little agreement on a best method to prepare sewage sludge samples for the measurement of their heavy metal contents. Clearly, additional work is needed to resolve the conflicting reports and to identify such a method. ( a ) Dry Oxidation. Bergman e t aZ.19 have used the dry ashing procedure of Ritter et a2.M to prepare sludge samples for the measurement of their cadmium, 36

37

S. W. Jennis, S. A. Katz, and T. Mount, Am. Lab., 1980, 12, 18. C. J . Ritter, Am. Lab., 1982, 14, 72.

Environmental Chemistry

86

Table 2 Ranking of dissolution methods f o r sewage sludges Best

Intermediate

Worst

Ritter 34

dry ashing HF digest. HNOJHCIO, digest.

aqua regia digest.

HNO, extract.

Delfino 2o

aqua re& digest.

HNO, digest. HNO,/H,O, digest.

dry ashing low temp. ash

Articola’o

HPDV* with aqua re& HNO,/dry ash/HF dry ashlfusion

HNO,/HClO, digest. HNO,/H,O, digest.

H 2 0 2digest.

Carrondo3’

HN0,/H20, digest. D & Ht HNO,/HClO,/HF digest. HNO,/H,SO, digest.

Jenniss 36

HPDV* with HNO,

HNO JH ,O digest. HNO, digest.

dry ashing

Katz ’’

HPDV* with HNO,

HNO,/H,O, digest. HNO, digest.

dry ashing

Ritter 37

dry ashing

dry ashing

HPDV* with HNO,

* High pressure decomposition vessel. -/ Dilution and homogenization. lead, nickel, copper, zinc, sodium, potassium, calcium, and magnesium levels by atomic absorption spectrometry. The results of replicate heavy metal determinations in sewage sludge samples from two Ohio communities show a precision corresponding t o coefficients of variation ranging from 8.6% for 3.5 p.p.m. of cadmium to 2.3% for 11 100 p.p.m. of zinc. Tyler18 prepared liquid sewage sludge and sludge cake for ICAP spectrometry by dry ashing one gram samples in Pyrex beakers for 12 hours at 475 OC, treating the ashes with 2 ml of concentrated hydrochloric acid, evaporating to dryness on a steam bath, and leaching the residues with 25 ml of 6M hydrochloric acid. The iron was removed from the hydrochloric acid solutions by extractions with three 25-ml portions of diisopropyl ether. On the basis of replicate analyses of eight samples collected from each site over a five-month period, she concluded that, with the possible exceptions of zinc and chromium, the metals present in the sludge from a single treatment plant (cadmium, zinc, nickel, lead, copper, manganese, cobalt, molybdenum, chromium, iron, aluminium) vary significantly, and that several analyses of sludge over a period of time are necessary t o obtain accurate values for sludge loading rate calculations. Hanni and have chosen dry ashing prior to atomic absorption spectrometry as ‘an exact and routine method’ for the determination of nickel in sewage sludges. They dried either 100 ml of liquid sludge or five grams of wet sludge before mineralizing i t at 5OO0C for from one to three hours. The ashes

’*

E. Hanni and R . Ch. Daniel, Mitt. Geb. Lebensmittelunters. Hyg., 1982, 7 3 , 94.

Determination of Heavy Metals in Sewage Sludge

87

were solubilized by boiling for 20 minutes with 1 0 m l of concentrated hydrochloric acid. On the basis of three different sludge samples, the precision of the method was reflected in coefficients of variation of from 2.3% t o 4.6%. Agreement with the results obtained by colorimetry was excellent. ( b ) Wet Oxidation. Martin and K ~ p reported p ~ ~ on a procedure for the determination of selenium in sewage sludge by which the sample was digested with nitric acid and hydrogen peroxide, treated with nickel nitrate, and evaluated by furnace atomic absorption spectrometry. This digestion mixture was chosen to prevent losses by volatilization during sample preparation, and the nickel was added to stabilize selenium as a selenide during the charring step of the electrothermal atomization cycle. For the evaluation of their municipal digested sludge quality control sample,40 the US EPA recommended the nitric acid digestion contained in their methods manual.41 Subsequently, however, the US EPA21 identified nitric acid-hydrogen peroxide digestion as applicable to the preparation of sewage sludge samples for both the direct aspiration and furnace atomic absorption spectrometric determination of all elemental priority pollutants except mercury. For the determination of mercury, both the US EPA29 and the UK DOE22require an acid perm angan at e digestion , Tabatabai and Frankenberger14 used a nitric acid-perchloric acid mixture to digest sewage sludge prior to the determination of its phosphorus, arsenic, boron, beryllium, cadmium, cobalt, chromium, copper, iron, manganese, molybdenum, nickel, lead, selenium, silver, strontium, vanadium, and zinc contents by inductively coupled plasma emission spectroscopy. They refluxed a 400 mg sample of air-dried sludge with 2 ml of nitric acid and 2 ml of perchloric acid in a 30-ml Kjeldahl flask until digestion was complete, and then they removed the excess nitric acid by boiling with 3 ml of hydrochloric acid. Amrnonsl6 employed an aqua regia digestion t o prepare her sewage sludge samples for the determination of the cadmium, nickel, lead, copper, and zinc levels by atomic absorption spectrometry. She heated 500 mg samples of dried sewage sludge with 12 ml of aqua regia for 30 minutes in covered beakers on an electric hot plate set at medium. Two independent interlaboratory comparisons of the US EPA’s municipal digested sludge quality control samplem have been carried out. One of these studies was conducted by Smith8 for the South African National Institute for Water Research, and it involved 20 participating laboratories. The other was reported by Adelman et aZ.42 for the New Jersey Department of Environmental Protection, and it consisted of data from 16 laboratories. Samples were prepared by nitric acid digestion41 in the former, and the latter used the nitric acidhydrogen peroxide digestion2’ to prepare the samples. In both studies, the heavy metal levels were measured by atomic absorption spectrometry. The mean values 39 40

41

42

T. D. Martin and J . F. Kopp, At. Absorpt. New& 1975, 14, 109. United States Environmental Protection Agency, ‘EPA Quality Control Samples, Municipal Digested Sludge’, Cincinnati, September, 1976. United States Environmental Protection Agency, ‘Manual of Methods for Chemical Analysis of Water and Wastes’, Washington, 1974. H. Adelman, S. W. Jenniss, and S. A. Katz, Am. Lab., 1981, 1 3 , 31.

Aluminium Arsenic Cadmium Chromium Copper Iron Lead Manganese Mercury Nickel Silver zinc

Element

Mean

-

2010-7110 0-88.9 2.49-39.1 115-294 831-1360 3810-28 500 305-733 172-23 8 0-36.1 164-233 0-203 1190-1450

95% C.L.

US EPA values40

4557.6 16.972 20.772 204.46 1095.3 16 155 518.76 204.98 16.315 198.31 80.583 1323

--

Table 3 Results of sludge analysis intercomparisons

4332 19.1 210 1055 17 198 559 213 195 1367

Mean

15.0-23.2 150-270 899-1 21 1 14 603-19 793 455-664 197-228 156-234 1177-1558

-

34 17-5 247

95% c L.

NIWR valuesa

4850 6.57 18.5 183 94 1 14 500 5 16 197 11.1 165 69.4 1190

Mean

2070-7630 0-32.0 12.6-24.4 107-259 6 89-1 190 9720-19 300 350-682 147-247 5.1-17.1 111-219 15.8-1 23 818-1560

95% C.L.

NJ DEP values4=

3

v,

U.

3

r

n

*r

-+

3

;

0

h

3

z.

00

00

Determination of Heavy Metals in Sewage Sludge 89 and 95% confidence limits for a dozen heavy metals determined in these interlaboratory studies are compared with the reference valuesm in Table 3. It appears that the wet oxidation procedures coupled with atomic absorption spectrometry are, for the most part, capable of generating accurate results.

Elemental Determinations. - The measurement of heavy metal levels in sewage sludges can be accomplished by essentially any method of elemental analysis. In considering the best approach t o such measurements on a routine basis, speed and simplicity as well as sensitivity and selectivity are major factors. More sophisticated techniques are needed for measurements directed t o ultratrace levels or for the determination of chemical form or chemical speciation. Atomic absorption spectrometry with flame or flameless atomization is the technique most frequently used for the routine measurement of heavy metals in sewage sludges, The utilization of anodic stripping voltammetry and potentiometry with ionselective electrodes has been minimal as has the use of colorimetry. The multi-element capability of activation analysis and its excellent sensitivity for most elements make this technique attractive for sludge analysis, but the long turn-around time and the limited availability of reactor or accelerator facilities make it impractical for the routine determination of heavy metals in sludges. Both energy dispersive and wavelength dispersive X-ray fluorescence spectrometry offer the advantages of multi-element determinations, availability, and speed. Atomic emission spectroscopy, particularly inductively coupled plasma emission spectroscopy with its multi-element capability and broad dynamic range, shows promise of becoming very useful for the determination of heavy metals in sewage sludges. A tom ic A bsorp tion / Atom ic Emission Spectrometry. Most regulatory agen~ies~5-~9 require, or at least recommend, the use of atomic absorption spectrometry for compliance monitoring of heavy metals. This technique meets the prerequisites of speed, simplicity, sensitivity, and selectivity. Once instrumental operating parameters have been established and calibration has been achieved, measurements can be made in minutes by a technical assistant. The non-flame procedures require more skill than does flame atomization, and direct aspiration is less complicated than standard additions. Nonetheless, these procedures are well within the abilities of technical personnel with modest direct professional supervision. The sensitivities and detection limits for the heavy metals frequently determined by atomic absorption spectrometry are listed in Table 4. Selectivities are excellent, but atomic absorption spectrometry is subject to matrix and chemical interferences, Matrix matching, matrix modification, background correction and a rigorous programme of quality control are necessary to assure the reliability of the results. The applications of flame and non-flame atomic absorption spectrometry to the determination of heavy metals in sewage sludge are summarized in Table 5. Tylerlg has applied inductively coupled plasma emission spectrometry t o the determination of heavy metals in sewage sludge. She was able to measure simultaneously 10 metals (aluminium, cadmium, chromium, cobalt, copper, lead, manganese, molybdenum, nickel, and zinc) at a rate much faster than that possible with sequential atomic absorption measurements. Tabatabai and

Environmental Chemistry

90

Table 4 Sensitivities and detection limits obtained in flame atomic absorption spe~trometry~~ Element

Aluminium Arsenic Barium Calcium Cadmium Chromium Copper iron Mercury Magnesium Manganese Nickel Lead Antimony Selenium zinc

Reciprocal sensitivity mg I-' 1%

Detection limit

1.o 0.15

0.02 0.02 0.01 0.001 0.002

0.4 0.05

0.03 0.1 0.1

0.2 10 0.01 0.1

0.15 0.7

0.5 0.25 0.02

mg 1-'

0.003 0 .oo 1 0.01 0.2 0.0001 0.002 0.002 0.01 0.04 0.1 0.001

Table 5 Determination of heavy metals in sewage sludge b y atomic absorption spectrometry Author Adelman e t al. 4 2 Ammans16 Artiola-Fortuny and Fuller l o Bergman et al. Carrondo et al. 3 5 Delfino and EndersonZ0 Hanni and Daniel3' Jennis e t al. 36 Johnson et al. 44 Katz et al. l 7 Keinholz et al. Knechtel and Fraser' Martin and K ~ p p ~ ~ Ritter et al. 34 RitteP Smith'

Metals determined Al, As, Ba, Cd, Ca, Cr, Cu, Fe, Pb, Mg, Mn, Hg, Ni, K, Se, Ag, Na, Sn, Zn Cd, Cu, Pb, Hg, Ni, Zn Cd, CoyCr, Cu,Fe, Pb, Mn, Ni, Zn Cd, Ca, Cu, Pb, Mg, Ni, K, Na, Zn Al, Ca, Fe, Mg Cd, Ca, Cr, Cu, Mg, Ni, Ag, Na, Zn, K, Fe, Mn Ni Cd, Pb Cd, Cu, Pb, Hg, Zn Cd, Cr, Cu, Fe, Pb, Ni, Zn As, Cd, Cu,Hg, Mo, Ni, Pb Ni Se Cd, Cu, Pb, Ni, Zn Cd, Pb Al, Cd, Cr, Cu, Fe, Pb, Mn, Ni, Zn

G. F. Kirkbright, 'Atomic Absorption Spectrometry', Technical Report Series No. 197, pp. 141-165, International Atomic Energy Agency, Vienna, 1980. 44 D. E. Johnson, E. W. Kienholz, J. C. Baxter, E. Spangler, and G. M . Ward, J. Animal. Sci., 1981,52, 108.

43

Determination of Heavy Metals in Sewage Sludge

91 Frankenberg14 have also used inductively coupled plasma emission spectroscopy for the measurement of heavy metals (silver, arsenic, boron, beryllium, cadmium, cobalt, chromium, copper, iron, mercury, manganese, molybdenum, nickel, lead, selenium, strontium, vanadium, and zinc) in sewage sludge. Neutron ActivationlPhoton Activation Analysis. Chattopadhyay6 has used multielement instrumental photon activation analysis to determine up to 34 elements in raw and digested sewage sludge samples collected from four treatment works. Bremsstrahlung photons from the conversion of 15, 20, 22, 35, and 45 MeV electrons in tungsten were used to irradiate the samples, A large number of nuclides were produced through (y, n), (y, 2n), (7, np), (y, p), (y, d), etc., reactions. Their ?-spectra were measured and recorded with a high resolution detector-4000 channel analyser system. The elements determined were: Cd, Cs, In, Sb, T1, Ag, As, Ca, C1, Co, K, Mn, Na, Rb, Sc, Si, Sr, Te, Mg, Ti, Zr, Ba, Ce, Cr, Fe, Hg, Mo, Ni, Pb, Se, Sn, Zn, Bi, and V. In the course of these analyses, Chattopadhyay observed that the sewage sludges from non-industrialized cities contained lower levels of toxic metals than those from industrialized cities. Chattopadhyay3* has also developed a combination of instrumental neutron activation analysis and instrumental photon activation analysis for multi-element determinations in a variety of sewage sludges. Up t o 50 elements have been measured by this combination of techniques. Where the techniques were complementary, agreement was good. Instrumental photon activation analysis supplemented instrumental neutron activation analysis in that cadmium and lead could be determined by the former technique. Ryan e t al.45 have determined up to 44 elements in sewage sludge samples by instrumental neutron activation analysis. Their procedure made use of three different irradiation times and four different decay periods for recording yspectra. Using monostandard instrumental neutron activation analysis, Kim et al.' measured the concentrations of 14 elements (Ag, Au, Ba, Br, Co, Cr, Cs, Fe, Hg, Na, Rb, Sb, Se, Zn) in the effluent and suspended matter from sewage treatment works. A single irradiation followed by a single y-spectrum was used for the determination of the 14 elements in each sample. Other Methodologies. %nardM has simultaneously measured the concentrations of zinc, cadmium, lead, copper, and bismuth in both domestic and industrial effluents by anodic stripping voltammetry. The samples were prepared by digestion with nitric acid in high pressure decomposition vessels, and the electrochemical reactions were carried out in a pH 5.5-5.8 sodium acetate solution. The results were competitive with those obtained by conventional flame atomic absorption spectrometry in terms of precision and accuracy, and superior in terms of sensitivity and detection limits. Sposito e t aL4' have used a cadmium ion selective electrode to measure cadmium ion activities in aqueous extracts of sewage sludge, The purpose of these measurements was to evaluate the cadmium-fulvic acid complexes 4s

D. E. Ryan, D. C. Stuart, and A. Chattopadhyay, Anal. Chim. Actu, 1978, 100,87.

46

J. T . Kinard, J. Environ. Sci Health, 1977, A12, 531,

47

G. Sposito, F. T. Bingham, S. S. Yadav, and C. A. Inouye, Soil Sci. SOC. Am. J., 1982,

46,Sl.

E nvironmentd Chemistry

92

extracted from the sludge rather than to determine the cadmium content of the sludge . Wavelength dispersive X-ray fluorescence spectroscopy was used by Rethfeld32 for the determination of heavy metals in sewage sludge, The samples were dried, ground, and pressed into tablets. The X-ray spectra were recorded and evaluated for the tin, cadmium, silver, arsenic, lead, thallium, zinc, copper, nickel, and chromium contents of the samples.

3 Selected Procedures for Sludge Analysis Recognizing the need t o provide guidance to the laboratories engaged in the analysis of sewage sludge, the regulatory agencies have identified procedures for the collection, preservation, and preparation of the samples, for the measurement of specific heavy metals, and for the establishment of quality control programmes. These procedures, for the most part, call for sample preparation by acid digestion and measurement by atomic absorption spectrometry.

US EPA Procedures. - The US EPA2' has recommended the adoption of an interim method for the analysis of elemental priority pollutants in sludge, The nitric acid-hydrogen peroxide digestion procedure described in this recommendation is directed to the preparation of sludge samples for the measurement of their heavy metal contents by atomic absorption spectrometry. With the exception of mercury, which requires digestion with acid permanganate and measurement by the cold vapour technique as described below, thishigestion is applicable t o all other elemental priority pollutants. These pollutants are: antimony, arsenic, beryllium, cadmium, chromium, copper, lead, nickel, selenium, silver, thallium, and zinc. The recommended digestion procedure is given below, Weigh and transfer to a 125 ml conical Phillips beaker a 1.0 g portion of the sample which has been dried at 6OoC, pulverized, and thoroughly mixed. Add 5 ml of 1:l nitric acid and cover with a watch glass. Heat the sample at 95 OC and reflux t o near dryness. Allow the sample to cool, add 4 ml of concentrated nitric acid, and again reflux to near dryness. After the second reflux step has been completed and the sample has cooled, add 1 ml of 1:l nitric acid and 3 ml of 30% hydrogen peroxide. Return the beaker to the hot plate to initiate the peroxide reaction. Care must be taken with the start of effervescence that losses do not occur or the reaction is not too vigorous. Heat until effervescence subsides and cool the beaker. Continue the addition of 30% hydrogen peroxide in 1 ml increments with warming until the effervescence is minimal or the general sample appearance is unchanged. Do not add more than a total of 10 mi of hydrogen peroxide. If the sample is being prepared for the furnace analysis of antimony and/or direct aspiration of antimony, beryllium, cadmium, chromium, copper, lead, nickel, and zinc, add 2 ml of 1: 1 hydrochloric acid, return the covered beaker t o the hot plate and reflux for an additional 10 minutes. After cooling, filter through Whatman No. 42 paper (or the equivalent) and dilute to 50 ml with deionized distilled water.

Determination of Heavy Metals in Sewage Sludge

93 If the sample is being prepared for the furnace analysis of arsenic, beryllium, cadmium, chromium, copper, lead, nickel, selenium, silver, thallium, and zinc, or the direct aspiration of silver and thallium, add 1 ml of 1:1 nitric acid, return the covered beaker to the hot plate and reflux for an additional 10 minutes. After cooling, filter through Whatman No. 42 paper (or the equivalent) and dilute to 50 ml with deionized distilled water.

After digestion, the heavy metal contents of the sludge sample are determined in accordance with the following general directions. The recommendations contained in 'Methods for Chemical Analysis of Water and Wastes'29are summarized in Table 6 .

