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TECHNICAL REPORTS SERIES No. 137

Mercury Contamination in Man and his Environment

A JOINT UNDERTAKING BY THE INTERNATIONAL LABOUR ORGANISATION. THE AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS. THE WORLD HEALTH ORGANIZATION AND THE INTERNATIONAL ATOMIC ENERGY AGENCY

J WJ

INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1972 CONTAMINATION IN MAN AND HIS ENVIRONMENT

TECHNICAL REPORTS SERIES No. 137

MERCURY CONTAMINATION IN MAN AND HIS ENVIRONMENT

A JOINT UNDERTAKING BY THE INTERNATIONAL LABOUR ORGANISATION, THE FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS, THE WORLD HEALTH ORGANIZATION AND THE INTERNATIONAL ATOMIC ENERGY AGENCY

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1972 MERCURY CONTAMINATION IN MAN AND HIS ENVIRONMENT IAEA, VIENNA, 1972 STI/DOC/lO/137

Printed by the IAEA in Austria July 1972 FOREWORD

In May 1967, at a Symposium organized by the International Atomic Energy Agency in Amsterdam, the special problems of food and environ- mental contamination by mercury were discussed by world experts on the subject and by representatives of FAO, WHO and IAEA. One of the recom- mendations made by this meeting was that the international organizations of the United Nations family should assist in the collection and distribution of information on environmental mercury. Subsequently, the organizations concerned agreed that a handbook on mercury contamination would be es- pecially useful. This would deal with sources of mercury in relation to man and his environment; with physical and biological transfer processes that determine its distribution; with analytical methods for determining mercury and its compounds as environmental contaminants; with actual concentrations of mercury found in the environment, in living organisms and in man; and with its in animals and man. The present monograph, which is published under the auspices of ILO, FAO, WHO and IAEA, represents the result of these earlier deliberations, up-dated in the light of recent developments in this rapidly advancing field. The international organizations concerned wish to express their gratitude to all those who have taken an active interest in the publication and especially to those who have contributed directly to the technical content of the mono- graph itself.

LIST OF CONTENTS

1. INTRODUCTION 1 F.P.W. Winteringham

2. USE OF MERCURY COMPOUNDS IN AGRICULTURE AND ITS IMPLICATIONS 5 Kristina Rissanen and J.K. Miettinen

3. USE OF MERCURY AND ITS COMPOUNDS IN INDUSTRY AND MEDICINE 35 N. Saito

4. BEHAVIOUR OF MERCURY IN THE ENVIRONMENT 43 S. Jensen and A. Jernelov

5. MECHANISMS FOR OF MERCURY IN THE ENVIRONMENT ... 49 J.M. Wood, M. W. Penley and R.E. DeSimone

6. THE OF MERCURY IN MAN AND ANIMALS 67 F.C. Lu, P.E. Berteau and D.J. Clegg

7. DETERMINATION OF MERCURY BY INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS 87 V.P, Guinn

8. THE DETERMINATION OF MERCURY AND ITS COMPOUNDS BY DESTRUCTIVE NEUTRON ACTIVATION ANALYSIS 99 T. Westermark and K. Ljunggren

9. THE DETERMINATION OF TRACES OF MERCURY BY SPECTROPHOTOMETRY, ATOMIC ABSORPTION, RADIOISOTOPE DILUTION AND OTHER METHODS Ill C.G. Lamm and J. R&zicka

10. IDENTIFICATION OF MERCURIAL COMPOUNDS 131 J. O'G. Tat ton

11. THE RELIABILITY OF MERCURY ANALYSIS IN ENVIRONMENTAL SAMPLES: RESULTS OF AN INTERCOMPARISON ORGANIZED BY THE IAEA 137 J. Heinonen, D. Merten and O. Suschny