Table 6 Recommended conditions for the determination of elemental priority pollutants in sewage sludge by atomic absorption spectrometry Element Antimony Arsenic Beryllium Cadmium Chromium Copper Lead Nickel Selenium Thallium Zinc

Wavelength

Flame conditions

217.6 193.6 234.9 228.8 357.9 324.7 283.3 232.0 196.0 276.8 213.9

lean air-acetylene ASH, in argon-hydrogen nitrous oxide-acetylene air-acetylene nitrous oxide-acetylene air-acetylene air -acetylene air-ace t ylene H,Se in argon-hydrogen air-acetylene air-acetylene

Dry, char, atomize furnace cycle 30 s/125 "C, 30 s/800 "C, 30 s/125 "C, 30 s/llOO "C, 30 s/125 "C, 30 s/1000 "C, 30 s/125 "C, 30 $500 OC, 30 s/125 "C, 30 s/1000 "C, 30 ~1125 "C, 30 s/900 "C, 30 s/125 OC, 30 s/500 "C, 30 s/l25 "C, 30 s/900 OC, 30 s/l25 "C, 30 s/l200 "C, 30 ~ 1 1 2OC, 5 30 s/400 "C, 30 s/l25 "C, 30 s/400 "C,

10 42700 "C 10 s/2700 "C 10 s/2800 "C 10 s/l900 "c 10 s/2700 "C 10 s/2700 ' C 10 s/2700 "C 10 s/2700 "C 10 42700 "C 10 s/2400 "C 10 s/2500 "C

For both direct aspiration and furnace analysis procedures, including the use of methods of standard additions, preparation of standard solutions, notes on interferences, instructions on the addition of required reagents or matrix modifiers, and recommended instrument settings, see the individual element analysis pages in 'Methods for Chemical Analysis of Water and Wastes'. The use of background correction for furnace analysis t o correct for nonspecific absorbance and scattered light is required. Background correction is also required for direct aspiration unless it can be shown t o be unnecessary. All furnace analyses shall be conducted using methods of standard additions, while noting the described limitations of its use as given in 'Methods for Chemical Analysis of Water and Wastes', Also, since the method of standard additions may be required for direct aspiration, verification is required to justify its omission. Katz e t aL1' have used this digestion procedure to prepare sewage sludge samples for conventional flame atomic absorption spectrometry directed to the determination of cadmium, chromium, copper, iron, nickel, lead, and zinc. Adelman et aZ.42 have reported on an interlaboratory comparison in which 16

94

Environmental Chemistry

participants used this digestion procedure for the preparation of a sewage sample prior t o the measurement of its aluminium, arsenic, barium, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, mercury, nickel, potassium, selenium, silver, sodium, tin, and zinc levels by atomic absorption spectrometry. US EPA29 method 245.5 is applicable t o the determination of total (inorganic and organic) mercury in soils, sediments, bottom deposits, and sludge type materials, The samples may be oven dried at 60 OC without loss of mercury. The dried material is pulverized and mixed thoroughly, and triplicate 0.2 g subsamples are weighed into BOD bottles. The contents of the bottles are treated with 5 ml of aqua regia and heated for 2 minutes in a 95 O C water bath. The bottles are cooled, treated with 50 ml of high purity water, and 1 5 ml of 5 % m / V potassium permanganate solution, and returned to the water bath for 30 minutes. The samples are cooled, brought t o a volume of 100 ml, and treated with 6 ml of 12% m / V sodium chloride-124b m / V hydroxylamine sulphate solution, The bottles are individually treated with 5 ml of 10%m / V stannous sulphate suspension in 0.5N sulphuric acid and immediately connected t o the aeration train of the cold vapour apparatus. The mercury contents of the samples are determined by direct comparison of their maximum absorbances at 253.7 nm to those of the standards. The maximum is usually exhibited within 30 seconds when the circulating pump is operated at 1 1 min-'. As an alternative procedure for preparation of the samples, the contents of the BOD bottles may be treated with 5 ml of sulphuric acid, 2 ml of nitric acid, and 5 ml of 5% m / V potassium permanganate solution. The bottles are closed with aluminium foil and autoclaved for 1 5 minutes at 121 O C and 5 lb in-2, The autoclaved samples are cooled and brought to a volume of 100 ml with high purity water, Canadian DOE Procedures. - Like its US analogue, the Canadian DOE25 has recommended a procedure for the determination of heavy metals in soils and sediments. This procedure is applicable to the determination of aluminium, cadmium, cobalt, copper, iron, lead, manganese, molybdenum, nickel, vanadium, and zinc in these matricies, and it is also suitable for sewage sludges, Freeze dried samples are preferred to frozen and subsequently air-dried samples. Initial coarse (20-mesh) sieving is used to remove extraneous debris, and subsequent grinding of a subsample, obtained by coning and quartering, is used to obtain a representative material for analysis. Portions of this material, weighing 1-3 g, are transferred t o Teflon beakers and treated with 15 ml of concentrated nitric acid. The beakers are heated on a hot plate covered with a sheet of asbestos. After the contents have boiled for 2 minutes, 10 ml of perchloric acid are added, and the heating is continued until a white paste remains in the beakers. (Caution: perchloric acid should be boiled only in an approved hood; the beakers should not be permitted to boil dry.) Ten ml of hydrofluoric acid are added to the white pastes in the beakers, and the heating is continued until the residues dissolve. The hydrofluoric acid is boiled off, and the contents of the beakers are dissolved in 5 ml of concentrated hydrochloric acid and 20 ml of high purity water with gentle heating. The solutions are transferred t o 10 ml volumetric flasks and brought t o volume with high purity water. The heavy metals cited above are

Determination of Heavy Metals in Sewage Sludge

95

Table 7 Typical wavelengths and flame conditions used for metal determinations b y atomic absorption spectrometry in Canadian DOE recommended procedure Element Aluminium Cadmium Cobalt Copper Iron Lead Manganese Molybdenum Nickel Vanadium Zinc

Wavelength/nm

Flame conditions

209.3 228.8 240.7 324.7 248.3 283.3 279.5 313.3 232 .O 3 18.4 213.9

reducing nitrous oxide-acetylene lean air-acetylene lean air-acetylene lean air-acetylene lean &-acetylene lean air-acetylene lean air-acetylene rich nitrous oxide-acetylene lean air-acetylene rich nitrous oxide-acetylene lean air-acetylene

measured by conventional flame atomic absorp don spectrometry. Typical wavelengths and flame conditions are given in Table 7. The Canadian DOE2' has recommended a method for the determination of mercury in soils and sediments that is also applicable to sewage sludges. Representative portions of wet sample equivalent t o 1-2 g dry weight (moisture content is determined on another portion of the sample from the weight loss after drying for 24 hours at 105 " C ) are weighed into 100 ml volumetric flasks, cooled in an ice bath, treated with 10 ml of sulphuric acid, 5 ml of nitric acid, and 2 ml of hydrochloric acid, and incubated in a shaking water bath for 2 hours at 5O-6O0C. After this time, the contents of the flasks are cooled, cautiously treated with 1 5 ml of 6% m / V potassium permanganate solution in 1ml increments, and, 30 minutes later, 5 ml of 5 % potassium persulphate solution are added. The flasks are allowed to stand overnight. If the purple colour of the permanganate fades, more permanganate is added until the purple colour persists for 1 5 minutes. The contents of the flasks are treated with 10 ml of 6% m / V hydroxylamine sulphate-6% sodium chloride solution and brought to volume with high purity water. The mercury contents of the sample are determined by cold vapour atomic absorption spectrometry using stannous sulphate suspension as the reducing agent.

UK DOE Procedures. - A procedure for the determination of mercury in sewage sludge is available from the UK DOE.22 A wet sample equivalent to 250 mg of solid on a dry weight basis (moisture content is determined on another portion of the sample) is weighed into a 5 0 m l Kjeldahl flask, and the flask and its contents are cooled in an ice bath. The contents of the flask are treated with 10 ml of sulphuric acid, and the lightly stoppered flask is left in the ice bath for 1 hour. The flask is transferred to a 5 5 O C water bath and allowed t o remain there with occasional shaking for 24 hours. The flask is cooled, returned to the ice bath and treated with three 6 ml portions of 5 % m/V potassium per-

96

Environmental Chemistry

manganate solution with intermittent cooling in the ice bath. Additional 5 ml increments may be added as required to keep the solution deeply coloured. The flask is then allowed to stand at room temperature for 24 hours. After this time, 1 ml of nitric acid and 1 ml of 12% m/V hydroxylammonium chloride12%m / V sodium chloride solution are added to dissolve the suspended matter and decolorize the solution. The solution is transferred to a 250ml measuring cylinder and diluted t o 150 ml with high purity water. A 7 5 ml portion of this solution is transferred to the Drechsel bottle of the aeration train, and its mercury content is determined by cold vapour atomic absorption spectrometry. Other Procedures. - The Environment Protection Authority of Victoria48 has reported a 3 hour reflux at 7OoC with sulphuric acid-nitric acid mixture followed by overnight, room temperature digestion with potassium permanganate and potassium persulphate for the preparation of soil samples prior to the measurement of their mercury contents by cold vapor atomic absorption spectrometry. A 5-g sample contained in a 25 ml Erlenmeyer flask is treated, dropwise, with 8 ml of sulphuric acid-nitric acid ( 3 : 1) digestion mixture, the flask is connected to a condenser, and its contents refluxed in a water bath at 7OoC for 3 hours. The contents of the flask are cooled, treated with 3 ml of 10%m / V potassium permanganate solution and 2 ml of 10%m / V potassium persulphate solution, and allowed to stand overnight. If at the end of this period the purple colour of the perrnanganate is not present, another 3 ml of the 10% solution is added to the contents of the flask, and it is allowed t o stand for an additional 6 hours. The purple colour is discharged by the addition of 12% m/V sodium chloride-1 2% m /V hydroxylammonium sulphate solution. Solid phase, if present, is separated by centrifugation, and the supernatant liquid is transferred to a 5 0 ml volumetric flask. The solid phase is washed with water, and the washings are added to the contents of the volumetric flask. The contents of the flask are brought to volume with high purity water and evaluated for mercury content by cold vapour atomic absorption spectrometry. For the determination of aluminium, barium, beryllium, calcium, cadmium, chromium, cobalt, copper, iron, lead, magnesium, nickel, silver, vanadium, and zinc, a 2-g sample, previously dried at 110 ‘C, is transferred to a 250 ml Erlenmeyer flask, treated with 10 ml of nitric acid, and digested until brown fumes are no longer generated. Then 5 ml of perchloric acid are added, and the contents of the flask are digested for 5 hours at 8OoC. (See previous caution note on perchloric acid.) After this time, the contents of the flask are cooled, filtered through a glass fibre filter into a 5 0 ml volumetric flask, and after dilution, evaluated by atomic absorption spectrometry, 4 Disposal and Utilization of Sewage Sludge

To some, sewage sludge is a bothersome waste product; to others it is a valuable resource. Regardless of the perspective, insight into its elemental composition is important in assessing its potential for disposal o r utilization. 48

Environment Protection Authority (Victoria), ‘Chemical Analysis of Polluted Soils’, Publication 139, East Melbourne, November, 1981.

Determination of Heavy Metals in Sewage Sludge

97

Incineration. - Incineration is a popular means of sludge disposal in the US. Most of the organic material is destroyed in this process which yields a sterile ash comprising from 30 t o 60% of the original dry weight of the sludge. The sludge is frequently dewatered by vacuum filtration and incinerated in a multiple hearth furnace at temperatures between 800 and 1000 ‘C. Feed rates are of the order of 3 to 6 tons per hour, and the ultimate disposal of the ash is frequently by land fill burial. Furr et aZ.49 have measured the levels of 42 elements in the sludge ashes from 10 US cities. Instrumental neutron activation analysis was used to determine most of the elements, cadmium and lead levels were measured by anodic stripping voltammetry, and mercury and nickel were determined by atomic absorption spectrometry. Mellbye et aZ. 50 digested sludge ash samples with nitric-perchloric acid mixture, filtered out the silica, and determined molybdenum, cadmium, lead, aluminium, zinc, copper, nickel, manganese, chromium, calcium, magnesium, potassium, sodium, and iron in the filtrate by atomic absorption spectrometry. Gablersl determined barium, bismuth, boron, cadmium, cobalt, chromium, copper, gallium, lead, lithium, manganese, molybdenum, nickel, potassium, rubidium, silver, strontium, tin, vanadium, zinc, and zirconium by atomic absorption spectrometry after decomposition of the samples with hydrofluoric and perchloric acids. He used a borax fusion to prepare samples for the determination of aluminium, potassium, iron, magnesium, silicon, and titanium as well as to confirm cadmium, chromium, copper, and zinc by X-ray spectrometry. The heavy metals were determined in sewage sludge ashes from 14 cities in the US. Land Disposal. -Most sewage sludge is disposed of in or on the land, Liquid sludges, sludge cake, dried sludge, and sludge ash is either buried in sludge land fills and entrenchment sites or applied t o the land surface. Sometimes, codisposal with mixed municipal refuse in land fills is used, Pre-disposal analysis of the sludge is necessary to identify the heavy metal loadings of the disposal site. The procedures described in Section 3 are suitable for this purpose. Ocean Dumping. - During the last few years, ocean dumping of sewage sludge has been greatly reduced. As a result of federal regulation, this mode of disposal accounts for no more than 10%of the US sludge production, Nonetheless, these actions have already produced a 10 square mile ‘dead sea’ in the near shore water of the northern New Jersey coast. The same pre-disposal monitoring procedures recommended for sludges disposed of on land apply to those for ocean dumping. Cropland Applications. - The use of sewage sludge as an agricultural fertilizer or soil amendment is much more common in Europe than it is in North America. Hanni and rcported that 80 to 90% of the liquid sewage sludge A. K. Furr, T. F. Parkinson, T. Wachs, C. A. Bache, W. H. Gutenmann, P. C. Wszolek, I. S. Pakkala, and D. J. Lisk,Environ. Sci. Technol., 1979, 13, 1503. 5 0 M. E . Mellbye, D. D. Hemphill, and V. V. Volk, J. Environ. Quai., 1982, 11, 160. ’’ R. C. Gabler, ‘IncineratedMunicipal Sewage Sludge as a Potential Secondary Resource for Metals and Phosphorus’, Bureau of Mines Report of Investigation, RI 8390, Washington, 1979. 49

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Environmental Chemistry

produced in Switzerland is used as agricultural fertilizer, and Carrondo et aZ.35 estimate that 80% of the sludge from all inland sewage treatment works in the UK is disposed of on land, half of which is agricultural land, In the US, the land application of sewage sludge is subject to wide differences in opinion. New York State currently has a two year ban on the agricultural use of sewage sludge. In Ohio, on the other hand, approximately 30% of the sewage sludge produced in the state is applied t o cultivated soils. Of the sludge produced in the US some 20% is applied to the land.52 Pre-disposal sludge analysis can be carried out by the procedures described in Section 3 . Miscellaneous Applications. - In addition to utilization as an agricultural fertilizer, sewage sludge has been used as a nutritional supplement for cattle. Kienholz et aZ. l 1 and Ray et aLs3 have evaluated the retention of heavy metals by, and the quality of, meat from cattle fed diets containing up to 20% sterilized sewage sludge. The use of sewage sludge for the reclamation of disturbed land has been investigated by Sopper and Kerr.54 They found that sewage sludge can be used to revegetate and reclaim sites disturbed by mining activities in an environmentally acceptable manner. GablerS1 has investigated the recovery of valuable resources from sewage sludge ash. He evaluated three extraction procedures, and he identified a sulphuric acid leach for the recovery of heavy metals of economic value and of environmental concern.

5 Possible Consequences of Sewage Sludge Disposal and Utilization and the Need for Monitoring Among the several concerns associated with the disposal or utilization of sewage sludge are the toxic effects of heavy metals on plants and animals.55These toxic effects can be initiated by pollution of air, water, or soil with heavy metals released from the sludge. Pre-disposal/pre-utilization analysis of the sludge by the procedures contained in Section 3 are adequate for the identification of the potential hazards. Monitoring of the atmospheric, aqueous, and terrestrial environments as well as the plants and animals they contain is also necessary fully to determine t o what extent, if any, intoxication has taken place.

Soil and Water Contamination. - The land disposal, surface application or burial, of sewage sludge poses the potential for contaminating surface water, soil and ground water with sludge-derived heavy metals. The New Jersey Department of Environmental Protection2’ has developed a procedures manual for surface

’* 53 54

’’

United States Environmental Protection Agency, ‘Application of Sewage Sludge to Cropland: Appraisal of Potential Hazards of the Heavy Metals to Plants and Animals’, EPA/430/9-76-013, Washington, November, 1976. E. E. Ray, R. T. O’Brien, D. M. Stiffler, and G . S. Smith, J. Food Protect., 1982,45, 317. W. E. Sopper and S. N. Ken, ‘Criteria for Revegetation of Mined Land Using Municipal Sludges’, Institute for Research on Land and Water Resources, Reprint No. 78, Pennsylvania State Univ., University Park. United States Environmental Protection Agency, ‘Process Design Manual for Sludge Treatment and Disposal’, Cincinnati, September, 1979.

Determination of Heavy Metals in Sewage Sludge

99

water monitoring, and the Illinois Department of Energy and Natural Resources56 has published procedures for the collection of ground water data. Many of the procedures in the Canadian DOE’SAnalytical Methods Manual25are identified as applicable to surface and ground water samples, This manual,25 as well as that published by the Victorian EPA,48 also contain procedures for the determination of heavy metals in contaminated soils. Baedecker and Back5’ and Kehew and K n u d ~ e nreported ~~ on the effects leachate generation and migration have on ground water quality. Zindahl and S k ~ g e r b o ehave ~ ~ studied the behaviour of lead in soil, and Emmerich et al. 6o have investigated the movement of heavy metals in soils treated with sewage sludge. The effects of sludge application on heavy metal levels in the soil have been reported by Robertson et a1.,61 and Sikora et al. l2 have described the monitoring of a municipal sludge entrenchment site. Milde and Neumayr62 have reported soil and ground water decontamination during 20 years of inactivity at a surface disposal site for sewage sludge. The assessment of soil and water contamination is made, for the most part, by atomic absorption spectrometry. Water samples are frequently filtered and acidified at the time of collection. Little beyond dilution or matrix modification is required to prepare the water samples for measurements to determine heavy metal contents. Soil samples, on the other hand, are digested or extracted prior to the measurements of their heavy metal contents. Digestion procedures were reviewed in Section 2. Typical soil extractants are EDTA solutions, ammonium acetate solutions, and hydrochloric acid solutions. The Canadian DOE25 procedure for non-residual metals is applicable t o the determination of aluminium, cadmium, chromium, cobalt, copper, iron, lead, manganese, nickel, and zinc, This procedure calls for the treatment of a 10 g air-dried sample contained in a 125 ml wide-mouth plastic bottle with 100 ml of 0.5M hydrochloric acid and overnight mechanical shaking at room temperature followed by filtration ~~ through a 0.45 pm cellulose acetate membrane. The E P A - V i c t ~ r i aprocedure for acid extractable heavy metals specifies a 5 g sample of oven-dried soil stirred with 40 ml of 0.1 M hydrochloric acid for one hour at room temperature. The extract is filtered through an acid-washed glass fibre filter and used for the determination of aluminium, barium, beryllium, calcium, cadmium, chromium, cobalt, copper, iron, lead, magnesium, nickel, silver, vanadium, and zinc. Contamination of Plants. - The assimilation of sludge-derived heavy metals by plants grown in soils treated with sewage sludge is of concern from the standJ. P. Gibb, R. M. Schuller, and R. A. Griffin, ‘Procedures for the Collection of Representative Water Quality Data from Monitoring Wells’, State of Illinois, Department of Energy and Natural Resources, Champaign, 1981. 57 M. J. Baedecker and W. Back, Groundwater, 1979, 5 , 4 2 9 . A. E. Kehew and G. W. Knudsen, ‘Effect on Groundwater of the Cavalier, North Dakota, Sanitary Landfill’, North Dakota Geological Survey, Grand Forks. 6o W. E. Emmerich, L. J . Lund, A. L. Page, and A. C. Chang, J. Environ. Quul., 1982, 11, 174. 6 * K. W. Robertson, M. C. Lutrick, and T. L. Yuan, J. Environ. Quul., 1982, 11, 278. 6 2 G . Milde and V. Neumayr, Turn Ruckgang Schwermetallbelastung von Boden nach der Endigung einer Stadtischen Abwasserverrieselung’, Institut fur Wasser- Boden-und Lufthygiene des Bundesgesundheitsamtes, Berlin, 1981, pp. 737-758. 56

’*

E n vir o n rn e ntal Chemistry points of both phytotoxicity and entry into the food chain. The US EPA52has made an appraisal of the potential hazards of heavy metals t o plants and animals resulting from the application of sewage sludge to cropland. Sopper and Kerr54 have evaluated the vegetative uptake of sludge-derived heavy metals at sites reclaimed by sewage sludge application after being disturbed by mining activities. Furr et aZ.49 measured the absorption of arsenic, cadmium, cobalt, copper, iron, manganese, nickel, lead, and zinc in cabbage grown in soils mixed with the ashes of sewage sludges from 10 cities, Kirleis e t al. l 3 have reported on the heavy metal (cadmium, copper, iron, manganese, nickel, and zinc) content of groats and hulls of oats grown in soil treated with the sewage sludges from three cities. Similarly. Herak3’ measured the heavy metal contents of plants from six different soils treated with sewage sludge, and Hanni and measured the levels of nickel in grass, barley corn, barley straw, wheat and corn grown in sewage sludge amended soil. Using corn ( Z e a ) as the accumulator plant, Mellbye50 measured the uptake of cadmium, calcium, chromium, copper, magnesium, manganese, and lead from soil amended with sewage sludge ash. Atomic absorption spectrometry is used most frequently to measure the heavy metal levels in plants. Sample preparation is usually accomplished by acid digestion. The UK DOE24*63has provided general guidance for collecting samples of plant material and determining their heavy metal contents, 100

Contamination of Animals. - Entry into the food chain of sludge-derived heavy metals presents the potential of a toxicological hazard to higher animals, Miller and B ~ s w e l have l ~ ~ investigated heavy metal levels in tissues of animals whose diets contained vegetable matter grown in soils amended with sewage sludge. Wade e t al. have measured cadmium accumulation by earthworms inhabiting sludge-amended soils. Kienholz et a/. l1 supplemented the diet of feedlot steers with sewage sludge and evaluated the retention of sludge-derived heavy metals (arsenic, cadmium, copper, mercury, molybdenum, nickel, lead, selenium, and zinc) in blood, bone, brain, fat, kidney, liver, lung, muscle, and spleen. Johnson et a1.* reported a similar study with specific emphasis on the potential for cadmium, mercury and/or lead intoxication in humans. Ray et aZ.53measured calcium, cadmium, chromium, copper, iron, lead, magnesium, manganese, mercury, nickel, potassium, sodium, silver, and zinc in livers and kidneys of cattle receiving diets supplemented with sewage sludge, and Hallford et a2.65 have measured the levels of heavy metals in kidneys and livers of lambs produced by ewes consuming sewage sludge supplemented diets.

United Kingdom Department of Environment, ‘Atomic Absorption Spectrometry, 1979 Version’,HMSO, London, 1979. 64 S. E. Wade, C. A. Bache, and D. J. Lisk, Bull. Environ. Contam. Toxicol., 1982, 28, 557. 6s D. M. Hallford, R. E. Hudgens, D. G. Morrical, H. M. Schoenemann, H. E. Kiesling, and G. S. Smith, J. AnimalSci., 1982, 54, 922. 63

Determination of Heavy Metals in Sewage Sludge

101

The procedures for collecting, preparing, and analysing animal tissues for heavy metals have recently been reviewed by Sansoni and Iyengar,66,67Kirkbright,43 Stoeppler and Nuernberg,68 and Masironi and Parr.69 The Canadian DOE25 procedure for the determination of trace metals (cadmium, chromium, copper, nickel, lead, and zinc) in fish is applicable to other animal tissues. 6 Conclusions The heavy metal contents of sewage sludge determines both its utilization and its disposability. Even moderately high concentrations of heavy metals preclude recycling to the land as a fertilizer. The impact of heavy-metal laden fly ash on air quality prevents the incineration of such sewage sludges for energy recovery. Contamination of ground water by leachate makes landfilling of sewage sludge a risky proposition. Reliable methods of analysis are available for the determination of heavy metals in sewage sludge. These provide the information that is essential in making serious decisions about the best course to follow in utilizing or disposing of sewage sludge.

66

67

69

G. V. Iyengar and B. Sansoni, ‘Sample Preparation of Biological Materials for Trace Element Analysis’, Technical Report Series No. 197, pp. 73-101, International Atomic Energy Agency, Vienna, 1980. B. Sansoni and G. V. Iyengar, ‘Sampling and Storage of Biological Materials for Trace Element Analysis’, Technical Report Series No. 197, pp. 57-71, International Atomic Energy Agency, Vienna, 1980. M. Stoeppler and H. Nuernberg, ‘Critical Review of Analytical Methods for the Determination of Trace Elements in Biological Materials’, Conf. Proc., Internat. Workshop on Biological Specimen Collection, Luxembourg, April, 1977. R. Masironi and R. M. Parr, ‘Collection and Trace Element Analysis of Post Mortem Human Samples’, Conf. Proc., Internat. Workshop on Biological Specimen Collection, Luxembourg, April, -1977.

inorganic Deposits in invertebrate Tissues BY M. G. T A Y L O R A N D K . SlMKlSS 1 Introduction

Types of Deposits. - Inorganic deposits provide the basis for the skeletal structures of most groups of animals. Thus, among the vertebrates the bones and teeth consist of calcium hydroxyapatite incorporated in an organic matrix. Among the invertebrates the skeletons are typically calcium carbonate but there may be a great deal of variation in the crystal forms (aragonite or calcite) and in some phyla phosphates do occur (e.g., Brachiopods).' In the non-skeletal tissues a greater variety of inorganic deposits is found but renal stones, salivary calculi, and a variety of inorganic deposits in the joints have all been interpreted as examples of diseased states. Thus, the general view emerged that the main skeletal structures were made of calcium phosphate and calcium carbonate. These are extracellular deposits induced by a variety of specialized cells to form characteristically shaped structures. Any other inorganic deposits were generally considered to be pathological. This view has persisted well into this century but in the past 25 years there has been ever increasing evidence from electron microscopists that membranebound deposits of inorganic material occur in virtually every cell type in almost every phylum of the animal kingdom. These cells are not pathological in the sense of being either moribund or infected and one is therefore forced t o one of two conclusions. The deposits2 are produced either as part of a ubiquitous physiological function which, to date, has not been seriously studied by cell biologists, or they are by-products of inorganic biochemistry involving pathways that are only just being recognized, Both of these interpretations are radical in that they are not part of a generally accepted aspect of cell biology and yet there appears to be no alternative to them in explaining the fact that most organisms can accumulate relatively large concentrations of metals in their soft tissue^.^ These 'body loads' reflect, and in fact are often used t o monitor, the levels of metals in the environment. There is, therefore, an urgent need t o understand more clearly the biological processes that are involved in their formation and this review attempts to clarify three aspects of the problem. First, we review the available data on the occurrence of a variety of metal containing deposits of invertebrate soft tissues.

' E. T. Degens, Top. Cum. Chem., 1976, 64,1. ' N. Warabe, V. R. Meenakshi, P. L. Blackwelder, E. M. Kurtz, and D. G. Dunkelberger in 'The Mechanisms of Mineralization in the Invertebrates and Plants', ed. N. Watabe and K. M. Wilbur, Univ. South Carolina Press, 1976. K. Simkiss, M. Taylor, and A. Z. Mason, Mar. Biol. Lett., 1982, 3, 187.

Inorganic Deposits in Invertebrate Tissues

103

Second, we consider the information on the chemical form of these deposits since this provides a fundamental insight into the mechanisms that may be involved in their formation. Finally we try to relate these chemical properties to the biological evidence for cellular activities. I t appears from this review of the data that the inorganic deposits found in these invertebrate tissues are probably the end products of a small number of quite different cellular processes and we state this conclusion at the start in order t o simplify the understanding of the subsequent data. Basic Approaches. - The metals that are found in the soft tissues of invertebrates can be loosely classified by the hard-soft, acid-base, and class ‘a’ and ‘b’ concepts of Pearson and chat^^ I t is therefore extremely exciting t o find that this chemical classification is reflected in a similar biological grouping based on the morphology of the deposits. The hard acid, or class ‘a’ metals aregenerally found associated with oxygen donor ligands such as carbonates and phosphates. The resulting granules5 are found in specific cell types and they are characteristically membrane bound with a distinctive ‘concentric ring’ structure. These granules are generally in the range of 0.2-3.0 pm diameter but may extend up to as much as 250pm perhaps as a consequence of fusion between smaller granules. Despite the fact that they have a clear morphological shape the class ‘a’ inclusions do not have: a well ordered chemical structure and most are amorphous to X-ray and electron diffraction (Figure 1). The soft acid or class ‘b’ metals are often found within residual bodies in the cytoplasm of cells where they are associated mainly with sulphur donors. The deposits are again membrane bound but are smaller (0.5-2.0 pm diameter) and lack a distinct physical morphology (Figure 1). A number of the border-line metals may be present in either of the cellular deposits and it is also often easy to identify iron inclusions, which form a specific type of granule as do the urates and purines which are excretory products. The occurrence and number of granules that are found in an animal appear to reflect the physiological state of the organism and are often seasonal. The inclusions in yeasts and bacteria often contain polyphosphates and this has attracted considerable interest because of the importance of the energy stored in these phosphate bonds.6 Among protozoa kept in laboratory cultures the granules occur mainly in the stationary ‘growth phase’ and they can also be induced by starvation. Higher organisms only contain granules in specific organs and the digestive glands and renal tissues are the most common sources. This has led to a number of hypotheses proposing that the granules are related to the metabolic or excretory functions of these tissues. Frequently the class ‘a’ type of deposits are rich in calcium ions and a number of authors have related this to shell growth or repair,’ while a more recent awareness of the potential ( a ) R. G. Pearson, J. Am. Chern. SOC., 1963, 85, 3533; ( b ) S. Arhland, J. Chatt, and N. R. Davies, Q.Rev. Chern. SOC.,1958,12,265. B. E. Brown, Biol. Rev., 1982,57,621. I. S. Kulaev, in ‘The Biochemistry of Inorganic Polyphosphates’, Wiley, New York, 1979. A. Abolins-Krogis, Cell Tissue Res., 1976,172,455.

104

Environmental Chemistry

Inorganic Deposits in Invertebrate Tissues 105 toxicity of intracellular calcium has led to the suggestion that granules are basically sites of detoxification.8 Since the class ‘b’ metals are more reactive with many of the cytoplasmic proteins they are also potentially very toxic. The lysosoma1 degradation of these proteins and the occurrence of metals in residual bodies may also be important not only in detoxification but also in the formation of granular deposits. The variety of minerals that can be formed by biotic influences is, however, extremely large and Lowenstam’sg review should be consulted in order to appreciate the full range of these activities. The terminology for describing cellular deposits has not been rationalized and a list is therefore given in Table 1. This review deals mainly with the literature from 1970 to 1983 and uses the term granule as a general term for inorganic inclusions in invertebrate cells. Table 1 Synonyms used in the literature f o r ‘inorganic granules’ Granule Calcareous concretion Calcareous corpuscle Calcospherite Mineral concretion Intestinal inclusion Spherite

Refractive body Inclusion body Volutin Calcareous spherules Crystalline concretions Cadmosome Cuprosome

2 Metal Deposits

A variety of organisms have been shown to concentrate metals to levels many times greater than those of the environment3 (Table 2). Since some form of proportionality appears to exist between the concentrations of metals in the ecosystem and those of the animals inhabiting that environment a system of biological monitoring has been adopted in many countries. The advantages of this system are the ease of analyses, the integration of many influences (such as intermittent variations in environmental levels, seasonal variation, etc. ) and the concept of biological availability which automatically excludes inactive forms of the metals. Data on the accumulation of metals in invertebrates in such circumstances is reviewed by PhillipslO for aquatic ecosystems and by Martin and Coughtreyll for terrestrial and aerial influences. Since the concentrations of metals in organisms are often a t levels which would be lethal if they were present as the free ions, it is implicit in such studies that the metals must be compartmentalized or precipitated within the organism.

lo

l1

K. Simkiss, Calcif. Tissue Res., 1977, 24,199. H. A. Lowenstam, Science, 1981,211,1126. D. J . H. Phillips, ‘QuantitativeAquatic Biological Indicators’, Applied Science Publishers, London, 1980. M. H. Martin and P. J . Coughtrey, ‘Biological Monitoring of Heavy Metal Pollution’,

Applied Science Publishers, London, 1982.

Environmental Chemistry

106

Table 2 Concentration (mg kg-l dry wt.) of metals in soft tissues of some marine molluscs in relation to seawater concentrations3 Metal

Ag

A1 Cd co Cr

cu Fe Mn Ni Pb Zn Dry wt.

Bivalve (Filter feeding) Mytilus edulis 0.03 76 5.1 1.6 1.5 9.6 1700 3 .s 3.7 9.1 91 11.1%

Gastropod (herbivore) Littorina littorea 2.5 286 2

1.6 <2.5 70 435 26 3.2

9 99 25.6%

Cephalopod (carnivore) Alloteu this subulata 1 .o 72 1.4 0.7 <0.4 146 71 3 <1 0.2 a3 20.3%

Seawater

Concentration factor (approx.I

0.1 5

lo4 104 lo5 105 lo4 104 10 5 104 103 los 104

0.05 0.02 0.6

3 3 2 2 0.03 5

-

-

Studies on the distribution of metals within the tissues of aquatic invertebrates12 was pioneered by Bryan who not only identified the main organs involved but also indicated that the metals may exist in a variety of bound forms.13, l4 From these studies arose an interest in the metal inclusions of cellular granules and this data has been reviewed by C o o m b ~ and ’ ~ Brown.s The variety of metals found in granules are wide ranging and include the transuranics such as plutonium and americium. l 6 In considering this wealth of information we have decided to review the data selectively by considering metals individually and by relating the available information t o environmental perturbations, Potassium. - All cellular systems maintain a high intracellular potassium concentration through the effects of a Na/K exchange process involving membrane bound ATPase enzymes. The presence of high levels of p o t a s ~ i u r n ’in~ bone mineral was therefore used as an argument for the involvement of a ‘bone membrane’ in the process of ossification. The significance of potassium in the calcium phosphate type granules has not been explored fully. In the protozoan Tetrahymena pyriformis the K/P ratio varies according t o the nature of the D. A. Wright, Appi. Biol., 1978, 3 , 231. G . W. Bryan, J. Mar. Biol. ASS. U . K . , 1973, 53, 145. I4 G . W. Bryan, Phil. Trans. R . SOC.Lond., Ser. B , 1979, 286,483. I s T. L. Coombs and S. G. George, in ‘Proc. 12th Eur. Mar, Biol. Symp’, ed. D. S . McLuskey and A. J . Berry, Pergamon Press, Oxford and New York, 1978. J. C. Guary and R. Negrel, Comp. Biochem. Physiol., 1981, 68A, 423. 17 W. F. Neuman and W. K . Ramp, in ‘Cellular Mechanisms for Calcium Transfer and Homeostasis’, ed. G . Nicholas and R. H. Wasserman, Academic Press, New York and London, 1971. l2

l3

Inorganic Deposits in Invertebrate Tissues

107

growth medium. Thus Coleman et al. l8 found that the ratio increased when the organisms were reared in a casein, yeast extract medium supplemented with 0.3 mmol dm-3 calcium chloride or 0.3 mmol dm-3 strontium chloride, but in other nutrients they were relatively uniform. It is likely that further studies of potassium in granules would produce exciting results. Magnesium, Calcium, Strontium, and Barium. - Calcium and magnesium are frequently the major cations of all the oxygen anionic granules; carbonates, oxalates, and phosphates. The role of these granules in biomineralization, their characterization, and the mechanisms of deposition are reviewed in Section 3. Magnesium and calcium are essential metals for many physiological processes in addition to calcium’s obvious role in biomineralization. The presence of magnesium in intracellular granules has received relatively little attention although it sometimes exceeds the concentration of calcium several fold in marine molluscs. l9 This observation is interesting because magnesium is accumulated intracellularly at the expense of calcium in an analogous situation to K and Na. Thus although calcium can hardly be considered as an environmental pollutant intracellular calcium is carefully regulated at levels of 10”7-10-6mol dm-3.8 It is not surprising therefore that environmental calcium levels are important in influencing granule formation. Maintaining the fresh water snail, Helisoma duryi eudiscus,20 in different calcium concentrations showed that optimum growth of the animal occurred with 8 mg CaClz 100 cm-3 distilled water. At levels of 1, 16, and 32 mg CaClz 100 cm-3 higher numbers of granules were seen in the mantle edge, pulmonary region, and foot than at intermediate levels of calcium. These results could be interpreted as a need to store the element at lower calcium levels or detoxify it at higher levels. Uptake experiments with the radioactive isotope 45Ca in the freshwater snail, Limnaea stagnalis, showed that within an hour the isotope was distributed throughout the snail.21 Levels in soft tissues were initially high but at the end of the experiment most of the 45Ca had been incorporated into the shell suggesting that the soft tissues acted only as a temporary reserve. Other work22 has shown that the amount of calcium taken up in such experiments is usually greater than that lost in normal physiological processes. Coleman et aZ.23 have studied the effects of adding calcium and strontium to the medium on the composition of granules in Tetrahyrnena pyriforrnis. The addition of calcium (0.3 mmol dm-3 and 3 mmol dm-3) decreased Ca/P ratios in protease peptone medium but slightly increased them in a casein yeast extract medium, Sr/P ratios increased with increasing strontium concentrations usually at the expense of calcium but the results showed that there was some discrimination against the incorporation of strontium. Discrimination against the in vivo incorporation of strontium into

l8

l9 ’O

” 22

23

J . R. Coleman, J . R. Nilsson, R. R. Warner, and P, Batt, Exp. Cell Res., 1972, 74, 207. A. 2,Mason and J . A. Nott, Aquatic Toxicol., 1981, 1, 239. G. F. Haley and M. A. Gibson, Cun. J. Zool., 1971,49, 1001. P . Greenaway, J. E x p . Biol., 1971, 54,609. A. S. Gupta, BioL Bull., 1977, 1 5 3 , 369. J . R. Coleman, J. R. Nilsson, R. R. Warner, and P. Batt, Exp. Cell Res., 1973, 80, 1.

108

Environmental Chemistry

intracellular granules was confirmed by Simkiss= in his work on cellular uptake processes in the metal accumulating cells of the snail, Helix aspersa. He compared the incorporation of strontium with manganese, the cation most readily incorporated. In in vitro experiments with isolated granules and equimolar concentrations of the two metals the Sr/Mn ratio was 0.69 whereas in in vivo experiments the ratio was 2 x These results confirm that discrimination occurs at the cellular level which could be related to the size of the cation (Ca2+, 99 pm; Sr2*, 127 pm), or the cell's ability t o pump out strontium. The incidence of barium being detected in more than trace levels in granules is low. After including barium nitrate in the food of the ant, Formica polctena, barium was found in granules along with Ca, K, Mg, Mn, Fe, and Zn, although the exact concentration^^^ were not determined. Deposits, which are essentially pure barium sulphate, are found in desmids where they probably act as statoliths.= The rate of formation of these granules was related to the external concentration of barium and, as in many unicellular organisms, the deposits disappeared during exponential growth of the organisms. Aluminium and Silicon. - There is no known physiological role for aluminium but it has been detected in the phosphatic granules and lysosomes of many animals, although rarely in more than trace amounts. In the sea urchin Echinocardium, granules have been described which contain both aluminium and ~ i l i c o n .Some ~ insects, spiders, and gastropods also accumulate aluminium and sometimes incorporate it with ~ilicon.~'In association with Ca, Mg, Fe, Zn, Cu, and P (but not K) it was thought t o accumulate in the residual bodies of lysosomes. Despite this the accumulations were not thought t o be induced by environmental levels. Many organisms use silica as a major component of their skeletons and the process of silicification is therefore considered separately in Section 4. Vanadium. - The role of vanadium in biology is uncertain but recent speculation that Na/K ATPase activity might be controlled by vanadate ions28 has led to renewed interest in this element. Certainly laboratory experiments which included vanadium as VOS04 in the diet of some insects29 showed that vanadium can be incorporated in the granules formed by the midgut cells of ants. The nature of these accumulations has not been determined but analyses of the e.s.r. spectra of purified granules from the kidney of the mussel Mytilus edulis gave a signal assigned to that of the vanadyl ion.30 The vanadyllvanadate interaction remains a particularly intriguing aspect of this pathway.

24

25

K. Simkiss, J. Exp. Biol., 1981,94,317. A. Y. Jeantet, R. Martoja, and M. Truchet, C. R. Acad. Sci. Paris, Ser. D , 1974,278,

1441. A. J. Brook, A. Fotheringham, J . Bradley, and K. G. A. Jenkins,Br. Phycol. J., 1980,15, 261. 2 7 R. Martoja and M. Martoja, C. R. Acad. Sci Paris, Ser. D , 1973,276, 2951. L. C. Cantley, M. D. Resh, and G. Guidolti, Nature (London), 1978,272, 552. 2 9 A. Y. Jeantet, C. Ballan-Dufrancais,and R. Martoja, Rev. Ecol. Biol, Sol., 1977,14,563. 30 S. G. George, T. L. Coombs, and B. J. S. Pirie, Biochim. Biophys. Acta, 1982,716,61.

26

Inorganic Deposits in Invertebrate Tissues

109 Despite the sparse reports of the presence of vanadium in most organisms it does accumulate in large quantities in tunic ate^.^' For many years it was considered that it might be involved as an oxygen carrier comparable with haemoglobin or haemocyanin since it occurred in particular blood cells. Various workers have, however, been unsuccessful in demonstrating reversible O2 binding32 and this function is now disregarded. Nevertheless vanadium does accumulate and its uptake appears to be regulated, Danskin31 found that in some tunicate species vanadium was concentrated t o levels up t o lo6 fold greater than those in the environment. He found no seasonal variation and no increase in uptake at higher vanadium levels. The vanadium of ascidians is found in the granules of vacuoles in amoebocytes, signet ring cells, and compartment cells.33 The concentrations in the vanadophores of the vanadocytes of this species were described as scanty, but in the vacuoles the levels of vanadium were 1.0-1.2 mol dm-3. This concentration is apparently maintained by a low pH in reducing conditions. Most of the vanadium is present as a hydrated VII1species with sulphate as counterion. EXAFS studies indicate that in these situations vanadium is most likely coordinated by six oxygen atoms at an average distance of 199 pm.34 N.m.r. studies suggest that VII1 was capable of rapidly exchanging with water molecules.35 The biochemistry of vanadium in ascidian blood cells has been described by K ~ s t i n ~ ~ and its possible roles discussed. I t has been proposed that vanadium is taken up as the anionic vanadate, reduced t o the cationic VII1 and thus trapped at particular cellular sites. Chromium and Molybdenum. - Chromium has been detected at low concentrations in some granule^.^' Analyses of the phosphate granules of molluscan of the total mass or kidneys showed that chromium contributed 2 x 15x of the metal content.38 Chromium is frequently present in the environment as the relatively insoluble CrIII species which probably contributes t o its low bioavailability. By contrast molybdenum is present in seawater as the soluble MOO:- species. Since molybdenum is an essential metal for the enzymes involved in purine metabolism (xanthine oxidase) leading to the formation of uric acid and could be considered a phosphate analogue it is perhaps surprising that there is only one report of molybdenum in granules. In the digestive gland of the mollusc, Pecten r n a x i r n u ~ its , ~ ~presence in lysosomal bodies was considered to be an enzymic residue. 31

32 33 34

G . P. Danskin, Can. J. Zool., 1978,56, 547. I. G . Macara, G . C. McLeod, and K. Kustin, Comp. Biochem. Physiol., 1979,62A, 821. L. Botte, S. S. Cuppa, and M. D. Vincentus, Experientia, 1979, 35, 1228. T. D. Tullins, W. 0. Gillum, R. M. K. Carlson, and K:O. Hodgson, J. Am. Chern. SOC.,

1980,102,5670. R. M. K. Carlson, Proc. Natl. Acad. Sci. U.S.A., 1975,7 2 , 2217. 36 I. G. Macara, G. C. McLeod, and K. Kustin, Biochem J., 1979,181,457. 3 7 L. J. Doyle, N. J . Blake, C. C. Woo, and P. Yevitch, Science, 1978,199, 1431. 38 N. G. Carmichael, K. S. Squibb, and B. A. Fowler, J. Fish. Res. Board Can., 1979, 36, 35

1149. 39

C. Ballan-Dufrancais, A. Y. Jeantet, and S. Halpern, C. R . Acad. Sci. Paris, Ser. D , 1982,

294,673.