12. PRESENT LEVELS OF MERCURY IN MAN AND HIS ENVIRONMENT 143 A. V. Holden

13. PRESENT LEVELS OF MERCURY IN MAN UNDER CONDITONS OF OCCUPATIONAL EXPOSURE 169 M. Berlin

LIST OF AUTHORS'ADDRESSES 179

1. INTRODUCTION

F.P.W. WINTERINGHAM

The metallic element mercury has been known since ancient times and was certainly mentioned by Aristotle (350 B. C.). Its unique and curious properties of being a -like of high (13. 596 g+1 cm"3 at 0°C), its relatively high vapour (8 X 10"3 Torr at 40°C; 270 X 10"3 Torr at 100°C) and its consequent volatility (e. g, many times greater than that of the DDT), its ready ability to dissolve (amalgamate with) certain such as , , and , sometimes with the formation of definite compounds (e. g. NaHg^, and its obtuse angle of contact with glass and other "non-wettable" surfaces (depression of the internal mercury surface by a glass capillary) have attracted the attention of philosophers and scientists for hundreds of years. The elusive behaviour of spilled mercury globules is known to almost every schoolchild. The toxic properties of mercury and its com- pounds have also been known since ancient times, and mercuric chloride was one of the first used in clinical medicine. The element occurs in nature in its native elemental form. Indeed, it was the discovery of the mirror-like liquid at the bottom of local drink- ing wells at Idrija in Yugoslavia which led to its and extraction there more than three centuries ago (personal communication from Profes- sor L. Kosta). It occurs in forms especially as (mercuric sulphide, HgS). Cinnabar is found in widely separated regions such .as Almaden (Spain), Idrija (Yugoslavia), Monte Amiatra (Italy) and in lesser quantities in , California, Mexico, Mainland and . Accord- ing to Partington ( Textbook of Inorganic , Macmillan & Co. Ltd., , 1933), Pliny reported that 10 000 lb of cinnabar came annual- ly from Spain in his day. Its volatility and occurrence in account for its presence throughout the environment and it can be detected in all living tissues. It is, therefore, a clear example of a natural constituent of the environment which only becomes a in virtue of the special conditions of concentration or location as a result of man's activities. The total global production of mercury in 1966 (excluding USSR, China and CSSR) was 726 0 metric tons and no significant trend was dis- cernible during the preceding quinquennium (UN Statistical Yearbook, New York, 1968). However, this does not represent the upper limit of potential or environmental contamination as a consequence of man1 s activities. If the average mercury content of crude oil is taken to be about 0. 1 ppm then some 200 tons of mercury may be expected to be released annually into the atmosphere from the combustion of petro- leum fuels alone. From the known average mercury content of snow or rainwater it has been estimated that the surface of the earth receives an- nually some 100 000 tons through precipitation from all mercury sources including natural ones (SAHA, J. G. , Significance of mercury in the en- vironment, Residue Reviews, 1972, in press). Mercury in one form or another lends itself to a wide variety of uses in addition to its well-known use in and . Major

1 applications include those as an industrial chemical catalyst, in the manufacture of electrical batteries, , relays and , in the -alkali industry, in marine paints, in paper and pulp manu- facture, as a in agriculture, in dentistry, pharmacy and in medicine. Its powerful inhibitory action against basal metabolic processes com- bined with special conditions of location or concentration as a result of man's activities have had tragic consequences from time to time interms of human health. Notable were the many confirmed cases of mercury - ing, a considerable number of which were fatal, in Minamata and , Japan, as a result of eating or caught in coastal and river waters contaminated as a result of an industrial discharge. Such instances have emphasized the growing problems of environmental contamination by undesirable chemicals, both in fact and in the public mind, and to which the forthcoming UN Conference on the Human Environment stands witness. The particularly extensive studies of mercury have demonstrated clearly the importance of an overall and balanced approach to such pro- blems. This so-called integrated approach must consider all possible sources of the contaminants, including any natural ones, their physical and chemical fate, behaviour in ecological systems and food webs, and their possible acute and chronic effects not only on human health but on wildlife and the complex of living organisms constituting the exposed ecosystem. Finally, it must consider benefits in terms of human health, comfort and welfare. Thus, in the case of mercury the relatively high mercury concentrations found in some aquatic samples led to an early suspicion that the use of agricultural mercury was mainly re- sponsible. However, further study of the sources revealed that industrial discharges were the principal causes of contamination. Similarly, careful study of mercury concentrations in fish, including samples of fish caught as long as 90 years ago, as well as studies of natural mercury in the marine environment have shown that the relatively high levels* of mercury in tuna fish are unlikely to be due to man's activities. They are •more likely to be due to the natural mercury content of the marine en- vironment and the physiology of the fish species concerned (HAMMOND, A. L. , Science r71_ (1971) 788). The element mercury lends itself to neutron radioactivation analysis with great sensitivity so that unequivocal identification of the element and its determination in a wide range of biological and environmental - samples at concentrations of the order of 1 part in 109 have been greatly facilitated. In addition, the availability of radioactive of mercury such as mercury-203 has enabled the movement, fate, meta- bolism and excretion of the element to be studied in living organisms and in food chains with unrivalled specificity and sensitivity. Notable applica- tions of these techniques have been made by Professor Miettinen and his colleagues in Finland, by Professor Kosta and his colleagues in Yugoslavia, and by Professor Jervis and his colleagues in Canada. Such studies have provided a wealth of information on the behaviour of mercury in the environment and in living organisms including man. Of particular interest and significance has been the demonstration that inorganic mercury may undergo biological methylation with the formation of methyl mercury and this organic metabolite is, in fact, more toxic