110 Environmental Chemistry Manganese. - Manganese is intermediate in size between magnesium and calcium (Mg2+, 72 pm; Mn2+, 82 pm; Ca2+, 99 pm) and is frequently incorporated into the phosphate granules. The isotope YliMn is one of the important nuclides discharged from power stationsm since manganese is not only capable of substituting for Ca2+ and Mg2+ but is also a cofactor for many enzymes. I t is not surprising, therefore, that bivalve molluscs41 and polychaete worms42 may concentrate manganese over environmental levels by up to lo4 in the digestive gland and up to lo6 in the kidney. The kidney of the mollusc Pinna n o b i l i ~ ~ ~ contains amorphous phosphate granules that may be as much as 18%manganese. These granules occur as nephroliths in the lumen of the kidney but the surrounding nephrocyte cells also contained granules of the same composition. High manganese levels were also found in kidney granules isolated from two species of bivalve. molluscs, bay and calico scallops collected from seas off the coasts of North and South Carolina.38 Manganese made u p 5 . 5 and 13.8%of the metal content in the two species. Carmichael e t al. laterM reported on the uptake and distribu don of manganese by the clam Mercenaria mercenaria after exposing them to 54Mn in seawater for five days. Accumulation of the metal was highest in the kidneys and 34.3% of the activity was found in the granules which had a composition similar to that found for the scallops (Ca, Mg, Mn, Zn, P). In similar experiments with zinc and cadmium only 5.6 and 1.3%of the metal, respectively, was eventually located in the granules. In vitro experiment^^^ with isolated granules incubated for three hours with 54Mn and lWCd showed that the numbers of receptor sites for both metals was similar (14.9 and 1 5 . 1 nmolM2+/ mg granule, respectively ). Scatchard plots showed that there was competition between the metals for binding sites. These experiments supported the view that in the cell class 'a' metals are accumulated and possibly detoxified in the granules whereas class 'b' metals are more likely t o be detoxified by binding to proteins. The most extensive studies on the incorporation of manganese into the intracellular granules of the hepatopancreas have been made on the snail, Helix aspersa. These granules are normally composed of calcium and magnesium phosphates and pyrophosphates. In these experimentsM a series of pairs of radioisotopes of metals was injected into the haemocoel of the snail and their uptake into the hepatopancreas and incorporation into the granules compared. The metals used were Mn, Fe, Co, Zn, Sr, Cd, and Hg and the uptake of manganese into the hepatopancreas exceeded that of any of the other metals by factors of between three for iron and 352 for strontium. The isolated granules gave factors of from six for iron to 4075 for strontium demonstrating the facile incorporation of manganese into the granules. Simkiss concluded that the pyrophosphate granules in the snail represented an ideal detoxification system for class 'a' R. J . Pentreath, J. Mar. Biol. Ass. U.K., 1973,53, 127. W.C.Bryan, J. Mar. BioL Ass. U.K.,1973, 53, 145. '' G. W. Bryan and L. G. Hummerstone, J. Mar. Biol. Ass. U.K., 1973,53, 859. 43 F. Ghiretti, B. Salvato, S. Carlucci, and R. Depieri, Experientiu, 1972,28, 232. 44 N. G.Carmichael, K. S. Squibb, D. W. Engel, and B. A. Fowler, Comp. Biochem. Physiol., 1980,65A,203. 4 5 N. G. Carmichael and S. C. Bondy, Experientiu, 1981,3 7 , 7 5 2 . 40

"

Inorganic Deposits in Invertebrate Tissues

111 cations since metal pyrophosphates are not only very insoluble but also show little change in binding strength from one metal ion t o another. Pyrophosphates are produced in cells in many anabolic processes but they are usually rapidly hydrolysed so that their presence in these metal containing granules suggests a specific metabolic role. Further studies& showed that the morphology of the granules depended on the physiological state of the animal, i.e., whether starved or fed. In starved animals the granules are spherical and manganese is incorporated throughout the granules. In fed animals, however, the manganese was deposited on the surface of preexisting granules from which it was assumed that fed animals were more metabolically active and so deposited more pyrophosphate. These hepatopancreas granules are subsequently exocytosed from the cells and occur in the faecal strands thus demonstrating that the granules can provide a ready route for the removal of excess metal from the snail. In a subsequent ultrastructural study4’ it was shown that the granules are extruded at approximately 48 h after a large dose of manganese. This fact was exploited to study the formation of new granules. Manganese did not seem t o initiate granule formation since all the centres of the granules were composed of calcium, magnesium, and phosphorus but there was clear evidence that manganese was added on the intravesicular side of the membrane. It was suggested that the membrane might be acting as a site for controlling the translocation of metal ions as a way of modifying the local pH t o favour mineralization or as a site that was active in the synthesis of pyrophosphate on its inner surface. Above all it demonstrates the cell’s ability to regulate effectively the class ‘a’ metal ions presented at pollutant levels.

Iron. - Although iron is often incorporated into the phosphate-type granules there are also some deposits in which it is the major component. Iron is essential for virtually all forms of life and is stored intracellularly in the ferritin molecule.48 Iron in the FeII form exists in the cytoplasm but it is oxidized to FelI1 when it enters the ferritin molecule, The molecule acts as a storage system which effectively detoxifies the free ion by ensuring that cytoplasmic proteins are not disrupted by non-specific binding. Insoluble deposits of iron are known as haemosiderin and apparently result from the intracellular breakdown of ferri tin. Iron granules have been described in many crustacea. Opra4’ studying the hepatopancreas of the crayfish Procam barus cZarkii found iron was deposited in specific secretory cells (F) which were active in biosynthesis. Miyawaka et a l s o followed up Ogura’s study by using histochemical techniques and deduced that iron was present as iron(II1) in a colloidal form bound possibly t o mucopolysaccharides or mucoproteins. Since there are also high levels of calcium in these cells he suggested that the presence of both these metals may be related to the moult cycle. K. Simkiss, K. G. A. Jenkins, J. McLellan, and E. Wheeler, Experientia, 1982,38, 333. A. Z.Mason and K. Simkiss, Exp. Cell Res., 1982,139, 383. 4 8 P. M. Hamson and R. J. Hoare in ‘Metals in Biochemistry’, Chapman and Hall, London, 46 4’

49

1980. K. Ogura, Ann. ZooL Jpn., 1959, 32, 133. M. Miyawaki, M. Matsuzaki, and N. Sasaki, Kumamoto J. Sci., 1961,5,161.

Enviro nm en tal Chemistry 112 Martins1 also implicated iron in the moult cycle of Cancer irroratus. Large quantities of iron were accumulated not only in the hepatopancreas but also in the gdls although this was lost at ecdysis. Metal levels in Crangons2 from a polluted environment were monitored through the moult cycle. Only iron and zinc were accumulated although the iron was found in granules in the hepatopancreas. ~ ~ found to have large The amphipod Stegocephaloidis c h r i ~ t i a n e n s i swas octahedral red brown deposits lying in rows along the gut caeca. Staining revealed the presence of both iron(I1) and iron(II1). There were no other cations and uncertain protein content. Although the deposits had a distinct morphology they were not birefringent. Birefringent iron (111) granulesH were found t o be associated with chromolipids and melanins in the epithelial cells of prosobranch mollusc kidneys. A detoxification mechanism was suggested by Lowe, considering that the iron had been compartmentalized in the lysosomal vacuolar system of the secretory cells. Exposing the mussel, Mytilus edulis, to 59Feas iron(1II) hydroxide resulted in A 17-fold and a 25-fold concentration in the viscera (hepatopancreas and a 35-fold increase in accumulation was observed when 59Fe was present in ferritin and transferrin, respectively.s The mode of uptake, (pinocytosis) was similar in all three compounds but the site of absorption for iron(II1) and ferritin was at the gills with subsequent transport by amoebocytes whereas transferrin was absorbed via the gut. Ferritin and transferrin were effectively immobilized in membrane bound vesicles in various tissues with little evidence for their degradation or excretion. Iron(II1) hydroxide was similarly contained but ca. 30% was excreted into the byssal threads. Extracellular iron deposits in the connective tissue of the echinoderm gut have been chara~terized.~‘Analyses showed that the deposits were iron(II1) phosphates. Molar ratios were close to 1:l and deviations could be accounted for by the presence in much lower concentrations of Mn, Cu, Zn, and Pb. The deposits were cumulative with age and the authors speculated that the deposits were derived from the oxidation of iron(I1) salts ingested under reducing conditions. It is interesting that Kulaev and co-workers have found polymeric metal phosphates in micro-organisms and proposed a structure for iron phosphate. Additional metals may also be present (Mg, Ca, Mn). Kulaev6 believes these deposits are a detoxificatory and regulatory mechanism constituting reserves for phosphate and metal ions,

” 52

s3 54

55

56

57

J . L. M. Martin, Comp. Biochem. Physiol., 1973,46A,123. B. E l k a h and C. Chassard Bouchard, Cah. Biol. Mar., 1978,19,459. P. G. Moore, J. Exp. Mar. Biol. Ecol., 1979,39, 223. D.M. Lowe and M. N. Moore, J. Mar. Biol. Ass. U.K., 1979,59, 851. S. G. George, B. J . S. Pirie, and T. L. Coombs, J. Exp. Mar. Biol. Ecol., 1976,2 3 , 71. S. G. George, B. J . S. Pirie, and T. L. Coombs in ‘Proc. Int. Conf. on Heavy Metals in the Environment’, Publs. Natl. Res. Council Canada, 1977,2, 887. J . B. Buchanan, B. E. Brown, T. L. Coombs, B. J . S. Pirie, and J . A. Allen, J. Mar. Biol.

Ass. U.K.,1980,60,631.

Inorganic Deposits in Invertebrate Tissues 113 Iron granules are also present in the epithelial cells of the dorsal wall of the ~ ~ occur in three forms, namely radular sac of Cryptochiton ~ t e l l e r i .They crystalline and paracrystalline ferritin and amorphous deposits believed t o be haemosiderin. Their presence is assumed t o be related t o the mineralization of chiton teeth which are curiously composed of a form of magnetite (Fe203.FeO). in the formation of radula teeth in the chiton the iron present as ferritin in the superior epithelial cells is considerably transformed in the process, Reduction of a third of the iron(iI1) is accompanied by structural changes. Webb59 has reviewed the bioinorganic chemistry of iron and related it to metabolic pathways producing iron oxide deposits. These are found in many invertebrate phyla, bacteria, rnollusca, arthropoda, and annelida, and also as iron sulphides in bacteria. Magnetite particles (40-120 nm) are found in membrane bound magnetosomes in certain bacteria where they are believed t o have a direction sensing role. A strange observation was made by Fowler et aL6' in their investigation on the effects of mercury on the clam Mercenaria mercenaria. The clams were maintained for 6 days in tanks t o which mercury(I1) chloride was added. At the lowest level (0.1 p.p.m. Hg) there were no obvious differences from controls in the mantle epithelial cells but at higher levels (up to 10 p.p.m. Hg) there were electron dense cytosomes which when analysed by X-ray probe were found t o contain iron as well as mercury, in a ratio 1 : l . No counterion element such as P or S was detected. Atomic absorption analyses of the total tissues gave Fe/Hg ratios varying from 51 in controls to 3 (1 p.p.m. Hg). Cobalt. - The requirement for cobalt in most organisms is low and in unpolluted sites the concentration of cobalt in the soft tissues of a gastropod were of the order of 7 nmol g-' dry tissue6' while levels in a burrowing bivaIve from an area contaminated with mine tailings were 2 pmol g-' dry tissue.62 The chemical form of the cobalt is largely unknown and reports of cobalt in granules are few, Hignette63 found cobalt in the nephroliths of two molluscs, Pinna nobizis and Tridacna maxima, and he considered these species to be good bioindicators of metallic or radiological pollution. Laboratory experiments injecting snails with cobalt solutions23 or feeding ants cobalt(I1) chloride25have shown that the metal becomes incorporated in the phosphate granules of the snail's hepatopancreas and the intestinal epithelium of ants.

Nickel. - Nickel is only rarely detected in granules but nickel (1 pmol g-') has been found in granules from the kidney of the clam Mercenaria mevcen~ria.~' Copper. - Intracellular deposits of copper are found in the absorptive (R) cells .~~ in the hepatopancreas of the freshwater crayfish Procam barus ~ Z a r k i i The occurrence of these deposits was enhanced when the crayfish were kept in tanks 59

6o 61

63

K. M. Towe and H. A. Lowenstam, J. Ultrastruct. Res., 1967, 17, 1. J. Webb in 'Biomineralisation and Biological Metal Accumulation', ed. P. Westbroek and E. W. de Jong, D. Reidel, Dordrecht, The Netherlands, 1983. B. A. Fowler, D. A. Wolfe, and W. F. Hettler, J. Fish,Res. Board Can., 1975, 32, 1767. G. W. Bryan, G. W. Potts, and G. R. Forster, J. Mar. Biol. Ass. U.K., 1977, 57, 379. G. W. Bryan and L. G. Hummerstone, J. Mar. Biol. Ass. U.K., 1978, 58, 401. M. Hignette, C. R , Acad. Sci. Paris, Ser. D , 1979, 289, 1069.

114

Environmental Chemistry

containing 20 mmol dm-3 copper sulphate from which it was concluded that they were influenced by environmental levels.50 Copper is, however, toxic to crayfish and the LD50 at 96 h was 48 mmol dm-3.64 The hepatopancreas of the lobster Homarus americanus is the site of synthesis of the copper-containing protein h a e m ~ c y a n i n which , ~ ~ acts as the respiratory pigment of many arthropods, I t appears likely, therefore, that the storage of copper in this tissue is related to haemocyanin formation. Certainly during the moult cycle there are considerable movements of copper and other metals between the hepatopancreas and the blood of the shrimp Crangon.& In other crustacea the blood proteins are apparently catabolized during periods of starvation when the copper from the haemocyanin is apparently skored in the h e p a t ~ p a n c r e a s Ecdysis .~~ may, therefore, be affecting the storage of copper in the vesicles of the R cells partly as a result of nutritional deprivation.68 ~ ~ levels were comIn a study of the amphipod Corophium v o l ~ t a t o rcopper pared in animals from clean and contaminated sites. The body levels of animals from the contaminated sites were three times those of specimens from the clean waters. Ultrastructural examination of the caeca of the midgut revealed the presence of intracellular granules which were shown on analysis to contain copper and sulphur with traces of iron and occasionally calcium. Only after starvation were similar granules seen in the amphipod from the clean site. The ability to retain copper in intracellular granules was considered t o give the amphipod a tolerance to the high copper levels in the environment. The granules were eventually excreted when the cells matured and migrated to the end of the tubule where they disintegrated. Tolerance t o copper and lead by the isopod Asellus was investigated by Brown.m, 71 Experiments in which animals were exposed for 10-14 days showed that copper tolerant animals survived in copper sulphate solutions (7.7 pmol dm-3) while the controls died. In experiments of feeding leaves soaked with copper sulphate the isopods which had acquired a tolerance to lead still died. Analysis at the end of the experiment showed that only tolerant animals accumulated copper from food whereas the control animals accumulated higher levels from solution. Ultrastructural studies and X-ray microanalysis showed that copper was found in spherical granules and in more granular deposits in the hepatopancreas of the copper tolerant species. The lead but not copper tolerant animals also had the intracellular copper inclusions while in the control population the copper was found throughout the cell in non-membrane-bound deposits. Brown concluded that the mechanism of copper tolerance was related t o the ability t o accumulate and detoxify copper in the granules in the hepatopancreas. The studies on lead are reported below.

64

65

J. H.Hubschman, Cmstaceunu, 1967,12,3 3 . E. G. Senkbeil and J . C. Wriston, jun., Comp. Biochern. Physiol., 1981,68B,163.

J. S. Djangmah, Comp. Biochem Physiol., 1970,32, 709. M. A. Alikhan, Comp. Biochem Physiol., 1972,42A,823. E. Adams, K. Simkiss, and M. Taylor, Comp. Biochem. Physiol., 1982,72A,73. 69 J. D. Icely and J. A. Nott, Mar. Bid., 1980,5 7 , 193. 70 B. E. Brown, WaterRes., 1976,10, 555. 71 B. E. Brown, FreshwaterBiol., 1977,7,235.

66 6’

Inorganic Deposits in Invertebrate Tissues

115

Barnacles (Balanus balanoides)" collected from the same polluted sites as the amphipods just described were also found to have membrane-bound granules composed of copper and sulphur with smaller amounts of potassium and calcium in the parenchyma cells of the midgut. The barnacle also accumulates zinc but this is found in a different type of granule which suggests that different mechanisms are operating for the two metals; phosphate granules for class 'a' zinc and sulphur granules for class 'b' copper. Heavy metal levels have also been studied in two marine gastropods, a carnivore Thais lapillus and a herbivore Littorina littorea from the coasts around Wales. The copper levels were always higher in the carnivore than in the herbivore and significantly higher in the digestive gland and gonad compared with whole body levels.n The bioaccumulation of copper has also been studied in Littorina from around the coast of France.% Copper granules were only rarely found in the digestive gland confirming the observation that the digestive gland is not an organ which accumulates copper in this species. Copper deposits were found in the form of CuS needles inside lysosomes in specific cells of the connective tissue. These accumulations increased with age but were independent of environmental levels. Subcellular fractionation studies of the digestive glands of the mussel75 Mytilus galloprovincialis that had been kept in tanks which contained copper (0.7 pmol dm-3) for 3 days showed not only a significant increase in the whole digestive gland but also in each cellular fraction. In the cytosol copper was bound to a low-molecular-weight thionein-like protein. After transferring the animals t o copper-free water these copper-protein levels increased initially but then fell with a subsequent increase of copper in the lysosomes. Ultimately the copper was concentrated in residual bodies which were then exocytosed. There were therefore two forms of copper in the digestive gland (1) copper thionein-like protein and (2) copper in membrane bound vesicles (lysosomes or residual bodies) and it was proposed that there was a metabolic relationship between the two fractions. After feeding houseflies (Musca d o r n e s t i ~ awith ) ~ ~ sucrose-containing copper sulphate (0.03%) copper deposits were found in the midgut cells. The copper ~ ~ were similarly raised in a levels increased with age. Larvae of D r o ~ o p h i Z awhich medium enriched with copper( 11) sulphate also accumulated copper in the organelles of the midgut cells described as cytolysomes. The abundance of granules in this case was related to the copper concentration in the diet. The granules were membrane bound and associated with sulphur. There was no evidence for the presence of lysosomal enzymes in the granules in Drosophila although the granules in Musca were thought t o be derived from Golgi components and lysosomes.

G . Walker, Mar. Bid., 1977,39,343. M. P. Ireland and R. J. Wootton, Environ. Pollut., 1977,12,27. 74 M.Martoja, V. T. Tue, and B. EIkaim, J. Exp. Mar. BioL Ecol., 1980,43,251. '' A. Viarengo, G. Zanicchi, M. N. Moore, and M. Orunesu, Aquatic Toxicol., 1981,1, 147. 76 B. K. Filshie, D. F. Pouhon, and D. F. Waterhouse, Tissue Cell, 1971, 3, 77. 7 7 R. L. Tapp, J. Cell Sci., 1975,17,449. 72

73

116

En viro n m en tal Chemistry

Zinc, Cadmium, and Mercury. - Zinc is often found to be a component with calcium and magnesium of the phosphate type granules37*38, 79 and occasionally it is actually the major cation.s0 I t is also an important constituent of many of the sulphur granules where it often occurs with cadmium, mercury, and copper, Cadmium and mercury are found only rarely in phosphate granules24 and they generally occur in membrane bound secondary lysosomes or residual bodies.54,81,82These observations are in accord with the different chemistry of Group IIA and IIB elements. Zinc is an essential trace metal and although it is less toxic than cadmium and mercury, George and Pirie found in a study of zinc metabolism that the mussel (My tiZus eduZis)= was distressed at levels greater than 15 pmol dm-3. Inshore levels are in the range 1.5-308 nmol dm-3. In laboratory experiments over a seven day period there was almost direct proportionality in the accumulation of zinc into soft tissues and cell free haemolymph. The levels in the soft tissues were an order of magnitude greater and it was found that 30% of the body load was concentrated in granules in the kidney. The granules have a heterogeneous composition but are either mainly zinc, iron, and sulphur with low phosphorus and calcium, or high phosphorus and low sulphur with zinc as the major cation with some calcium. The granules were excreted in the urine. Zinc in the soft tissues of the barnacle (BaZanus baZanoides)* is apparently correlated with the levels of zinc in the environment. I t is the major cation in the granules of the parenchyma tissues surrounding the midgut which are phosphatic but also contain traces of calcium, magnesium, potassium, copper, and iron in decreasing amounts. Cadmium can apparently occupy the same metabolic pathways as zinc in some animals. Thus, exposing Mytilus eduZis* t o this metal resulted in 85% of the metal accumulating in the kidneys in membrane bound granules that would normally have contained zinc. Sub-cellular fractionation and differential centrifugation demonstrated, however, that 80% of the cadmium remained in the cytosol. Gel permeation chromatography showed that most of the cadmium eluted with a low-molecular-weight protein of the thionein type. These results imply that the isolation procedures used may release the components of the granules. They also suggest that the granules are lysosomal in origin since acid phosphatase activity was also recovered in the cytosol. The role of lysosomes in the metabolism of zinc, cadmium, mercury, and copper has emerged in several studies in different species. In laboratory experiments in which the cockroach, BZateZZa, was exposed to mercury both as mercury(I1) chloride and methylmercury the metal was eventually located in the membrane bound residual bodies. 82, 85 789

P. Prentq, Cell Tissue Res., 1979,196,123.

’’ S. Dunlop and G. Chapman, Environ. Res., 1981,24,264. G.Walker, P. S. Rainbow, P. Foster, and D. J . Crisp, Mar. Biol., 1975,30, 57. A. J.Morgan and B. Morris, Histochem., 1982,7 5 , 269. a2 C. Ballan-Dufrancais, A. Y. Jeantet, and C. Quintat, C. R Acad. Sci. Paris, Ser. D, 1975, 288,847. 83 S. G.George and B. J. S. Pirie, J. Mar. Biol. Ass. U.K.,1980,60,575. 84 S. G.George and B. J . S. Pkie, Biochim. Biophys. Acta, 1979,580, 234.

80

Inorganic Deposits in Invertebrate Tissues

117

Fowler e t aLm found mercury became associated with iron in electron dense cytosomes in the mantle epithelial cells of the clam, Mercenaria mercenaria, when it was exposed to mercury(I1) chloride in the laboratory. No anionic component of these deposits was reported. The mercury appeared initially to suppress the iron content but as the mercury levels increased the iron levels were restored t o control levels, The effects of cadmium on freshwater and marine decapods have been compared in a number of studies. In the freshwater crayfish, Pontastacus leptod a ~ t y l u s ,it~ was ~ , ~suggested ~ that cadmium induced the Golgi body to produce primary lysosomes in the midgut gland. The secondary lysosomes subsequently contained cadmium with zinc and copper all associated with sulphur. After exposure of the crab Carcinus maenus t o cadmium in the laboratory,88 the metal was found in granules of the midgut gland where it was also associated with zinc, copper, and sulphur with traces of calcium and phosphorus, Further experiments which kept the clam, Mercenaria mercenaria, in seawater containing cadmium and zinc showed little accumulation of these elements in granules.44 in a separate experiment with isolated granules it was shown that cadmium will bind to phosphate type granules showing that the observed discrimination presumably occurs at the cellular The chemical properties of cadmium are likely t o favour protein binding and in earthworms (Lumbricus ~ ~ polluted sites the metal becomes rebeEZus and Dendrobaena r ~ b i d a )from associated with the chloragocyte cells lining the peritoneum. In these cells the trace metals accumulate in cytoplasmic granules called chloragosomes which may contain cadmium and sulphur with minor amounts of Ca, Pb, Zn, and P. Lead. -This is another of the metals with no known biological function although it is readily incorporated into biological tissues where it often substitutes for calcium.89 There have been several reports of the incorporation of lead into cytoplasmic granules particularly in species living in polluted environments, The levels of lead found from field and laboratory studies of animals are invariably related to the environmental levels.g0 The uptake and distribution of metals in earthworms from base metal mining areas has been studied by Irelandg1 who established correlations between calcium and lead levels. An ultrastructural study of earthworms taken from a polluted environment located lead in chloragosomes and debris vesicles in the chloragocytes. X-Ray microanalysis showed that the lead was associated with phosphorusg2 but the interpretation of this was difficult since phosphate buffers had been used in the preparation of the tissues. Subsequent studies have confirmed, however, that the chloragosomes contain lead in phosphate granules.81 Normal chloragosomes are A. Y. Jeantet, C. Ballan-Dufrancais, and J. Ruste, Biol. Cell., 1980, 39, 325. C. Chassard-Bouchaud, C. R. Acad. Sci Paris, Ser. D , 1980,290, 347. " C. Chassard-Bouchaud, C. R. Acad. Sci. Paris, Ser. D, 1982, 293, 261. " C. Chassard-Bouchaud, C. R. Acad. Sci Paris, Ser. D , 1982, 294,153. a9 U. Sturesson, Ambio, 1976, 5, 253. 9 0 M. P. Ireland, Comp. Biochem. Physiol., 1975, 52B, 551. 91 M. P, Ireland and R. J. Wooton, Environ. Pollut., 1976, 10, 201. 9 2 M. P. Ireland and K. S. Richards, Histochemistry, 1977, 51, 153. 85

86

En viro nmental Chemistry 118 composed of calcium phosphate with some magnesium and zinc.93 No carbonate has been detected though the cells are believed to have a physiological function in the regulation of Ca2+and HC03- ions in the blood and coelomic fluid. In a series of experiments with the copper tolerant isopod, AseZZus meridianus, Brown demonstrated that tolerance to copper also conferred a tolerance to She located lead with copper in the ‘cuprosomes’ of the hepatopancreas of this animal.% The ‘cuprosomes’ are essentially sulphur granules with calcium as a minor component. The addition of lead t o the culture medium of the protozoan Tetrahymena p y r i f ~ r m i s results , ~ ~ in the uptake of precipitated lead into food vacuoles at low lead levels. Higher lead levels inhibited food vacuole formation and cell growth but lead also appeared to penetrate the cell surface so that eventually it occurred in lysosomes. Lead also accumulates in marine invertebrates. George and Pirie analysed tissues of Mytilus edulis taken from a non-polluted environment and found that more than 70% of the body load of lead occurred in the kidney.83 Earlier experiments relating the form of lead t o the uptake and distribution showed that lead citrate was taken up three to four times faster than lead nitrate a t the same concentration (0.5 mol dm-3).% The lead was retained in granules in the kidney which also contained calcium, phosphorus, iron, potassium, and chlorine. In the gut lead was associated with less electron dense amorphous material. Lead is thus considered to be detoxified in granules prior to excretion. Schulz-Baldesg7 reported on the kinetics of lead uptake into MytiZus and the mechanisms by which the lead is incorporated into membrane-bound vesicles within the excretory cells of the kidney. 3 Ligand Binding I t will be apparent from the review of the distribution of metals in intracellular granules that two main types of ligand appear to be involved. The hard acid metals are found associated with oxygen donor ligands while the soft acid metals appear t o be mainly bound t o sulphur donors.

Oxygen Donor Ligands - Carbonate, Phosphate, Sulphate, and Oxalate Granules. - These are the main components of most intracellular granules and they are typically present as calcium and magnesium deposits into which varying amounts of other metals may become incorporated. The literature on the possible functions of these granules is confused for three reasons. First they are often seen only as organic remnants in decalcified sections of histological material.98 Sccond they are often considered simply as inorganic granules and

”

95 96

97

A. J . Morgan and C. Winters, Histochemistry, 1982, 73, 589. B. E. Brown, Nomure (London), 1978, 276, 388. J . R. Nilsson, Celt Sci., 1979, 39, 383. T. L. Coombs, Proc. Anal. Div. Chem. SOC.,1977, 14, 219. M. Schulz-Baldes, in ‘Proc. Int. Conf. on Heavy Metals in the Environment’, Publs. Natl. Res. Council, Canada, 1977, 2, 211. S. P. Hopkin and J. A. Nott, J. Mar. Biol. Ass. U.K., 1979, 59, 867.

Inorganic Deposits in Invertebrate Tissues 119 their functions are deduced from their anatomical location* rather than from their chemical composition. This has given rise t o an extensive literature that is often difficult to interpret in terms of CaC03 and Ca3(PO& interactions. Finally, the two most common deposits of carbonate and phosphate are actually the salts of the two most common inorganic buffering systems in biology. This probably accounts for their occurrence but it also leads t o considerable speculation on the role of acid-base balancelW in the maintenance of these deposits. Superimposed on these speculations are the additional concepts that (a) intracellular and extracellular calcium levels are both regulated by living organisms and usually maintained at a several thousand-fold difference (lo-’ and mol dm-3 respectively), (b) bicarbonate metabolism is related t o the transport of respiratory gases, and (c) phosphate is regulated by cells and maintained as the basis of energy storage as adenosine triphosphate. All of these concepts have been variously invoked to explain particular aspects of the functions of intracellular granules. Intracellular granules have a characteristic morphology such that decalcified granules frequently have a concentrically layered organic component which is variable in composition and form. It has therefore been suggested that it is a matrix for mineral deposition (as in shells) or that the deposition is intermittent. There are in fact many conflicting reports in the literature on the nature, function, and mechanism of granule formation. Many of these differences may be attributed to differences between species, environmental factors, habitats such as marine, freshwater, or terrestial and even seasonal variations. Many of the species may hibernate and aestivate or have regular periods of growth and reproduction. Stress factors such as damaged shells, acidosis, etc. also appear to affect granule deposition. These influences have undoubtedly complicated the interpretation of the structuring of the organic matrix but most workers agree that it is composed of sulphated or acidic mucopolysaccharides associated with a sparse protein component, There are no reported cases of the successful remineralization of one of these organic ghosts in vitro. The functions of most intracellular granules are not clear but much of the impetus for these studies has arisen from studies of the mechanisms of shell repair in molluscs. lol Wagge investigated the nature and function of the calcium granules in shell regeneration and reviewed much of the earlier literature. The ability to repair damaged shells has long been recognized and correlated with the observation that calcium is stored in specific cells in che digestive glands. The method by which the calcium was mobilized was unknown especially since Siolilo2 reported that there was no increase in the blood calcium levels. Early reportslo3 suggested that the calcium was present as calcium phosphate in a protein sphere but Wagge found a negative response t o tests for phosphates but

A. Abolins-Krogis,Symp. Zool. SOC.London, 1968, 22, 75. J . W. Campbell and B. D. Bayan, in ‘The Mechanisms of Mineralization in the Invertebrates and Plants’, ed. N. Watabe and K . M. Wilbur, Univ. South Carolina Press, 1976. 101 L. E. Wagge, Q. J. Microsc. Sci., 1951, 92, 307. ‘ 0 2 H. Sioli, Zool. Jahrb. Abt. Allg. Zool. Physiol. TiGre, 1935, 54, 507. B. J . Krijgsman, 2. VgZ. Physiol., 1928,8 , 187. ”

loo

En viro nmen taE Chemistry

120

because they reacted with dilute acids believed them to be amorphous calcium carbonate. His tests involved the dissolution of the granules in acids in which carbonates are readily soluble and was really based on the fact that changes in pH can dramatically change K s p CaC03. The solubility of phosphates is, however, also pH dependent and as the phosphate anion is protonated it becomes more soluble [equation (l)].

~

+

+

0 H+ ~+ H ~P O-~ ~ -H+ + H,PO~-

+ H+ =+H ~ P O ~

FretterfW found that following the administration of 32P it became localized in the granules of the hepatopancreas, while Burton105 analysed the granules from Helix pomatia and found they were basically phosphate with up t o 10% carbonate. Wagge's study appeared to show that these calcium reserves could be transported by amoebocytes (wandering cells) in the snail's haemolymph to the sites where calcium was required, thus establishing that the digestive gland granules could act as a c;ilcium store. He believed that the calcium and protein were ingested separately by the amoebocytes and found high levels of alkaline phosphatase in the digestive gland and the mantle. Sumnerlo6 investigated the distribution of hydrolytic enzymes in the cells of the digestive glands of certain lameilibranchs and gastropods and found acid phosphatase in the calcium granules of Helix aspersa in the digestive gland. Kapur and Gibson107,108studying the freshwater gastropod Helisoma duryi eudiscus did not find calcium in the amoebocytes but found calcium in large granules in the connective tissue of the mantle edge as well as in other tissues. These granules consisted of concentrically arranged layers of calcium which dissolved in sulphuric acid t o recrystallize as monoclinic crystals characteristic of calcium carbonate (sic). Tests for inorganic phosphate were negative. Thus it seemed that the granules in the mantle consisted of calcium carbonate. There was an organic matrix of sulphated mucopoly saccharide also found by A b o l i n s - K r o g i ~who ~ ~ ~observed changes in the number of granules in the mantle Ca cells during shell repair. The extracellular granules are in close contact with the haemolymph which as Burton has shown is supersaturated with respect t o calcium carbonate. 110The ionic composition of the blood is variable between species, individuals, and the various physiological states of the animal (hydration, feeding, hibernating, e t c . ) and terrestrial molluscs show a wide tolerance to a whole range of ionic concentrations. Studies on the buffering capacity of the blood show, however, that the changes in concentration of calcium and bicarbonate are qualitatively those that would be expected if the blood were in equilibrium with solid calcium carbonate. Many of these problems can be resolved if it is accepted that in molluscs there are two entirely different populations of cells capable of forming intraV. Fretter, Q. J. Microsc. Sci., 1952, 93, 1 3 3 . R . F. Burton, Comp. Biochem. Physiol., 1972,43A, 655. l o 6 A. T. Sumner, J. Zool., 1969, 158, 277. ' 0 7 S. P. Kapur and M. A. Gibson, Can. J. Zool., 1967.45, 1169. I o 8 S. P. Kapur and M. A. Gibson, Can. J. Zool., 1968,46,481. l o g A. Abolins-Krogis, Ark. Zool., 1963, 1 5 , 461. R. F. Burton, Comp. Biochem. Physiol., 1970, 37, 193. '04

105

Inorganic Deposits in Invertebrate Tissues

121

cellular granules. If granules are extracted from a variety of different tissues it can be shown that one group is highly soluble, calcareous and occurs in a diversity of sites, while the second group is insoluble, phosphatic and largely restricted to the hepatopancreas.lll This concept has been extended by Mason and Nottfg who reviewed the literature to show that the calcareous type occurs in pore cells in the connective tissues of the mantle, head, and foot where it appears t o supply ions for shell repair and the control of electrolyte and acidbase balance, The phosphatic type of granule is restricted to certain 'basophil cells' and it is this form that appears to accumulate the great variety of trace metals. While many workers were putting their efforts into the role of the granules in biomineralization, especially with respect to calcium, different groups were concentrating their studies on the inorganic phosphate granules in microorganisms. The work has been excellently reviewed in a book by Kulaev,6 where the chemistry, biochemistry, biological function and evolutionary aspects are extensively discussed, I t has been suggested that the polyphosphate deposits represent an atavistic biochemistry which predates ATP so that polyphosphates can fulfil many of the functions that would normally be associated with ATP, e.g., phosphorylation. Organisms with both systems can switch between the two.112 It has also been proposed that polyphosphate granules can serve a role in storing or detoxifying phosphate as well as metals such as Ca, Mg, Mn, and Fe. It has been considered that polyphosphates will be present only when there is an energy surplus. Storing phosphates as polyphosphates has the advantage that there is little effect on the osmotic pressure of the organism. The fact that granules disappear during exponential growth, i. e ., when the cell's metabolic activity is high due t o cell division, gives credence to this interpretation of the deposits as an energy store. An investigation by Rosenberg and Munk combined both aspects of metal and phosphate metabolism in Tetrahymena pyriformis. Magnesium, calcium and phosphate were rapidly accumulated from the medium and deposited in pyrophosphate granules. When the cells were then placed in a phosphate free medium the process was reversed. The granules completely disappeared and calcium was lost against a concentration gradient. Only after the granules had disappeared was there an increased efflux of magnesium. These experiments give the granules a multifunctional role involving energy storage and the detoxification of various ions. An interesting example of an entirely different system for forming intracellular granules in the protozoa occurs in the coccolithophorids~f4In these organisms a highly structured plate of calcite (a coccolith) is formed in a vesicle prior t o being exocytosed onto the surface of the organism. These systems have been studied in great detail in relation to the organic matrix of the coccolith, the

"' K. Simkiss, Symp. SOC.Exp. Biol., 1976, 30, 423. 'I2

'13

'I4

I. S. Kulaev, S. E. Mansurava, E. B. Burlakova, and V. F. Dukhavich, Biosystems, 1980,

12,177. H. Rosenberg and N. Munk, Biochim. Biophys. Acta, 1969, 184, 191. D. Klavenes and E. Paasche, 'Physiology of Coccolithophorids', in Biochem. and Physiol. of Protozoa', 1979,2nd ed. Vol. 1.

Environmental Chemistry

122

ultrastructure of the cell, the sources of C 0 3 2 - ions and the involvement of Ca2+ in contrast to Mg2+and Sr2+ions. Sulphate granules are known to occur in two groups of organisms. The desmids form deposits of barium sulphateZ6while the ephyra larvae of jellyfish form a statocyst of calcium sulphate that acts as a gravity detector.l15 Deposits of oxalates are extremely common in many plants but have also been found as renal concretions in the mollusc Pecren maximus.

-

Oxygen Donor Ligands Analyses and Structure. - The absence of good analytical data has been a major obstacle to the understanding of intracellular granules. Most of the early work was based on a variety of histochemical methods but they suffer from problems of low specificity and physical adsorption of the reagents. The detection of metals by anatomists is almost entirely done by electron probe X-ray analysis of thin sections of tissue or isolated granules. The method is limited t o elements 2 > 10 and is at the present time only semi-quantitative. Full analyses of isolated granules are few. The weight loss after heating to ca. llO°C, which is considered to measure the water content, ranges from 18 t o 31%.78*117-119 The organic content is more variable and values range from ca. 2.5% to more than 60% of wet weight. Analyses of granules from Tetrahyrnena pyriformis by different workers have given very different estimates of organic content. Coleman18 suggested that the low result (2.5%) given by Rosenberg compared with their estimate from in situ probe analysis of ca. 62% was due t o the isolation procedures of Rosenberg who used ethanol and ether. Isolation of granules without the use of organic solvents is possible and Helix aspersa gives the comparatively low value of 7% organic matter.’18 The molar ratios of the inorganic ions from analyses of isolated granules are given in Tables 3 and 4 and the heterogeneous nature of the granules is obvious. The binding capacity of metal ions as equivalent divalent cations has been used to calculate metal/phosphorus ratios. The first figure is given as the M2+/P ratio and the second figure is corrected to take account of other anions present. These ratios can be compared with those of known calcium phosphate compounds given in Table 5, which are presented as an aid to interpretation of the analyses taking into account mass and charge balance. It is seen that many of the metalphosphorus ratios are ca. 1, which could indicate the presence of HPO2- or P20$-, Ratios closer to 1.67 suggest a hydroxyapatite species and further characterization of these is clearly indicated. The presence of phospholipids and other binding sites also needs to be considered in interpreting these data. Pyrophosphate has been confirmed as a constituent of granules from Tetrahyrnena117

115

‘16 ‘‘I

‘I8

D. B. Spangenberg, in ‘Mechanisms of Mineralization in the Invertebrates and Plants’, ed. N. Watabe and K. M. Wilbur, Univ. South Carolina Press, 1976. J . Overnell, J. Exp. Mar. B i d . Ecol., 1981, 52, 173. H. Rosenberg, E x p . Cell Res., 1966, 41, 397. B. Howard, P. C. H. Mitchell, A. Ritchie, K. Simkiss, and M. Taylor, Biochem J., 1981, 194, 507. G. Walker, P. S. Rainbow, P. Foster, D. J. Crisp, and D. L. Holland, Mar. Biol., 1975, 33, 161.

1.0

1.0

1.31

0.37 0.24

1.0

1.0

1.0 1.0 1.0 1.0

0.57 0.97 0.29 0.33

0.10

1.0

1.0

0.06

0.003 0.81 1.67 X 0.2 10-3

1.55

0.99 1.04 1.05 1.21

0.92

1.78 1.77

0.03

&

Equivalent*

-

Lower levels of Cr, Cu, Cd . cf. Table4

1.83 Some copper also detected

0.94 1.05 Full analysis 1.07 cf. Table 4 1.15 c$ Table4 full analysis

0.96 cf. Table 4

MZ+JP M2+JP Comments

0.77 0.75

0.01

C1-

1.78

0.07

S2-

3.92

0.09

0.82

Pz0,4-

/

0.15 4.54

1.58 1.92 2.83 1.27

0.53

PO,3- C0,'-

\

0.94 0.38

0.07 0.09

0.09

K+

.

0.34

0.02

0.02 9.88

0.06

Mg2+ Ca2+ BaZ+ Mn2+ Fen+ Znz+ Na+

A

Molar ratios relative to Ca

* Ratio of meta1:phosphoru.safter balancing charges of other ions; (a) ref. 117; ( b ) ref. 105; (c) ref. 118; @) ref. 167; ( e ) ref. 43; (f) ref. 78; ( g ) ref. 119; (h) G. L. Becker, C. H. Chen, J. W. Greenawalt, and A. L. Lehninger, J. Cell Biol., 1974,61,316; (i) ref. 38.

Lumbricus terrestris (f) Balanus balanoides ( g ) Gzllinectes (h) Argopecten irradians (i)

Tetrahymeqa pyriformis (a) Helixpomutia ( b ) Helix aspersa (c) Pecten maximus ( d ) Pinna nobilis (e)

Species

4

Table 3 Analysis of isolated granules

0.89 0.17

Argopecten irradians ( c ) Pecten maximus ( d ) Lumbricus terrestris ( e ) Allobophora longa ( e )

1.0 1.0 1.o 1.o

1.0

1.0 1.0

0.19 0.90

0.18

1.21 0.27 0.18

Mn2+ Zn"

K'

0.28 3.23 0.02

0.55 0.39 0.69 0.22

Na+

A

0.07

A13+

Molar ratios relative t o Ca

(c) ref. 38; ( d ) ref. 167; ( e ) ref. 93.

0.35

Pinna nobilis ( b )

(a) Ref. 18; ( b )ref. 63;

0.71 0.70

Mg2+ Ca"

Tetrahymena pyriformis (a)

Species

/.

Table 4 X-Ray microanalysis of granules

0.10

0.83 0.03 0.86

1.32 1.89 1.79 1.47 1.04

2.62 2.3 7

Pod3-

1.45

0.06

0.18

1.10 1.83 0.86 1.13

1.16

0.65 0.72

A

Comments

4

0.83 Mean of 63 analyses 0.95 Mean of 10 granules from 3 cells. cf: Table 3 1.13 7 elements analyses cf: Table 3 cf: Table 3 1.03 cf. Table 3 0.92 cf Table 3 0.32 Note high sulphur content

M2+/P

Equivalent

h

%

g

Y

9. Y

Inorganic Deposits in Invertebrate Tissues

125

Table 5 Ca :P ratios of various minerals Ca/P = 0.5 Ca/P = 1 Ca/P = 1 Ca/P = 1.33 Ca/P = 1.5 Ca/P = 1.67 Ca/P = 1 Ca/P = 1 Ca/P = 1 Ca/P = 0.67 Ca/P = 0.20 Ca/P = 1.45

Mone tite Brushite Octa-calcium phosphate Whitlockite Hydroxyapatite

Amorphous calcium phosphate (mixture of several phosphates)

and Helix aspersa118 by the use of specific enzyme assays, Oxalate has been determined in granules from the kidney of the scallop Pecten rnaxirnus.l16 The granules from Helix pornatia contained phosphate and carbonateloSand granules from different species of cestodes have shown that they are mostly carbonate with some phosphate,lm It is often difficult t o interpret the published analytical data since all the elements present are not analysed or accounted for and often there is poor definition of units. Frequently, analyses do not add up to ca. 100% and the presentation is often confused with some results only being mentioned in the discussion, Many workers appear overwhelmed by the complexity of the granule composition but the mineral part, although not having a fixed composition, may well have a characteristic one. An understanding of the mechanisms of granule formation can only advance when there is a complete knowledge of their composition which can point the direction for further chemical and biochemical analyses and when full analyses have been made the results have often been extremely rewarding, Thus, the presence in granules of pyrophosphates, which are usually rapidly hydrolysed in the cell, suggest that there is a control over this process either by an inhibition of enzyme activity or by the influx of excess metal ions which overload the system and precipitate the metal pyrophosphates. Pyrophosphate granules also enable one to compare the results of analyses by different techniques, Tetrahyrnena granules analysed by chemical methods gave a M2+/P ratio of 0.96,11' which compares well with the X-ray probe results of 0.95 k 0.12.18Analyses of Ca, Mg, Mn, P, and S in granules (nephroliths) of the lamellibranch Pinna nobilis by HignettelZ1 using a crystal detector attached t o the electron microscope gave results within 2% of each element when compared with the chemical analyses of G h i ~ - e t t iThese . ~ ~ results show a remarkable consistency of composition and good agreement between quite different techniques. By contrast the analyses of granules38from Argopecten gave a Ca :P ratio of 1.69 by chemical methods but a ratio of from 1.04 to 1.38 by X-ray probe analysis. T. von Brand, M. U. Nylen, G. N. Martin, and F. K. Churchwell, J. Parasif., 1967, 53, 683. l Z 1 M. Hignette, in Colloque GABIMCNRS La Rochelle, 1978, 195; see also ref. 63. 120

126

Environmental Chemistry

Species differences in composition have been detected by Morgan in his analyses of chloragosomes in annelids. Equivalent divalent metal (Ca, Zn, K) to phosphorus ratios were 0.97 in L. terrestris compared with 1.14 in A. longa, but sulphur levels in the latter were considerably higher.93 It has been suggested that in these granules the sulphur may be derived from thiamine. This is interesting since thiamine pyrophosphate is a cofactor for the decarboxylation of oxyacids. Morgan suggests that there may be a physiological link between the activity of calcium in chloragocytes and the excretion of calcium carbonate by the adjacent calciferous glands. One of the most fruitful techniques for determining both the anionic species in isolated granules and also the structural arrangements in the material is infrared spectroscopy, Carbonates, oxalates, phosphates, and sulphates have distinctive spectra. Infrared and Raman spectra of amorphous orthophosphates, mixtures of carbonates and phosphate in amorphous calcium carbonate phosphates and amorphous calcium pyrophosphates have been published by Termine and Lundy122 who also discussed the differences in the spectra due t o hydrated species and different cations (Ca2+, Mg2+,and Cu2+). The few studies reported have been very useful in elucidating the types of anions present in granules, In an investigation of granules from Helix aspersa,I18 infrared spectra of the granules were compared with those of hydroxyapatite, brushite (CaHP04. 2H2O), and calcium pyrophosphate. No bands specific t o hydroxyapatite were found. The spectrum was, however, identical to that of calcium pyrophosphate, with bands at 920 and 745 cm-' which are assigned to the symmetric and assymetric vibrations of P-0-P. Again there appear t o be species differences since infrared spectra of granules from Mytilus eduZis showed none of these diagnostic features,30 Calcareous deposits123 from the renal sacs of molgulid tunicates were found t o contain urates, oxalates (1324 and 16201650 cm-') and carbonates (14-45-1450, 876, and 712 cm-l) by identification of the spectra. Hignette similarly studied the composition of granules from the kidney of two molluscan species. Infrared spectra of granules from Pinna nobi1isl2l showed the presence of a mixture of phosphate (1050 cm-l), oxalate (1630 and 1320 cm-I), and weak bands for carbonate components, although only phosphate bands occurred in the granules of Tridacna. N o uric acid was detected in either species. Ghiretti has analysed similar nephroliths from Pinna nobizis and found only phosphate and some organic bands in the infrared spect r ~ r n The . ~ ~granules were amorphous to diffraction studies but after heating gave a pattern for whitlockite, (Ca3P04)2, and magnesium oxide. This and the release of carbon dioxide gas confirmed the infrared results. 121 Heat induced crystallization patterns were similarly determined in granules from several species of ~ e s t 0 d e s . IThe ~ ~ granules are composed of calcium, magnesium, carbonate with smaller but variable quantities of phosphate depending in part on the isolation procedures. Granules stored for two t o three years did not become crystalline. Dolomite patterns appeared after 7 days at 18OoC 12'

124

J . D. Termine and D. R. Lundy, Calc. Tissue Res., 1974, 1 5 , 55. M. B. Scaffo and H. A. Lowenstam, Science, 1978,200, 1166. T. von Brand, M. U. Nylen, G . N. Marten, F. K. Churchwell, and E. Stites, Exp. Parasit., 1969, 2 5 , 291.