2 and more stable in vivo than some inorganic forms of the element. Mercury can become concentrated in contaminated seed--mammalian predator food chains, and analyses of fish-muscle tissues have indicated that concentration factors of 1000 or more may obtain in marine and aquatic systems. The inhibition of photosynthesis of phytoplankton by low concentrations of mercury has also been indicated. Despite the wealth of information now available on the occurrence and significance of mercury in the environment many outstanding problems remain to be solved as usefully indicated in Dr. J. G. Saha's comprehensive review of the subject (Significance of mercury in the environment, Residue Reviews, 1972, in press). The International Atomic Energy Agency, through its co-ordinated research programs on food and environmental contamination and colla- borative programs with other UN Agencies, was placed in a good position to encourage an overall appraisal of environmental mercury problems and their study, as shown in the authoritative contributions which follow. To conclude: The problems of mercury in the environment and their study have demonstrated the great importance of the so-called integrated approach and of taking fully into account the benefits as well as the risks associated with man's exploitation of this important element. For ex- ample, this approach has strongly suggested that some uses such as those in dental medicine or the prescribed uses as agricultural fungicides are without significant risk and provide considerable benefits. On the other hand, certain industrial uses and discharges require very careful moni- toring, control and possible restriction. The elaboration of maximum permissible concentrations in food a!nd drink, acceptable daily intakes by man and "action levels" must also take fully into account the natural occurrence of this element and its variations. The studies of the natural distribution of mercury in food and environment have also shown how man must always have been, and forever must be, exposed to this ele- ment. It is only on the basis of balanced and comprehensive studies that any necessary counter-measures and controls can be identified and de- veloped on a rational basis. The views and conclusions of this introduction are not necessarily those of the International or UN Agencies concerned.

3

2. USE OF MERCURY COMPOUNDS IN AGRICULTURE AND ITS IMPLICATIONS

Kristina RISSANEN and J.K. MIETTINEN

2.1. INTRODUCTION

Mercury fungicides have been used for treatment of seeds against fungi for more than half a century. In view of the large scale of use, their safety record is notably good, except for the alkyl mercury compounds, especially methyl mercury, whose agricultural use has been recently discontinued in several countries because of its accumulation by terrestrial food-chains. High levels of methyl mercury in the fish of industrialized regions, as well as severe accidents in which hundreds of people in developing countries have died due to the consumption of cereals treated by methyl mercury or to the consumption of animals which have consumed such treated cereals, have also recently focused attention on mercury fungicides. The present review of the agricultural use of mercury fungicides is concerned with their persistance in nature, their concentration into and their effect on living things, man included.