Inorganic Deposits in Invertebrate Tissues

127

and after 5 minutes at 400 'C. Hydroxyapatite patterns first appeared at 400 'C. Magnesium ions facilitate and stabilize the amorphous phase of calcium phosphate. The technique of EXAFS also shows promise for elucidating the local structure of metals present in granules, for unlike other methods for structure determination, it does not need crystalline material. The availability of solid-state n.m,r. should prove especially valuable in studying the nature and structure of phosphate granules. The Formation of Inorganic Granules. - The theoretical aspects of the chemical and physical processes involved in biomineralization were discussed in detail at a recent Dahlem K ~ n f e r e n z e n land ~ ~ the molecular mechanism of carbonate, phosphate, and silica deposition were reviewed by Degens.' Despite this, major problems remain in considering the formation of intracellular granules for the process poses the following major problems. Why are the deposits amorphous? What are the factors that prevail whereby a solid phase is produced? How are foreign cations incorporated? Is the system of ligand binding dictated by cellular influences or is it a direct consequence of chemical reactivities? Those factors which inhibit the formation of crystalline solids will favour the formation of amorphous precipitates but in both cases supersaturation is obviously the first requirement so that for a sparingly soluble salt CA equation (2) applies, where Ksp is the thermodynamic solubility product and (C'), and

(A-)e are the activities of C+ and A- in equilibrium with pure solid phase (CA)solid at a given temperature. In biological fluids the first practical problem is in knowing the activities of the ions which are dependent on the ionic strength of the solutions. Estimates are available for extracellular fluids but within cells the ionic strength is less certain. Even with in vitro studies using simple solutions metastable supersaturation may persist for a long time in the absence of a nucleating material and the presence of crystal poisons may further complicate the rates of nucleation or crystal growth. In addition the presence of more than one pure solid phase will further complicate deductions from supersaturated stares. What is clear is that local concentrations of ions must reach supersaturation levels and then precipitate under conditions that are unfavourable for crystallization. These are difficult conditions to simulate in vitro but Kitano126-128has studied a large number of influences that affect the formation of calcium carbonate deposits. The presence of a variety of cations, anions, organic materials and physical conditions such as the degree and rate of stirring can all influence the polymorph formed. Thus, without organic material the presence of

lZ6

12*

G. H. Nancollas, ed. 'Biological Mineralization and Demineralization', Dahlem Konferenzen, Springer-Verlag, 23, 1982. Y.Kitano, Bull. Chem. SOC.Jpn., 1962, 35, 1973. A. Tokuyama, Y. Kitano, and N. Kanamori, Bull. Sci. Eng. Div., Univ. Ryukyus (Maths. Nat. Sci,), 1973, 1 6 , 9 0 . Y. Kitano, N. Kanamari, and S. Yoshioka, in 'The Mechanisms of Mineralization in the Invertebrates and Plants', ed. N. Watabe and K. M. Wilbur, Univ. South Carolina Press, 1976.

Environmental Chemistry

128

magnesium favours the formation of aragonite whereas with an organic component the magnesium carbonate forms a solid solution with calcite and dolomite. It is suggested that calcium binding to the organic material allows magnesium t o be coprecipitated. Other cations such as Sr, Ba, Mn, Ni, Cu, Zn, and Cd are readily incorporated or adsorbed on t o calcium carbonates. Thus, the absence of these cations in carbonate granules implies that they must be excluded from the cells where carbonate granules are found. Phosphate ions have an even greater effect on the precipitation of calcium carbonate and Burton129 suggested that in many molluscs this anion was responsible for maintaining the metastable state of the blood. Similar conclusions were reached by Bachra et al. 1 3 0 9 1 3 1 using a variety of serum ultrafiltrates and seeding them with various calcium minerals. The precipitates found depended on the carbonate/phosphate ratios as well as the calcium levels and the presence of other ions such as Mg2+ and P2074-. Higher phosphate levels led to the precipitation of amorphous calcium carbonate phosphate. Competition for anions disfavours crystallization and the nature of the granules thus depends on the relative proportions of the anions generated locally. Amorphous deposits are more soluble than crystalline components and therefore would be more favourable energetically for the mobilization of ions in invertebrates. Carbon dioxide, phosphates, and pyrophosphates are generated in cells by many metabolic pathways and are regulated by enzyme activities. The cellular precipitation of carbonate requires a low phosphate level and an elevation of HC03- activity. Precipitation involves the release of carbon dioxide [equation (3)l and carbonate levels could be maintained by carbonic anhydrase [equation (4)l. The activity of carbonic anhydrase at the site of granules involved in 2HC03- -t Ca2+=1 CaC03 COz

+ COz 4- H 2 0

+ H20 =+HC03- -I- H+

(3) (4)

the regulation of calcium has been investigated in the mantle of the freshwater mussel, A n o d o n t a cygnea. 132, 133 Zinc was detected on the surface of the granules and it was suggested that it was derived from ‘fossilized’ carbonic anhydrase. Pyrophosphates are generally rapidly hydrolysed by pyrophosphatase. Pyrophosphatase requires magnesium for activity and hydrolyses inorganic pyrophosphare. Other divalent cations such as Mn, Co, and Zn can act as cofactors in the absence of magnesium but in the presence of magnesium they act like calcium as inhibitors.’% Zinc is active in the hydrolysis of pyrophosphates of other phosphates such as ATP. I t has therefore been suggested that the true substrate and the activity of the various metals is related to the release of is MgP2072-,135 R. F . Burton and R. T. Mathie, Experientia, 1975, 31, 5 4 3 . B , N. Bachra, 0. R . Trantz, and S. L. Simon, A r c h Biochern. Biophys., 1963, 103, 124. 1 3 ’ B. N. Bachra, Ann. N.Y. Acud. Sci., 1963, 109, 251. 1 3 2 M. Istin and J . P. Girard, Calcif. Tissue Res., 1970, 5 , 247. 1 3 3 N. Roinel, F. Marel, and M. Istin, CaIcif. Tissue R e s . , 1973, 11, 163. 134 M. Kinitz and P. W. Robbins, in ‘The Enzymes’, Vol. 5 , ed. P. D. Boyer, 2nd Ed., Academic Press, London and New York, 1961. 1 3 ’ M. Dixon and E. C. Webb, ‘Enzymes’, 3rd Ed., 1979. I3O

Inorganic Deposits in Invertebrate Tissues

129

-CRYSTAL SURFACE

. HYDRATION LAYER

,BOUNDARY LAYER TRUE Figure 2 Ion interactions between crystals and their surrounding solutions. A. Ions such as Na+, K+,C1- diffuse reversibly depending on concentrations. B. Ions of higher charge such as Mg2', Sr2', C0s2- cnter hydration layer, concentrate and contribute t o charge neutrality due t o high surface area and surface energy of crystal. Steady state achieved within hours. C. Ions pass through hydration layer and exchange with ions on crystal surface. D. Isomorphous substitution of Ca2+ in crystal interior. Equilibration achieved in months and irreversible. (Based on W. F. Neuman and M. W. Neuman, 'Chemical Dynamics of Bone Mineral', Univ. Chicago Press, 1958)

phosphate bound to the metals.136 Granule formation is often associated with membranes derived from the endoplasmic reticulum or the Golgi complex. I t is possible, therefore, that the variable levels of organic matter that are found in granules could well represent membranes associated with the production of anions since it clearly does not have an epitaxial role. The granules of many organisms are heavily hydrated and this is probably important in their formation and function. A model has been proposed (Figure 2) t o indicate the processes that may occur in the growth of crystals and the way that foreign ions may be incorporated into such structures. Sulphur Donor Ligands. - There have been few studies t o characterize granules in which metals are associated with sulphur. This is partly because the granules are more difficult to isolate and partly because losses by diffusion are more likely. Most studies have therefore involved the use of the X-ray probe attached 136

K. M. Welsh, A. Jacobyansky, B. Springs, and B. S. Copperman, Biochemistry, 1983, 22,2243.

130

Environmental Chemistry

to the electron microscope. The granules are usually described as being homogeneous in such studies and the metals are usually bound t o sulphur in an organic form. Other anions are generally absent but mixed granules are known. The major role of these deposits appears to be related to the regulation and detoxification of class 'b' metals such as copper, zinc, cadmium, mercury, and lead. analysed the deposits in the midgut epithelium of the pupae of the fruit fly, Drosophila melanogaster, after feeding the larvae with a medium enriched with copper(I1) sulphate. The copper could only be detected histochemically with sodium diethyldithiocarbamate. The only other element detected in the electron probe microanalyser was sulphur. Quantitative analyses of eight granules showed considerable variation in the Cu/S ratios (Z= 4.63 f 1.40) although it was recognized that some copper may have been lost in the fixation process. Granules containing copper have been isolated from the barnacle, Balanus balanoides. The copper granules were homogenous with a lower electron density than the zinc granules which are also found in these cells. X-Ray microanalysis showed that copper and sulphur were the major elements but smaller peaks f o r calcium and potassium were also found. Comparison of the spectra with standards of copper(I1) sulphide powder showed that they had a lower copper content relative t o CuS. The granules which were green turned black on heating to 32OoC. They did not stain with rubeanic acid but were soluble in KCN (0.1 mol dm-3), concentrated nitric acid, and NaOCl (0.1 mol dm-3). The granules were amorphous to electron diffraction. It was concluded that the copper was bound to sulphur in an organic complex which was resistant to hydrolytic enzymes. Granules with similar properties have also been found in the amphipod Corophiurn volutator. 69 M a r t ~ J a ' ~investigating copper accumulation in Littorina Zittorea found copper associated with sulphur in needle like deposits. The copper did not stain with rubeanic acid but Raman spectroscopy showed a band at 474 cm-' assigned to Cu-S. A spectrum based on a standard of copper(I1) sulphide showed a comparable band. These deposits were considered to be residual bodies of lysosomes produced by the degradation of haemocyanin. In haemocyanin copper is bound to 138 b a t a mechanism was proposed to explain the association with sulphur. When HgClz or CH3HgC1 were administered in the diet of the cockroach, Blatella, mercury was found with copper, zinc and sulphur as dense deposits in l y s o s o m e ~ .139 ~ ~ , Quantitative electron probe microanalysis revealed the stoicheiometry given in equation ( 5 ) , where (x 4- y 2 ) = 1 and n varied

+

137

'"

J. M. Brown, L. Powers, B. Kineaid, J . A. Larrabee, and T. G. Spiro, J. Am. Chem. Soc., 1980,102,4210. J. A. Larrabee and T. G . Spiro, J. Am. Chem. SOC., 1980,102,4217. C. Ballan-Dufrancak, J . Ruste, and A. Y. Jeantet, B i d CeZZuZ., 1980, 39, 317.

Inorganic Deposits in Invertebrate Tissues

13 1 independently of the mercury storage process. Sometimes the HgZnCuS inclusions were surrounded by calcium phosphates. The deposits were considered to be an organic molecule possibly a metallothionein with additional copper present in a separate form. Studies exposing crabs, Carcinus maenas,88 to cadmium as CdClz gave similar results in that cadmium was found in deposits together with Cu, Zn, and S and some calcium phosphate. ‘Cadmosomes’** are found as intracellular deposits in the chloragocyte cells of earthworms living in contaminated soils. Analyses showed that calcium and sulphur were present with lower levels of zinc, lead, calcium, and phosphorus, The Cd/S relative mass ratios were ca. 0.40 and the (Cd Zn)/S atomic ratio was 0.11. The deposits were electronlucent and it was suggested thac they were derived from a form of metallothionein. This view was supported by the work of George and Piriew who exposed mussels (M. edulis) to cadmium for three months. The (Cd Zn)/S atomic ratio in this case was 0.34. Similar granules from the copper tolerant i ~ o p o d s ’were ~ mostly copper and sulphur though lead and calcium were also detected. Sulphur granules are clearly important in the detoxification of toxic metals which thereby become isolated intracellularly in compartments before being extruded. Although it is generally considered that these deposits have an organic component it is exceedingly difficult to characterize it, especially since many of the granules are the residual bodies left after lysosomal digestion. George30 has reported some results of these analyses but acknowledged that more work is required. The variability of the histochemical results suggesting that copper can be detected sometimes or not at all with rubeanic acid or intermittently with sodium diethyldithiocarbamate suggests that copper is bound in complexes with different stabilities. Thus the age of the granules may represent different stages of lysosomal degradation with resulting differences in ligand composition. Many workers describe the metal sulphur deposits as metallothionein-like but from the lysosomal association it would appear that they are often the breakdown products of these molecules. Metallothioneins have been well characterized from vertebrate and invertebrate tissues. They have a molecular weight of 6000 which on gel permeation chromatography elutes at an apparent molecular weight of ca. 10000. They have a high cysteine content representing a third of the total amino-acid residues which can bind 6-7 g atom metal. Although metal binding t o thioneins can be variable, metal sulphur ratios are usually of the order 0.3 for cadmium or zinc and 0.5 for copper. Copper sulphide has a Cu/S atomic ratio of 1 or 2 depending on the oxidation state of copper. Thus metal/S ratios outside this range mean either that the metal is bound t o other ligands or that much of the sulphur is not involved in binding. The metal/S granules clearly present a complex picture involving the physiological requirements of the organism and the need for protection from the toxic effects of group ‘b’ metals. The mechanisms of binding to sulphur sites in proteins appears to be universal though there remains the uncertainty of whether the deposits are residual lysosomal bodies related to the turnover and degradation of these complexes. Different species appear t o use different organs to achieve these ends. Much more work is obviously required to characterize these sulphur containing deposits and the immunoassays that are currently being

+

+

Environmental Chemistry

132

developed for metallothioneinslm may well help to clarify some of these questions. 4 Silica Deposition

At pH 8, 98% of silicic acid in aqueous solution occurs in a non-ionized form and it is this molecule that is thought to act as the precursor of silica (Si02), structures. Since these deposits are amorphous hydrated molecules of unknown molecular weight they are often shown as Si02.nH2O which emphasizes the relationship with silicic acid. The solid deposits formed by diatoms are often referred to as ‘opaline’ and as ‘opal’ in other organisms. Silica deposits are mainly formed in plants and ‘lower organisms’. In plants the silicic acid is polymerized extracellularly with little or no morphogenesis. The deposits often appear to act as abrasives that deter herbivores. In other organisms the silica is formed intracellularly (diatoms, chrysophytes, choanoflagellates, radiolaria, testate amoebae, and sponges) but these deposits may be extruded and ‘glued’ together extracellularly to form elaborate structures (chrysophytes, choanoflagellates, testate amoebae). In all cases where the silica is deposited intracellularly there is elaborate control of the shape of the inorganic deposits t o form structures that can be bewilderingly complex. The form in which silicon is transported around organisms and in which it enters cells is not known. I t is generally agreed that silica deposits are formed in special vesicles derived from the Golgi system, but since this association is often difficult to demonstrate, the membrane bound areas that deposit silica intracellularly are usually referred to as a silicalemma around silica deposition vacuoles (S.D.V.). Silica deposition is normally associated with an organic matrix. In diatoms the process has been summarized as (1) uptake of Si(OH)4 from the medium, (2) synthesis of silicalemma from membrane units, ( 3 ) transport of silicon from the cytoplasmic pool, (4) polymerization of silicon within the vacuole, and ( 5 ) synthesis of secondary organic material, mainly carbohydrates, which are added to the developing walls.142The organic matter plus water of hydration may account for 36-90% of the diatom wall that is synthesized in this way and the organic coating appears t o be important in protecting the siliceous ‘frustule’ from dissolution. Amorphous silica is unstable and dissolves in fresh and seawater. Frustules from killed cells dissolve faster than those from living diatoms and acid cleaned structures dissolve faster than intact structures.143 This increased resistance t o dissolution has been variously attributed to polysaccharides, proteins, lipids, or trace elements such as iron and aluminium. In testate amoebae and chrysophytes a fibrilIar matrix occurs in the silica forming vacuoles. In the first of these groups a complex silicon-sulphomucinI4O 141

14*

143

J . S. Garvey, R. J . Vander Mailie, and C. C. Chang, Methods Enzymol., 1982, 84,121. T. L. Simpson and B. E. Volcani, ‘Silicon and Siliceous Structures in Biological Systems’,

Springer Verlag, New York, 1981. B. E. Volcani, in ‘Silicon and Siliceous Structures in Biological Systems’, ed. T. L. Simpson and B. E. Volcani, Springer Verlag, New York, 1981. J . C. Lewin, Geochim. Cosmochim. Acta, 1961, 21, 182.

Inorganic Deposits in Invertebrate Tissues

133

basic protein is formed apparently from a fibrillar precursor and this becomes the organic cement of the test matrix.lW In the chrysophyceae coated vesicles are often seen attached to the edges of the forming vacuole and silica deposition occurs in a vesicle that is filled with fibrillar material.145 Among the sponges a prominent proteinaceous filament is formed around which silica is subsequently deposited.lM The composition of this axial filament is only partially known although its carbohydrate and amino-acid content has been determined. 147 If spicules are broken and etched in hydrofluoric acid it can be shown that they have a concentric ring structure that disappears on heating t o 600°C.148 The Si02/H20 proportion of these spicules ranges from 2-5 and the residue (4.3% m / m ) after hydrofluoric acid treatment contains C (0.3-0.8%),Si (17.5%), Na (13.7%), K (2.6%), and Al (3.1%).149 The specific gravity varies from 2.04 to 1.96 and the refractive index from 1.438t o 1.445.150 In recent years there has been considerable interest in the possibility that silicification, matrix biosynthesis, and calcification processes may have some common basis. This has been stimulated by the discovery of a unique class of sponges the Sckrospongiae. These organisms form siliceous spicules that become embedded in an organic matrix that then becomes enclosed in a massive aragonite skeleton. Once the silica becomes embedded in calcium carbonate it becomes much more soluble.151 Among radiolarian protozoa a skeleton may be formed of silica while the quite closely related Acantharia produce a skeleton of strontium sulphate. * 52 5 Urates

Many of the mineralized inclusions in excretory organs are found t o contain uric acid, urates, or related compounds. Uric acid is the most important constituent of nitrogenous excretion in insects, birds, reptiles, and some molluscs and its biological advantage over urea or ammonia as a nitrogenous waste stems from its insolubility.153 Uric acid is also formed by a separate metabolic pathway involving purine metabolism. The insolubility of uric acid means that it frequently occurs in deposits which have the typical morphology of granules with a F. W. Harrison, D. Dunkelberger, N. Watabe, and A. B. Stump, J. Morphol., 1976, 150, 343. 14’ C. B. McGrory, Br. Phycol. J., 1976, 11, 197A. 146 W. C. Jones, in ‘Biologie des Spongiaries’, ed. C. Levi, and N. Boury-Esnault, Cell Intern. C.N.R.S.,Paris, 1979, 291, pp. 425-447. 14’ R. E. Shore, Biol. Bull., 1972, 143,689. 148 R. Garrone, T. L. Simpson, and J. Pottu-Boumendil, in ‘Silicon and Siliceous Structures in Biological Systems’, ed. T. L. Simpson and B. E. Volcani, Springer Verlag, New York, 1981. 149 D. W. Schwab and R. E. Shore, Biol. Bull., 1971, 140,125. D. W. Schwab and B. Wahl, Nuturwissenschuften, 1956, 43, 513. W. D. Hartman, in ‘Silicon and Siliceous Structures in Biological Systems’, ed. T. L. Simpson and B. E. Volcani, Springer Verlag, New York, 1981. 0.R. Anderson, in ‘Silicon and Siliceous Structures in Biological Systems’, ed. T. L. Simpson and B. E. Volcani, Springer Verlag, New York, 1981. l S 3 V. B. Wigglesworth, in ‘The Principles of Insect Physiology’, Chapman and Hall, London, 1972.

144

134

Environmental Chemistry

concentric ring structure. Often several granules are fused. Frequently the uric acid is only one component of the granules which may also contain calcium carbonate, calcium oxalate, and calcium and magnesium phosphates and, as in other phosphate granules, other metal ions may accumulate. The granules are found in the fat bodies, Malpighian tubules, and excretory systems of various arthropods. Some observers have noted a breakdown in structure prior to egestion suggesting that some components are reabsorbed in the rectum. The uric acid levels in prosobranch molluscs such as Littorina ZittorealS4have been shown to be related t o the activity of uricase, the enzyme which degrades uric acid to allantoin. The activity of this enzyme is related t o tidal rhythms.lS5 Urate granules can be demonstrated histochemically in the nephrocytes of pulmonate molluscs. ls6 The composition of the rings alternated between uric acid and guanine with a nucleus of calcium urate. Phospholipids and mucopolysaccharides were also found within the granule. mull in^^^^ isolated uric acid granules from cockroach tissues with urate cells and analysed them by infrared spectroscopy using model compounds for comparison. All the samples had a band at 1680cm-l, which was assigned to the uric acid carbonyl group, but none of the spectra corresponded exactly t o the model compounds and he concluded that they were composed of a mixture of potassium and sodium urates with free uric acid. Bands at ca. 3500 cm-' distinguished mono- and di-hydrates or uric acid. Analyses also showed the presence of non-urate nitrogen. A complementary study by Ballan-Dufrancais et uZ.lS8 looked at the purinic granules in several species of insects, spiders, and pulmonates by Raman laser microprobe. The strength of this technique is that the probe can be focused onto a very small area 1-2 pm diameter. The granules from the insects BZateZZa germanica and Schistocerca gregaria showed that uric acid was present with potassium and sodium urates . By contrast granules from the spider, Epeira, were pure guanine, whereas the granules from Helix kidney were composed of uric acid, potassium and sodium urates, and xanthine. Earlier electron probe studies had shown that the granules were heterogeneous having not only a purinic component but also Ca, Mg, P, and C1. The heterogeneous nature of granules from excretory organs was confirmed by Scaffo and L ~ w e n s t a m ' *who ~ investigated the composition of the calcareous deposits in the renal sac of a molgulid tunicate. From infrared spectra major peaks a t 1324 and 1620-1650 cm-l were assigned to calcium oxalate (CaC204. 2H20). Further peaks at 1445-1450 and 876 and 712cm-1 and the ready dissolution of the granules in 0.1 mol dm-3 HC1 suggested that another component was calcium carbonate. Analysis showed also traces of Mg, S , and C1. HubertlS9 studied the granules in adipose cells of Cylindronilus teutonicas Pocock and found that the larger granules (2-18 pm) were urates but the very J. Daguzan, C, R. Acad. S c i Paris, Ser. D , 1970, 279, 3131. J . Daguzan and P. Razet, C, R . Acad. Sci. Pans, Ser. D, 1971, 272,2800. C.Gostan, Ann. Biol. T., 1965, IV, 481. D. E. Mullins, Comp. Biochem. Physiol., 1979, 62A,699. 1 5 8 C. Ballan-Dufrancak, M. Truchet, and P. DhamelinCourt, Biol. Cellul., 1979, 36, 51. l S 9 M . Hubert, C. R. Acad. Sci Paris, Ser. D . , 1975, 281, 151. 155

"'

Inorganic Deposits in Invertebrate Tissues

135

small granules were calcium phosphates. In the granules found in the ovary of the same species only calcium was detected. A study of several species of Archaeogasteropods (MoZZusca prosobranchia)lm showed that the occurrence of uric acid varied from species t o species,

6 Conclusions In 1976 it was argued that sufficient ultrastructural studies had been published on invertebrate tissues to justify the recognition of a phenomenon of inorganic granule formation in membrane bound vesicles of a wide variety of cells.*ll In order to establish that phenomenon a Table was published showing the variety of animals and cells that contained these 'calcium granules', A modified form of that compilation is reproduced in an abbreviated form as Table 6. Its

Table 6 Some examples of intracellular 'calcium'granules"' Phylum Protozoa Coelenterata Platyhelminthes Trematoda Cestoda Mollusca Gastropoda Lam ellibranchia Arthropoda Crustacea Onychophora Diplopoda lnsecta

'Organ'

Species Proroden morgani Renilla reniformis Aurelia aurata

-

Scleroblasts Statoliths

Cyathocotyle bushiensis Taenia taeniaeformis

Excretory

Ferrisia wautieri Helix pomatia Mercenaria mercenaria

Connective tissue Hepatopancreas Mantle

Orchestia cavimna Callinectes sapidus Peripatus acacioi Pleuroloma sp. Pogonognathellus longicornus Rhodnius prolixis Cercopis sanguinea Blatella germanica

Posterior caecum Hepatopancreas Intestine Ovary Intestine Malpighian tubule Intestine Male accessory

-

aim was two-fold. First it attempted to show that calcium granules occurred in most phyla and in a great variety of tissues. Thus the ability to form calcium granules was not a pathological condition, in fact, on the contrary it appeared to be a normal activity of many living systems. The second aim was to try and indicate that all calcium granules were not the same and at least two fundamentally different ligands (one carbonate based and the other involving phosphates) provided basically different functions and involved quite dissimilar 160

W.Delhaye, Cah. Biol. Mar., 1976, 17, 305.

136

Environmental Chemistry

cellular systems. The calcium carbonate system appears to be involved in the storage and recycling of these ions through the body fluids while the phosphatic system is more involved with the binding of other metals onto these deposits and the excretion of these granules from the body, This duality of the ‘calcium granules’ system has been extensively reviewed in molluscs by Mason and Nott.lg Ultrastructurally the calcium granules form in membrane delimited vesicles or vacuoles. These are frequently associated with the Golgi complex (e.g., the mollusc Pomacea paludosa2 and diplopod Cylindroiulus Zondinensisl6l) or with the endoplasmic reticulum ( e . g . , the homopteran insect Philaenus s q u a m ~ ~ i s ’ ~When * ) . initially secreted the granules appear t o be associated with organic material which may be involved in their induction. As the granule increases in size it may have an irregular outline suggestive of numerous needlelike crystals but it usually becomes much more regular in shape as it enlarges and frequently shows electron clear and opaque concentric layers. Granules of this type are common in many molluscs and arthropods. There appears, therefore, to be considerable uniformity in the ultrastructural evidence for how the calcium granules are formed. The details of membrane function remain obscure but there is general agreement about the organelles involved and their possible function. Since this phenomenon was identified, however, there has been a large increase in the recognition of other metalcontaining inclusions in cells. Thus a number of reviews have extended the listing of references on ‘intracellular granules’ to include other metals so that Coombs and GeorgeIs cite Pb, Zn, Cu, Mn, Fe, Mg, and V deposits while Brown’ includes three extensive tables of ‘metal-containing granules’. These latter reviews provide valuable reference sources to the literature but their aims are entirely different from those behind the compilation of Table 6 , The information in such compilations is certainly not describing a single phenomenon nor is it grouping together comparable cellular activities. Thus in order to rationalize such information and to assist in putting it into an environmental context it is important t o recognize the cellular systems that are likely to be involved in metal-stressed organisms. There are currently three cellular mechanisms that are recognized as regulating divalent cations within the cytoplasm. The first is the mitochondrion which is known to transport calcium inwards via a carrier with a K , of about 3 pM. Intramitochondrial granules of calcium phosphate are frequently found in cells adjacent t o sites of biomineralization but it is not clear whether the granules are related to the calcification process or whether they are simply the products of a cellular system that responds to transient influxes of ~ a 1 c i u m . lA~ similar ~ problem of interpretation occurs with the metallothionein proteins which appear t o regulate the copper and zinc metabolism of the cytoplasm. These sulphydrylrich proteins will also bind strongly onto Cd, Hg, Ag, and Au. These inducible proteins act as a fast responding feedback system controlling the intracellular

16‘ 462

163

M. Hubert, C. R . Acad. Sci. Paris, Ser. D,1979, 289, 749. J . Gouranton, J. Celt Biol., 1968, 37, 316.

A. L. Lehninger, in ‘Biomineralization and Biological Metal Accumulation’, ed. P. Westbroek and E. W. de Jong, D. Reidel, Dordrecht, New Netherlands, 1983.

Inorganic Deposits in Invertebrate Tissues 137 concentration of free metal ions.164The metallothioneins have a half-life of only 2-3 days in viuo and are turned over by the lysosomes; organelles rich in proteolytic enzymes that form part of the recycling and phagocytic systems of the cell. In metal-stressed cells the lysosomes or the residual bodies derived from them are frequently found to accumulate metals. In certain circumstances, e.g,, the , ~ ~mineral ~ concretions copper deposits in the midgut of larval D r o ~ o p h i Z athe in the Malpighian tubules of Musca dornestica,166and the granules in the kidney of Pecfen,l6’ and remnants of lysosomal enzyme activity are easily detected within these deposits. Ferritin molecules have a similar short half-life and during conditions of iron overload a breakdown product, haemosiderin, is found in membrane-bound vesicles within the cell. Thus, all these examples of intracellular deposits of metals appear t o involve different pathways within the cell, One of the aspects of this problem which we have tried to emphasize is that little progress can be made in clarifying these pathways until these deposits are characterized by a fuller understanding of their chemistry. Perhaps the best example of the confusion that arises by speculation in this area is in relation to the availability or not of calcium phosphate granules for calcium carbonate metabolism in molluscs. The literature is full of theories invoking the interconversion of these granules without any reference to the chemical processes involved. In fact it would appear that the first phase in the study of inorganic granules in invertebrate cells is now drawing t o a close. The phenomenon is well documented and the variety of deposits is clear. There are now as a consequence three clear aspects that need further investigation,

The Penetration of Metals into Organisms and Cells. - This is a fundamental problem that is of particular importance t o environmentalists. The ‘classical’ view that biological membranes are hydrophobic whereas metal ions are hydrophilic is probably an oversimplification in that metals exist both in the environment and within the body in such a variety of complexes that many can probably penetrate cell membranes at a reasonable rate. Having penetrated the organism they will, of course, encounter a wide variety of ligands that presumably dictate their subsequent fate,168 Precipitation or Binding. - Three types of ligand can be considered as existing within the organism. The first of these are anions which act as sinks, trapping a variety of cations by processes of adsorption and coprecipitation. The physiologically active anions are likely to be buffers (HCOs-/CO?-; HPOt-/PO:-) excretory products (e.g., urates) or products of biosynthetic pathways (e.g., P20:-) and the metabolic activities of the various cells will clearly dictate which of these are available, The second type of ligand is the regulatory protein which has a rapid turnover and is capable of being parasitized by other metals, MetalloM. Vasak and J. H. R. Kagi, in ‘Biominerafizationand Biological Metal Accumulation’, ed. P. Westbroek and E. W. de Jong, D. Reidel, Dordrecht, The Netherlands, 1983. 1 6 5 R. L. Tapp and A. Hockaday, J. Cell Sci., 1977,26,201. 166 R. S. Sohal, P. D. Peters, and T. A. Hall, Tissue Cell, 1976, 8,447. S. G. George, B. J. S . Puie, and T. L. Coombs, J. Exp. Mar. Biol. Ecol., 1980,42,143. 168 R . J. P. Williams, Phil. Trans. R. Soc. London, Ser. B , 1981, 294, 57. 164

’“

138

Environmental Chemistry

thioneins are the obvious example of this type of molecule but it is t o be expected that a large number of other examples will be characterized in the next few years. Proteins of this type are ‘turned over’ by lysosomes but metals, being incapable of enzymatic digestion, will accumulate at these sites, Finally one has to recognize the possibility that a whole range of proteins and metabolic pathways may be ‘protected’ or have such specificity that only one metal is ever involved with them.169 This appears to be the only explanation for the fact that in animals from polluted sites interfering metals such as Cd do not accumulate in cells specialized for example in the metabolism of copper in the synthesis of haemocyanin, The concepts of ‘general metal binding’ and ‘specific metal binding’ underlie many of the approaches to detoxification on the one hand and biomineralization on the other. In the secretion of the calcite plates of the coccolithophorids strontium and magnesium are excluded from the d e p o ~ i t s l ’ ~ and a similar phenomenon occurs in the formation of silica deposits in the Chrysophyceae. 145 In both these latter cases materials appear t o be transported to the mineral forming vacuoles by smaller granular bodies (e.g., coccolithosomes). Clearly there is a great deal of specificity in the transport of these bodies and these ‘protected pathways’ appear to have a physical identity within the cell. Accumulation Sites. - These are of importance for three reasons. First, by their occurrence they draw attention to the whole phenomenon of metal accumulation within cells. Secondly, it is implied from their existence that they isolate potentially hazardous metals from the sites where they may do damage, Certainly it appears that it is these deposits which people are estimating when they use organisms as environmental monitoring systems. Thirdly, they may be transient accumulation sites that are excreted or removed from the cells at various rates. This is clearly a major advantage t o the organism and a serious difficulty for those who wish to use organisms for estimating environmental levels of various metals. Unfortunately little is known about this aspect of granule behaviou r. I t will be apparent, therefore, that further analyses and metabolic studies are required before a fuller understanding can be gained of the unusual properties of metal deposits in intracellular granules of invertebrate tissues.

‘69 ‘’O

K. Simkk and A. Z. Mason, Environ. Mar. Res., in the press. P. L. Blackwelder, R. E. Weiss, and K. M. Wilbur, Mar. Biol., 1976, 34, 11.

Author Index Abolins-Krogis, A., 103, 119, 120 Adams, E., 114 Adelman, H., 87 Agard, E. T., 59 Akagi, H., 73 Akimoto, H., 29,48 Alabaster, J. J., 56 Aldaz, L., 18,21 Aldridge, W. N., 58, 61 Alexander, L., 28 Ali, A., 46 Alikhan, M. A,, 114 Allard, D. W., 3 3 Allen, D. W., 76 Allen, J. A,, 112 Allison, I., 21 Altshuller, A. P., 30,43 Alyea, F., 20 Ammons, B., 80 Anderson, J. A., 39 Anderson, 0. R., 133 Andreac, M. O., 70 Andrews, C., 64 Anger, J. P., 59 Anlauf, K. G., 39 Antonovich, V. P., 69 Apling, A. J., 36 Aquino, R., 63 Arakawa, Y., 56, 59, 61 Arhland, S., 103 Artiola-Fortuny, J., 80 Ashmore, M. R., 45 Atkins, D. H. F., 24 Atkinson, R., 30 Attmannspacher, W., 6 , l l Bach, W. D., 41 Bache, C. A., 97, 100 Bachra, B. N., 128 Back, W., 99 Baedecker, M,J., 99 Baker, P. G., 61 Baldwin, A. C., 3 0 Ball, D. J., 24, 36 Ball, G. L., 63 Ballan-Dufrancais, C., 108, 109,116,117,130,134 Banzer, J. D., 63 Barber, F. R., 37 Barker, J. R., 30 Barnes, J. M., 59 Barnes, R. D., 64 Barth, K., 72

Barug, D., 64,75 Bates, R. R., 77 Batt, P., 107 Baxter, J. C., 80,90 Bayan, B. D., 119 Beck, Y., 38 Becker, K. H., 38 Beckett, P. H. T., 80 Beiber, W. D., 63 Beiter, C., 67 Bell, C. A., 5 Bell, J. N. B., 45 Bellama, J. M., 67 Bellmont, A. D., 11 Bengert, G. A., 61, 70 Bengtsson, B. E., 56 Bennett, J. P., 46 Berger-Wiersma, T., 67 Bergman, S. C., 80,83 Bernard, R. E., 36 Besemer, A. F. H., 72 Bingham, F. T., 91 Bischof, W., 14 Blackwelder, P. L., 102, 138 Blair, E. H., 59 Blair, W. R., 61, 67, 75 Blake, N. J., 109 Bleck, R., 5 Blumenthal, D. L., 39 Blunden, S. J., 61, 67, 69, 74, 75 Bock, R., 56, 59 Body, D. E., 46 Boettner, E. A., 63 Bokranz, A., 49 Bollen, W. B., 65 Bondy, S. C., 110 Booker, H. G., 2 Borden, T. R., 11 Born, H. J., 80 Born, L., 56 Boswell, F. C., 83 Botte, L., 109 Bowen, H. J. M., 65 Boyd, A. W., 47 Bradley, C. E., 24 Bradley, J., 108 Braegelmann, P. K., 46 Braman, R. S., 60 Brandt, C. S., 46 Branson, D. R., 59 Braude, G., 80 Braurnan, S. K., 63

139

Brecker, L. R., 62 Bridges, J. W., 58, 59, 60 Brierley, J. B., 58 Brinckman, F. E., 49, 61, 67, 70, 75 Brocco, D., 24 Broder, B., 17 Brook, A. J., 108 Brooks, J. S., 76 Brown, A. W., 58 Brown, €5. E., 103, 112, 114,118 Brown, J. M., 130 Brown, R. A., 59 Bruggeman, J., 72 Brunik, H., 72 Bruntz, S.M., 33 Bryan, G. W., 106, 110, 113 Buchanan, J. B., 112 Buchel, K. L., 56 Bufalini, J. J., 30 Bufalini, M. M., 3 0 Bull, A. T., 64 Bungarz, K., 56 Burch, D., 64 Burge, W. D., 80 Burlakova, E. B., 121 Burton, R. F., 120, 128 Byrd, J. T., 70 Cain, K., 58 Calvert, J. G., 24, 25 Campbell, J. W., 119 Cantley, L. C., 108 Cardarelli, N. F., 59, 65 Carey, J. K., 75 Carlson, R. M. K., 109 Carlucci, s., 110 Carmichael, N. G., 109, 110 Carrondo, M. J. T., 84 Carter, W. P. L., 3 0 Casida, J. E., 58, 59, 60 Castrilton, J., 59 Cecinato, A., 24 Cederwall, R. T., 3 , 4 0 Cenci, P., 73 Chadwick, R. C., 17 Chaim, S., 45 Challenger, F., 70 Chamberlain, A. C., 17 Chameides, W. L., 3, 9, 34

Author Index

140 Chan, M. W., 33 Chang, A. C., 99 Chang, C. C., 132 Chapman, A. H.,61,67, 73,75 Chapman, G., 116 Chapman, S., 1 Chappuis, J., 1 Chassard Bouchard, C., 112,117 Chatfield, R., 3, 10,11 Chatt, A.,80 Chatt, J., 103 Chattopadhyay, A., 80, 83,91 Chau, Y. K., 61,70 Chock, D. P., 43 Chockalingham, M. P., 18 Chromy, L., 67 Churchwell, F. K., 125, 126 Cleveland, W. S., 33, 39, 42,43 Cobet, A. B., 70 Coffey, P. E., 3, 5, 33 Coleman, J. R.,107 Coleman, W.M.,70 Conyers, E. S., 80 Cook, D. R., 18 Coombs, T. L., 106,108, 112,118,137 Cooney, J. J., 70 Copperman, B. S., 129 Corbin, H.B., 73 Cothern, C. R.,80,83 Coughtrey, P.J., 105 Cox, R. A., 24, 31, 36,45 Craig, P. J., 49,70,71 Cram, G. C., 59 Cremer, J. E., 58 Cremonhi, B., 73 Crkp, D. J., 116,122 C r k m a n , B. W.,41 Crosby, D. G., 61,67 Crowe, A. J., 67,76 Crutzen, P. J., 3, 31 Cunnold, D., 20 Cuppa, S. S., 109 Cyr, R., 47 Czeplak, G., 3 Daguzan, J., 134 Dalpra, C., 45 Daniel, R. Ch., 86 Daniels, S. L., 80 Danielsen, E. F., 4,5, 6 Danskin, G. P., 109 Darnall, K. R.,25, 30 Davies, A. G., 67,73 Davies, D.S.,59 Davies, N. R., 103 Davis, D. D., 14 Degens, E. T.,102

Deimel, H., 38 Delany, A. C., 14,21 Delfino, J. J., 80 Delhaye, W., 135 De Marrais, G.,41 DeMore, W.B., 48 Denison, P. J., 18 Dennett, R.,14 Depieri, R., 110 Dermejian, K. L.,25,30 Derwent, R. G., 5, 18,27, 31, 36 de Tirado, R. S., 59 Dhamelin-Court, P., 134 Dickson, K. L., 59 Dietz, G. R., 63 Dimitriades, B., 28, 30 Dixon, M., 128 Dizikes, L. J., 71 Djangmah, J. S., 114 Dobson, G . M. B., 1 Dodge, M. C., 28,30 Donagi, A., 38 Douland, H.,38 Doyle, L. J., 109 Dring, L. G.,60 Dudzinski, T.J., 13 Dutsch, H.U.,1, 1 1 Dukhavich, V. F., 121 Duncan, J., 65 Dunkelberger, D. G., 102, 133 Dunlop, S., 116 Dunn, P., 72 Dutkiewia, V. A., 7 Eastman, J. A,, 18 Eaton, W. C., 29 Eben, Ch., 64 Edlund, M. L., 75 Edmond, J.M.,13 Eggleton, A. E. J., 5, 24, 36 Ehhalt, D. H., 8 Elkakn, B., 112,115 Elkus, B., 43 Elliott, B. M.,58 Ellis, H.V.,49 Elzerman, A.W.,18 Emmerich, W. E., 99 Enderson, R. E., 80 Engel, D. W.,110 Engelhart, J. E., 67 Evans, C. J., 49,59,65 Fabian, P., 3, 10, 13, 14 Falconer, P., 13 Falk, H. L., 77 Falls, A. H., 30 Fanchiang, Y.T., 71 Fankhauser, R. K., 33 Farrel, J. B., 79 Farrington, D. S.,61 Farrow, L. A.,25