2.2. DEVELOPMENT AND SCALE OF SEED TREATMENT BY OR GANO MERCURIALS

2.2. 1. Development of methods

Inorganic mercury compounds had been in use for seed treatment against fungi since the end of the previous century, but the use of organo- mercurials was introduced in 1914 in the form of seed treatment with water of hydroxyphenyl mercury chloride (trade name Uspulun). This process, called "wet dressing", was difficult since water had to be removed in order to prevent premature germination. To avoid this problem, the "dry dressing" was developed in the middle of the 1920s. By this technique, the fungicide was mixed with an inert dry powder and in this form further mixed with the seeds in a special mill. Because of the resulting toxic dust, the handling of seeds was unhygienic. Today, however, modern technology has eliminated the hazard of toxic dust. The so-called "short wet" and "slurry" methods were developed in 1927 in which only 2-3 litres of liquid agent were necessary for 100 kg of seed. However, the moisture content was still so high that the seed had to be sown within a few days after treat- ment. This difficulty was overcome in the 1930s when "liquid dressing", with methyl or ethyl mercury compounds, was developed. This dressing requires only 100 to 200 ml of a 1% solution of the agent per 100 kg of seed. In Sweden, methyl mercury dicyandiamide was introduced in 1938 under the trade name Panogen, and the "liquid dressing" technique soon replaced other methods of seed treatment in that country because of its efficacy, safety and technical ease. Later, it also became popular in other European countries, and in the USA and Canada as well. of organomercurials and chlorinated hydrocarbons have also been on the market, but their use is now forbidden in many countries, e. g. Finland and Sweden.

5 The direct treatment of growing plants with organomercurials is a relatively new method. The use of phenyl mercury acetate in the prevention of rice blast began in Japan as late as 1952, but grew very rapidly until it was terminated around 1969. (See also FAO/WHO, Evaluation of Some Residues in Food, FAO/FL; 1967/M/ll/l, Rome 1968, p. 200 et seq., andFAO/WHO, Evaluation of Some Pesticide Residues in Food, FAO/PL-CP/15, Rome 1967, p. 188 et seq. )

2.2.2. Mercury compounds

Inorganic mercury (HgCl, HgCl2, HgO) has been little used. On the other hand, hundreds of organic derivatives have been used, all of the type R-Hg-X, in which R is an organic radical and X originates from an organic or inorganic substance possessing a dissociable atom. Examples of X, or substances which may produce X, are:

Inorganic acid radicals: chloride, bromide, , phosphate; Organic acid radicals: acetate, benzoate, salicylate; Amides: urea, thiourea, formamide, dicyandiamide; Phenolates: pentachlorophenolate, 8-hydroxyquinolinate; Mercaptides: 2, 3-dihydroxypropylmercaptide.

Although the anion can modify the properties of the compound, it is of less importance than the radical R. The classification of mercury fungicides is therefore based upon R, according to which the organomercurials are usually divided into the following three groups:

(1) Alkyl mercuric compounds:

Methyl mercury (CH3Hg-), Ethyl mercury (C2H5Hg-); (2) Alkoxyalkyl mercuric compounds:

Methoxyethyl mercury (CH3OC2H4Hg-), Ethoxyethyl mercury (C^HgOC^Hg-); (3) Aryl mercuric compounds: Phenyl mercury (CeHsHg-),

p-Tolyl mercury (CH3C6H4Hg-).

In each , the two most common radicals in use are mentioned.

2.2. 3. Chemical and physical properties

The of organomercurials in water and organic is primarily determined by the anionic (amidic) radical, although the radical R also has some influence. In general, the aryl derivatives are less soluble in water than the alkyl derivatives, given the same anion. Volatility is an important property of seed dressings. Generally, the alkyl compounds are the most volatile. However, volatility can be regulated to a certain extent by choosing the proper anion. In Table I, which is taken from Swensson and Ulfvarsson (1963), the volatilities of some organo- mercurials and of metallic Hg are presented as saturated vapour concen- trations (for references, see original).

6 TABLE I. SATURATED VAPOUR CONCENTRATION OF SOME ORGANO- MERCURY AT 20°C (SWENSSON AND ULFVARSSON, 1963)

Vapour concn Cation Anion (pg/m3)

Methyl-Hg Chloride 94000

Bromide 94 000

Iodide 90 000

Acetate 75 000

Hydroxide 10 000

Toluenesulphonate 15 000

Benzoate 2 000

Dicyandiamide 300

Ethyl-Hg Chloride 8 000

Bromide 7 000

Iodide 9 000

Dicyandiamide 400

Monohydrophosphate 50

Methoxyethyl-Hg Chloride 2 600

Acetate 2

Phenyl-Hg Acetate 17

Chloride 5

Nitrate 1

Methanedinaphthyldisulphonate 2

Metallic Hg 14 000

The C-Hg bond of the R-Hg- radical is chemically stable. It is neither split in water nor by weak acids or bases. However, as will be described later, only the alkyl-Hg bond is stable in soil and in most organisms. The C-Hg bond in alkoxyalkyl- and aryl-mercury compounds is easily broken, even in animals, thus producing inorganic mercury.

2.2.4. Dressing techniques

The techniques used for seed dressing in the middle of the 1960s were "liquid dressing", "dry dressing" and the "slurry method" (Ulfvarsson, 1969). They require different properties of the dressing agent, for the same agent may be excellent in one technique but poor in another, as was shown by Gassner (1951). "Liquid dressing" requires some volatility (at least 100 pg/m3 at 20°C; LindstrSm, 1958; Bombach, 1963) in order to guarantee

7 TABLE II. CHEMOTHERAPEUTIC INDEX (c/t) ACCORDING TO GASSNER (1951)

Dosis curativa, c Dosis toxica, t Chemotherapeutic Compound (ppm Hg) (ppm Hg) index, c/t

Methyl-Hg 15 72 0.21

Ethyl-Hg 23 56 0.41

Propyl-Hg 37 12 3.0

Butyl-Hg 44 10 4.4

Octyl-Hg 77 54 1.4

Methoxyethyl-Hg 36 73 0.50

Ethylene-Hg 42 80 0.52

Phenyl-Hg 34 37 0.92

Cyclohexyl-Hg 46 26 1.8

HgCl2 200 380 0.53

its homogeneous distribution via the vapour during storage. Higher volatility increases the risks. The less moisture involved, the less damage to the seeds. In "dry dressing", the homogeneity of distribution is aided by the inert carrier. The agents are usually rather non-volatile and have a fungicidal effect only in the moisture of the soil.

2.2. 5. Formulation and fungicidal effectivity

Gassner (1951) compared the effectivity of a number of mercury fungicides in protecting wheat against Tilletia tritici by the "short wet" and the "dry dressing" methods. In the process he determined the so-called chemotherapeutic index, which is the ratio dosis curativa/dosis toxica, dosis curativa being the lowest concentration of the dressing that prevents germination of the fungi and dosis toxica being the lowest dressing concen- tration that noticeably inhibits growth of the wheat. The effectivity of the dressing agent was determined by the structure of the R group, the X group being relatively insignificant. Some of Gassner's results are presented in Table II. As may be seen, methyl-Hg is the most effective dressing, (i. e. the index is lowest), while ethyl-Hg, ethylene-Hg and methoxyethyl-Hg are about two times and phenyl-Hg about five times less effective. Only these compounds are sufficiently effective in the "dry dressing" method. Ishiyama (1969) determined the effectivity of 58 R-Hg-X type compounds against the rice blast Piricularia oryzea and established the following order: phenyl > o- , p-tolyl > ethyl, methoxyethyl, dimethylphenyl > naphthyl. The anion (-X) had little influence, iodide being the most effective when

8 TABLE III. DISEASES OF CEREALS CONTROLLED BY SEED TREATMENT WITH MERCURY FUNGICIDES

Disease, Disease, Cereal common name scientific name

Wheat Bunt Tilletia tritici

Bunt Tilletia foetida

Snow mould Fusarium nivale

Seedling blight Fusarium spp.

Barley Covered smut Ustilago hordel

Leaf stripe Helminthosporium gramineum

Net blotch Helminthosporium teres

Seedling blight Fusarium spp.

Rye Snow mould Fusarium nivale

Stripe smut Urocystis occulta

Seedling blight Fusarium spp.

Oats Loose smut Ustilago avenae

Leaf spot Helminthosporium avenae

Covered smut Ustilago levis

Seedling blight Fusarium spp.

Maize Leaf spot Helminthosporium spp.

a Rice Blast Piiicularia oryzae

Stem rot Helminthosporium sigmoideum'

Brown spot Cochliobolus miyabeanusa

a Also sprayed during growth.

the radical R- was phenyl. The fungicides were thought to act at three stages of fungi development:

(1) Conidial germination; (2) Formation of the appressoria; and (3) Penetration of the plant surface.

Thus, for instance, phenyl mercury acetate prevents the latter two stages, but does not prevent conidial germination.