Feder, W.A., 45 Ferman, M. A.,40 Fetherston, W.T.,78 Fiedler, I., 80 Figge, K., 63 Filshie, B. K.,11 5 Finlayson-Pitts, B. J., 33 Fish, R. H.,58,59,60 Fishbein, L., 77 Fishman, J., 3 Flamm, D. L., 47 Fletcher, R. A.,46 Formstone, R., 76 Forster, G. R., 113 Foster, P., 116,122 Fotheringham, A., 108 Fowler, B. A., 109,110, 11 3 Fox, D. L., 28 Fox, M. M., 24 Frankenberg, W.T.,80 Fraser, J., 80 Freas, W. P., 33 Freiman, A.,67 Freitag, K. D., 59 Freitag, W.,63 Frenzel, L. M.,65 Fretter, V.,120 Fricke, W.,38 Fuller, W. H.,80 F u n , A. K., 97 Gabler, R. C., 97 Galbally, I. E., 18,21 Ganor, E., 38 Garland, J. A., 18 Garrone, R.,13 3 Gart, J. J., 77 Garvey, J. S., 132 Gay, B. W., 30 Gelinas, R. J., 30 G e m , J. L.,40 George, S. G., 106,108, 112,116,137 Georgii, H. W.,38 Getzendaner, M.E., 73 Gevers, E. Ch., 67 Ghiretti, F., 110 Gibb, J. P., 99 Gibson, M. A.,107,120 Gidel, L. T.,20 Gilbert, N.,47 Gillani, N. V., 39 Gillespie, T.J., 42 Gillies, D.G.,69 Gillum, W. O.,109 Girard, J. P., 128 Goddard, J. P., 67 Goldberg, E. D., 61 Golden, D. M.,30 Golovanov, P. V.,74 Good, M. L.,65,67,75 Goodwin, W. R.. 30 Gostan, G., 134

Author Index Gouranton, J ., 1 3 6 Graedel, T. E., 25, 3 3 , 1 2 Greenaway, P., 107 Greenfelt, P., 38 Gregory, G. L., 30 Griffin, R. A., 99 Griffiths, D. E., 58 Grim, S., 70 Guard, H. E., 7 0 Guary, J. C., 106 Guess, W.L., 6 3 Guichert, R., 38 Guidolti, G., 108 Gupta, A. S., 1 0 7 Gurnham, C. F., 78 Gutenmann, W. H., 9 7 Haagen-Smit, A. J., 2 4 Haagenson, P., 3, 5 , 14 Haqenson, P. L., 6 Halacka, K., 56 Hale, E. J., 64 Haley, G. F., 1 0 7 Hall, T. A., 137 Hallas, L. E., 70 Hallford, D. M., 100 Halpern, S., 109 Hameed, S., 10 Hammann, I., 5 6 Hampton, D., 64 Hampton, L., 36 Hanni, E., 86 Hanssen, J. E., 37 Hardon, H. J., 72 Harris, L. R., 64 Harrison, D. N., 1 Harrison, F. W., 1 3 3 Harrison, H., 3 , 1 0 , 1 1 Harrison, P. M., 111 Harrison, R. M., 34, 36 Hart, E. R., 77 Hartman, W. D., 1 3 3 Hartmannsgruber, R., 6, 11 Hawke, G. S., 39 Heagle, A. S., 45, 46 Hecht, T. A., 28 Heck, W. W., 4 5 , 4 6 Heggie, A. C., 39 Heller, J., 6 3 Hemphill, D. D., 9 7 Hendry, D. G., 25, 30 Henshaw, B. G., 75 Heron, P. N., 6 7 Herring, W. S., 11 Hesstuedt, E., 31, 34 Hettler, W. F., 113 Hicks, B. B., 18 Higginbottom, C., 7 0 Hignette, M., 113, 125 Hill, R., 65 Hintzc, W., 75 Hipskind, R. S., 6 Hoare, R. J., 111 Hochrnan, H., 6 7

141 Hockaday, A., 1 3 7 Hodge, V. H., 61 Hodgson, K. O., 109 Hoffman, J. F., 65 Hofstra, G., 46 Hogan, A., 5 Holdemann, J. D., 1 3 Holland, D. I&.,1 2 2 Hollingsworth, Z., 6 3 Holman, C. D.. 34, 36, 37 Hoodless, R. A., 61 Hopkin, S. P., 118 Horak, O., 8 3 Horsman, D. C.,45 Hoshino, M., 29 Houzeau, A., 1 Hov, O., 27, 31, 34 Howard, B., 1 2 2 Hubert, M., 134, 1 3 6 Hubschman, J. H., 114 Hudgens, R. E., 100 Huey, C.,70 Hummerstone, L. G., 110, 113 Huneault, H., 61 Hursey, P. A., 56 Hursthouse, M. B., 6 7 Husain, L., 3, 7 Husar, J. D., 39 Husar, R. B., 39 Hyde, R., 39 Icely, J. D., 114 Igumnov, A. S., 74 Imai, S., 29 Innes, J. R. M., 77 Inoue, G., 29 Inouye, C. A., 9 1 Ireland, M. P., 115, 1 1 7 Isaksen, 1. S. A., 31, 34 Istin, M., 128 Iverson, W.P., 6 1 , 6 7 , 70, 75 Iwai, H., 56, 5 9 , 6 1 Iyengar, G. V., 101 Jackson, J. A., 61 Jacobson, J. S., 2 4 , 4 5 Jacobyansky, A., 129 Jaffee, R. J., 25, 28 Jeantet, A. Y.,108, 109, 116,117,130 Jeffries, H. E., 28, 29, 34 Jehle, D., 74 Jeltes, R., 38 Jenkins, K. G. A., 108, 111 Jenniss, S. W., 80,85, 8 7 Jermer, J., 75 Jewett, K. L., 61, 67 Johnson, D. E., 80,90 Johnson, W. B., 3 , 2 5 Jones, J. E., 80 Jones, P. A., 49 Jones, W. C., 1 3 3

Joseph, D. W., 3 9 , 4 0 Junge, C. E., 3, 20, 23 Kagi, J. H. R., 1 3 7 Kamens, R. M., 28, 29 Kanamari, N., 1 2 7 Kapur, S. P., 1 2 0 Karl, T. R., 41, 4 3 Karpel, S., 6 3 Kasten-Jolly, J., 58 Katsumura, T., 65 Katz, S. A., 8 0 , 8 5 , 8 7 Kehew, A. E., 9 9 Kelleher, T. J., 45 Kelly, J. J., 13, 2 1 Kelly, N. A., 40 Kerr, J. A., 25 Kerr, S. N., 98 Kienholz, E. W., 80,90 Kiesling, H. E., 100 Kim, J. I., 80 Kimmel, E. C., 58, 59, 60 Kinard, J. T., 9 1 Kineaid, B., 1 3 0 Kinitz, M., 1 2 8 King, W.J., 41 Kirkbright, G. F., 90 Kirleis, A. W., 80 Kirschner, S. L., 78 Kitano, Y.,127 Klavenes, D., 1 2 1 Klein, L. A., 78 Klein, M., 77 Kleiner, B., 33, 39, 42 Kley, D., 7 Klimmer, 0. R., 72 Klotzer, D., 7 4 Knechtel, J. R., 80 Knudsen, G. W.,99 Komora, V. F., 7 4 Kopczynski, S. L., 30 Kopp, J. F., 8 7 Korth, M. W., 2 8 Kramar, O., 7 0 Krey, P. W., 4 Krijgsman, B. J., 119 Krueger, A. J., 1 Kuster, K., 7 4 Kuhlman, M. R., 29 Kulaev, I. S., 103, 1 2 1 Kulkarni, V. I., 75 Kummer, W. A., 48 Kuntz, R. L., 3 0 Kurtz, E. M., 1 0 2 Kustin, K., 109 Laidlaw, R. A., 75 Laird, A. R., 6 Lamb, B., 18 Lamb, R. G., 6 Landseidel, H., 75 Lang, M., 7 8 Larrabee, I. A., 1 3 0 Laye, P. G., 64

Author Index

142 Leeuwangh, P., 64 Lehninger, A. L., 1 3 6 Leighton, P. A., 9 Lenschow, D. H., 2 1 Lester, J. N., 84 Levitt, S. B., 4 3 Levy, H., 7 , 2 0 L e w h , J. C., 1 3 2 Liberti, A., 2 4 Linden, E., 5 6 Lindquist, F., 38 Ling, Ch.,11 Lioy, P. J., 3 9 , 4 0 , 4 2 Lisk, D. J., 9 7 , 100 Liu, s. c., 7 Lloyd, A. C.,25, 3 0 Label, J., 38 Logan, J. A., 2 Long, W.D., 4 7 Lonneman, W. A., 30 Lovelock, J. E., 24, 36 Lowe, D. M., 1 1 2 Lowenstam, H. A., 105, 113,126 Ludwig, F. L., 3 . 4 0 Luijten, J. G. A., 5 6 , 6 0 Lund, L. J., 99 Lundy, D. R., 1 2 6 Lusis, M. A., 39 Lutrick, M. C.,99 Lux, D., 80 Maas, E. V., 46 McAfee, J. M., 4 7 Macara, I. G., 1 0 9 McCartney, H. A., 3 4 , 3 6 Macchi, G., 69 McElroy, M. B., 2 McFarland, M., 7 McGrady, M. M., 69 McGrory, C. B., 1 3 3 Mcllveen, J. F. R., 34 McLellan, J 111 McLeod, G. C., 109 McMahon, T. A,, 18 McRae, G. J., 30 McRae, J. E., 3 9 , 4 3 McTaggart-Cowan, J. D., 21 Maegerlein, S., 64 Magos, L., 59 Maguire, R. J., 6 1 , 64 Mahlman, J. D., 7, 2 0 Manabe, S., 5 6 Mansurava, S. E., 1 2 1 Marel, F., 1 2 8 Marten, G. N., 1 2 6 Martin, A.,37 Martin, G. N., 125 Martin, J. L. M., 1 1 2 Martin, M. H., 1 0 5 Martin, T. D., 87 Martinez, E. L., 33 Martinez, J. R., 25

.,

Martoja, M., 1 1 5 Martoja, R., 108 Masuoni, R., 101 M w n , A. Z., 1 0 2 , 1 0 7 , 111,138 Massaw, F., 7 3 Mathie, R. T., 1 2 8 Matsuzaki, M., 111 Maughan, R., 40 Mazaev, V. T., 7 4 Means, J- C., 7 0 Meeks, S. A., 30 Meenakshi, V. R., 102 Megie, G., 11 Meinema, H. A,, 6 7 Mellbye, M. E., 9 7 Meyer, E. L., 33 Meyers, R. E., 3 , 4 0 Meyling, A. H., 6 7 Mohnen, V. A., 5 , 2 0 Monaghan, C. P., 6 5 , 75 Moore, M. N., 112, 115 Moore, P. G., 112 Morgan, A. J., 116, 118 Morrical, D. G., 100 Morris, B., 116 Mott, K. E., 7 7 Mount, T., 8 0 , 8 5 Moxim, W. J., 2 0 Middleton, P., 6 Midtbo, K. H.,31 Milde, G., 99 Miller, D. F., 30 Miller, E. M., 6 3 Miller, J., 8 3 Millers, F. J., 69 Millson, M. F., 49 Minzer, R. A., 1 Mitchell, A., 39 Mitchell, I., 7 7 Mitchell, P. C. H., 1 2 2 Miyawaki, M., 111 Mukammal, E. I., 4 2 Mullins, D. E., 1 3 4 Munk, N., 1 2 1 Murano, K., 4 2 Nagashha, T., 29 Nancollas, G. H., 1 2 7 Nash, N., 78 Nastrorn, G. D., 1 3 Naveh, Z., 4 5 Nazarenko, V. A., 69 Nazario, C. M., 59 Neely, G. E., 46 Negerbradt, G. W.,47 Negrel, R., 106 Nelson, D. W.,80 Neuman, W.F., 106 Neuman, W.P., 75 Neumayr, V.,99 Nevshaya, E. M., 69 Newell, R. E., 1 3 Newmann, H. H., 42

Nieboer, H., 2 4 Niesar, K. H., 7 2 Nijman, W.,64 Nilsson, J. R., 107, 118 Nott, J. A., 107, 114, 118 Nuernberg, H., 101 Nylen, M. U., 125, 1 2 6 O’Brien, E. J., 6 5 O’Brien, R. J., 25 O’Brien, R. T., 98 Ogato, G., 46 Ogawa, Y ., 4 2 Ogura, K., 111 Ohlsson, S., 75 Okuda, M., 2 9 , 4 2 Oldfield, D., 72 Olsen, G. J., 7 5 Omar, M., 65 Ono, T., 59 Order, R. J., 75 Orunesu, M., 1 1 5 Oshima, R. J., 46 OverneU, J., 1 2 2 Ozcan, M., 67, 75 Paasche, E., 1 2 1 Pack, D. H., 36 Paetzold, H. K., 2 0 Page, A. L., 9 9 Pakkala, I. S., 9 7 Palosaari, N., 58 Palotta, A. J., 7 7 Parkinson, T. F., 9 7 Parr, R. M., 101 Parrk, G. E., 6 1 , 6 7 Parrish, P. R., 5 9 Parsons, C. L., 1 7 Pasceri, R. E., 39, 40 Patapoff, M., 48 Paur, R. J., 48 Pearson, R., 2 1 Pearson, R. G., 1 0 3 Pelon, J., 11 Penkett, S. A., 18 Penndorf, R., 1 Pentreath, R. J., 110 Perry, R., 84 Peters, J., 7 7 Peters, P. D., 1 3 7 Petrucelli, L., 7 7 Pettine, M., 69 Phillips, D. J. H., 1 0 5 Phillips, N., 2 0 Pinto, J. P., 10 Pirie, €3. J. S., 1 0 8 , 112, 116,137 Pitchford, R. J., 6 7 Pittock, A. B., 11 Pitts, J. N., 2 5 , 30, 3 3 , 4 7 , 48 Plum, H., 49, 7 5 Poller, R. C.,64, 73, 75 Pollock, W.,5

Author Index Polster, M., 56 Popl, M., 74 Possanzini, M., 24 Possiel, N. C.,33 Posthumus, A. C., 45 Potts, G. W., 113 Pottu-Boumendil, J ., 133 Poulson, D. F., 115 Pounds, E. K., 46 Powers, L., 130 Prather, M. J., 2 Pratt, R., 1 3 Prent, P., 116 Price, J. W., 60, 73 Prinn, R., 20 Prough, R. A., 60 Pruchniewicz, P. G., 3, 13 Quintat, C., 116 Quon, J. E., 42 Rainbow, P. S., 116, 122 Ramp, W. K., 106 Rapsomanikis, S., 70, 71 Ray, E. E., 98 Razet, P., 134 Reed, R. J., 4 Regener, V. H., 1 8 , 2 1 Reily, C. L., 45 Reinert, R. A., 46 Reisdorf, R. P., 49 Reiter, E. R., 4, 7 , 2 0 Resh, M. D., 108 Rethfeld, H., 8 3 Revlett, G. H., 42 Richards, K. S., 117 Ridley, W. P., 71 Ripperton, L. A., 29 Ritchie, A., 122 Ritchie, H. R., 78 Ritchie, R. R., 78 Ritter, C. J., 80,83, 85 Robbins, P. W., 128 Robertson, K. W., 99 Robinson, E., 18 Roinel, N., 128 Rose, A. H., 28 Rose, B. A., 78 Rosenberg, H., 121,122

Ross, E. D., 42 Ross, W. D., 58 Rothwell, R., 39 Routhier, F., 14 Roy, C. R., 21 Runeckles, U. C., 45 Rusheed, A., 7 Ruste, J., 117, 130 Ryan, D. E., 9 1 Saeger, H., 34 Sakagami, Y., 73 Sakamaki, F., 29 Salottolo, G. D., 24 Saltbones, J., 38

143 Saltman, B. E., 47 Salvato, B., 110 Sansoni, B., 101 Sasaki, N., 111

Savory, J. G., 75 Scaffo, M. B., 126 Schjoldager, J., 24, 37, 38 Schmidt, M., 1 3 Schmidt, U., 8 Schoenbein, C. F., 1 Schoenemann, H. M., 100 Schonberg, M., 4 Schuller, R. M., 99 Schulz-Baldes, M., 118 Schurath, U., 38 Schwab, D. W., 133 Seiler, W.,3 Seinen, W., 58 Seinfeld, J. H., 28, 30 Selwyn, M. J., 58 Senich, G. A., 62 Senkbeil, E. G., 114 Sennett, D. H., 33 Sexton, K., 39 Sexton, K. G., 28 Shapiro, M. A., 6, 7 , 2 0 Shedlovsky, J., 5 Shelar, E., 4 0 Sheldon, A. W., 67, 74, 76 Shibuya, K., 29 Shore, R. E., 133 Sickles, J. E., 29 Siebenlist, K., 58 Siedel, S. L., 61 Sikora, L. J., 80 Simkiss, K., 102, 105, 108, 111,114,121,122,138 Simon, S. L., 128 Simpson, T. L., 132, 133 Singh, H. B., 3, 25 Sioli, H., 119 Sivertsen, B., 37 Skewes-Cox, P. D., 30 Skinner, H. A., 73 Slesinger, A., 59 Slesinger, A. E., 65 Smith, A. W., 78 Smith, D. J., 59 Smith, G. S., 98,100 Smith, P. J., 59,65, 67, 69,76 Smith, R., 60,80 Soderquist, C. J., 61, 67 Sohal, R. S., 137 Solomon, S., 3 Sommers, L. E., 80 Sopper, W. E., 98 Spangenberg, D. B., 122 Spangler, E., 90 Spicer, C. W., 29, 30, 39, 40 Spiro, T. G., 130 Sposito, G., 9 1 Springs, B., 129

Sproule, J. S. G., 67 Sprung, J. L., 30 Squibb, K. S., 109, 110 Stahman, R. C., 28 Staley, D. O., 4 Stallard, R. F., 1 3 Stalmach, M. A., 60 Stankov, B. B., 21 Stasiuk, W. N., 33 Stedman, D. H., 18,21, 34 Stein, V. T., 74 Steinberger, E. H., 45 Stern, G., 80 Stetson, T. B., 6 3 Stevens, R. D. S., 39 Stewart, H. N. M., 36 Stewart, R. W., 10 Sticksel, P. R., 39,40 Stiffler, D. M., 98 Stites, E., 126 Stoeppler, M., 101 Street, B. W., 58, 61 Stuart, D. C., 9 1 Stump, A. B., 133 Sturesson, U., 117 Suess, A., 64 Sullivan, E. J., 36 Summerhays, J. E., 33 Sumner, A. T., 120 Sunde, J., 31 Sundstrom, G., 56 Svanberg, O., 56 Sverdrup, G. M., 29 Tabatabai, M. A., 80 Taketa, F., 58 Tapp, R. L., 115, 137 Taylor, M., 102, 114, 122 Taylor, R. S., 4 0 Termine, J. D., 126 Thayer, J. S., 70, 71 Thust, U.,64, 74 Tiefenau, H. K., 1 3 Tiefermann, M. W., 1 3 Tingey, D., 46 Tobias, R. S., 67, 69 Tokuyama, A. 127 Tombach, I. V., 33 Tompkins, M. A., 60 Tooby, T. E., 56 Toonkel, L., 4 Tout, R. E., 8 0 Towe, K. M., 113 Trachman, H., 59 Trantz, 0. R., 128 Truchet, M., 108,134 Tsay, U. H., 74 Tu, C. M., 65 Tue, V. T., 115 Tullins, T. D., 109 Tyler, L. D., 80 Uhacz, K., 67 Ulland, B. M., 77

A utho r In d ex

144 Valerio, M. G., 77 Vander Horst, A,, 4 7 van der Kerk, G. J . M., 56 Vander Mallie, R. J., 1 3 2 Van Diujn, J., 47 Van Dop, H., 38 Van Ham, J., 24 Vasak, M., 1 3 7 Velds, C. A., 38 Vernon, F., 61 Verschoyle, R. D., 58 Versluis-de Haan, G., 6 7 Viarengo, A., 115 Viezee, W., 3 Vincentus, M. D., 109 Vind, H. P., 6 7 Volcani, B. E., 1 3 2 Volk, V. V., 9 7 von Brand, T., 125, 1 2 6 Vonk, J. W., 64 Vukovich, F. M., 41 Wachs, T., 9 7 Wada, O., 56, 5 9 , 6 1 Wadden, R. A., 4 2 Wade, S. E., 100 Wagge, L. E., 119 Wahl, B., 1 3 3 Wakelyn, N. T., 30 Walker, G., 115, 116, 122 Walker, J. C. G., 3 , 9 Walker, N. P. C., 6 7 Waller, R. E., 36 Walter, T. A,, 3 0

Ward, G. F., 40 Ward, G. M., 8 0 , 9 0 Ward, G. S., 59 Warner, J. L., 33, 3 9 , 4 2 Warner, R. R., 107 Wartburg, A., 5, 14 Watabe, N., 1 0 2 , 1 3 3 Waterhouse, D. F., 115 Watkins, D. A. M., 7 3 Webb, E. C., 128 Webb, J., 1 1 3 Weber, T. A., 25 Wehner, W., 49 Weiss, R. E., 138 Welsh, K. M., 1 2 9 Wesely, M. L., 18 Westberg, H., 39 Wheeler, E., 111 White, W. H., 39 Wiebe, H. A., 39 Wiebkin, P., 60 Wiener, R. W., 28 Wigglesworth, V. B., 133 Wilbur, K. M., 1 3 8 Wilcox, R. W., 11 Wilkinson, R. R., 4 9 Williams, B., 60 Williams, M. E., 1 7 Williams, M. L., 5 , 36 Williams, R. J. P., 137 Williams, R. M., 18 Williams, R. T., 59, 60 Willis, C., 4 7 Wilson, K. R., 4 3

Wilson, W. E., 39 Winer, A. M., 25, 3 0 , 4 7 Winges, L., 30 Winters, C., 118 Wirth, H. O., 49 Wise, L. E., 75 Wisse, J. A., 38 Wofsy, s. c., 2 Woggon, H., 7 4 Wolfe, D. A., 113 Wolff, G. T., 39, 40, 42 Wong, P. T. S., 61, 70 Woo, C. C., 109 Wood, J. M., 7 1 Wootton, R. J., 115, 117 Wright, D. A., 106 Wright, G. D., 3 9 , 4 0 Wright, R. S., 29 Wriston, J. C., jun., 114 Wszolek, P. C., 9 7 Wukasch, R. T., 46 Yadav, S. S., 91 Yevitch, P., 109 Yoshioka, S., 1 2 7 Yu, T. H., 5 9 , 6 1 Yuan, T. L., 99 Zamierowski, E. E., 80, 8 3 Zanicchi, G., 115 Zuckerman, J. J., 49 Zullig, W.,11