9 TABLE IV. USE OF MERCURY FUNGICIDES FOR SEED DRESSING IN FIFTEEN EUROPEAN COUNTRIES IN SPRING 1965 ACCORDING TO GRANHALL (1965)

Arable Sown with Mercury Percentage that is mercury dressed of: Country Area land cereal dressed 1000 ha 1000 ha 1000 ha 1000 ha The cereal The arable land The total area

Sweden 41.000 3 290 1.470 1.180 80 36 3

Norway 30.900 850 240 120 50 14 0.4

Finland 30.500 2 680 1.020 300 30 11 1

Denmark 4.300 2 820 1.570 1.290 82 46 30

Great Britain 24.100 7 270 3.400 2.630 77 36 11

Ireland 6.900 1 360 390 250 64 18 4

Netherlands 3.400 1 030 510 370 73 36 11

Luxemburg 260 75 45 30 67 40 12

West Germany 24.300 8 500 4.990 2.740 55 32 11

Poland 30.400 16 180 8.630 2.460 29 15 8

Austria 8.300 1 750 860 670 78 38 8

Hungary 9.300 5 620 3.000 1.700 57 30 18

Switzerland 4.000 420 160 90 56 21 2

France 54.700 21 410 7.910 2.850 36 13 5

Turkey 77.700 25 200 10.700 5.780 54 23 7 2. 2. 6. Plant disease and rate of use

The purpose of the seed dressing is to prevent the diseases which are spread with the seeds and to protect the germinating seeds against secondary- . The most important cereal diseases which can be prevented by seed dressing are presented in Table III. According to an announcement made by the US Department of Agriculture, (USDA, March 9, 1970), alkyl mercury fungicides are registered in the USA for the treatment of seeds of the following plants: barley, wheat, oats, rye, corn, millet, milo, sorghum, rice, beans, peas, cotton, flax, peanuts, soybeans, beets, tomatoes and safflower. Mercury fungicides are also used in the spraying of fruit trees against various types of scab, and rice against blast Piricularia oryzea, stem rot Helminthosporium sigmoideum and brown spot disease Cochliobolus miyabeanus. In countries with a snow-cover lasting at least three months, the protection of stands of winter cereals against Typhula, Sclerotinia borealis and especially Fusarium nivale is important. Mercury fungicides are effective for this purpose but, at least in Finland, only mercury-free compounds are used (Jamalainen, 1964). Fungus diseases of turf can be controlled by alkyl-Hg and phenyl-Hg compounds (Ulfvarsson, 1969). Their use seems to be especially wide in Canada. In 196 7, 2. 8 metric tons of mercury were used in this form for the treatment of golf courses and home lawns (Fimreite, 1970a). Smart (1968) presented a list of about 40 plants and 50 corresponding diseases, and also mentioned the stage and the rate of fungicide application most effective in control of the disease. The amounts used vary greatly, but are usually between 0. 02 and 0. 3 g Hg/kg seed. In fruit treatment, the amounts vary from 10 to 400 g Hg/ha1, and in soil treatment (root crops), they are an order of magnitude greater (0. 5-10 kg Hg/ha). Until 1950, only inorganic compounds seem to have been used in soil treatment.

2.2.7. Concentrations and extent of use

The concentration of organomercurials in the differing fungicidal formulations varies greatly. Smart (1968) sets the following limits (all figures are percentages of Hg in formulation) for seed dressings: "dry" 0.2-10, "wet" 0.4-6, "slurry" 1. 3 - 6; for bulb dips: "liquid" 1. 5 - 6; for seed-potato dips: "liquid" 0. 5 - 6, "dry" 3-9; for sugar-can dip: 6; for glasshouse : 0. 35; for orchard canker paint: 2; for orchard spray: "dispersible powder" 0.6- 40, "emulsifiable concentrate" 0.4- 40; and for lawn: 3.4-6.7. The inorganic mercury fungicides (HgCl or HgO) used in the treatment of potato soil contain 10% Hg, while those for lawn treatment contain 45% Hg. Granhall (1965) reviews well the extent of mercury fungicide use in 15 European countries as of spring 1965. Table IV is taken from this review and it shows that a large percentage of all the cereals grown in Europe in 1965 was dressed. Healthy seeds were also preventively treated. These figures probably represent the highest level of use, at least in Scandinavia. In 1965, much attention was directed there to the death of caused by dressed seeds, and farmers accordingly reduced their use of mercury

1 1 hectare (ha) = 2,48 acres.

11 TABLE V. QUANTITIES OF MERCURY COMPOUNDS USED IN OR SOLD TO AGRICULTURE IN 1965 ACCORDING TO SMART (1968)

Metric tons Metric tons Country of Country of Hg compounds Hg compounds

USA 400 0.4

Denmark 3.5 Portugal 0.2

Germany 41 Turkey 22.5

Great Britain 20 Spain 7.1

Bulgaria 5 Sweden 2

Finland 1 Morocco 1

Italy 26 Israel 0.2

Poland 9 New Zealand 0.5

Austria 4 Japan 1600 (approx.)

fungicides. In Sweden in 1965, treatment was restricted to contaminated lots of seed. Dressing was also reduced in other Scandinavian countries. Lihnell (1969) stated the following figures for the use of mercury fungicides in 1968 (as percentage of seed sown): Sweden 25- 30, Norway-35 - 40, Denmark 80 and Finland 30. Smart (1968) gave figures for the agricultural use of mercury fungicides in 1965 in a number of countries (world total: 2100 t). These are presented in Table V. In the USA, only 120 t were used in 1968 (Nelson et al., 1971), while in Japan, in the same year, about 400 t were used (personal communication, 1969). About 16 t were used in Canada in 1965, and only 9 t in 1968 (Fimreite, 1970a). In Scandinavia, the use of mercury fungicides in 1968 was on about the same level as in 1965 (Lihnell, 1969).

2. 2. 8. Control of mercury residues

Some countries have established "tolerances" [maximum concentration of that is permitted in or on food at a specified stage of harvesting, storage, , marketing, etc (see FAO/WHO, Pesticide Residues in Food, Report of the 1969 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Group on Pesticide Residues, FAO Agricultural Series, 84, Rome 1970, p. 40)] which have been reported to vary from 0 to 0. 1 ppm (FAO/WHO, Evaluation of Some Pesticide Residues in Food, FAO/PL; 1967/M/ll/l, Rome, 1968, p. 208). In view of the lack of adequate data and toxicological information the 1967 Joint FAO/WHO Meeting on Pesticide Residues was unable to recommend tolerances for organo-mercury compounds but "By way of guidance, however, practical residue limits [maximum unintentional residues (FAO/WHO Pesticide Residues in Food, Report of the 1969 Joint Meeting of the FAO Working Party of Experts on Pesticide Re'sidues and

12 the WHO Expert Group on Pesticide Residues, FAO Agricultural Series, 84, Rome 1970, p; 39)] of 0.02 ppm to 0.05 ppm mercury, according to local conditions, are suggested" (see also Sections VII and VIII).

2. 3. ACCUMULATION AND EFFECTS OF MERCURY FUNGICIDES ON ORGANISMS

2.3.1. Micro-organisms

There are in the literature several works dealing with the effects of inorganic mercury on micro-organisms (e.g. Webb, 1966; Keckes and Miettinen, 1970). Less is known, however, about the uptake and effect of organomercurials. In the few studies performed in this area, the radio- 203 Hg (T-l/2 = 46. 59 d) has been quite useful. 203 Tonomura et al. (1968a, b, c) found in experiments with Hg that HgCl2 and phenyl mercury acetate (PMA) are rapidly adsorbed on Pseudomonas which was isolated from PMA-contaminated soil. Equilibrium with HgCl2 was attained in 20 min at 2 x 108 atoms Hg/cell, while with PMA and with ethyl mercury phosphate (EMP), equilibrium was more slowly attained at 1 x 108 and 0. 6 x 108 atoms Hg/cell, respectively. About half of the adsorbed HgCl2 and PMA could be leached out in the original form with diluted acid and identified by thin-layer chromatography. Approximately

75% of the PMA and 55% of the HgCl2 radioactivity was found to be in the cell-wall fraction (2000 g, 15 min), which corroborates the old hypothesis that the bulk of Hg in cellular organisms is bound by SH-groups on the surface of the cell (Meyer, 1964). It has been known since 1889 that cells pretreated with SH-compounds are more resistant to Hg-compounds, and that Hg inhibition can be prevented by adding SH-compounds to the medium (Webb, 1966). Many micro-organisms can adapt themselves to the presence of Hg- compounds. This acquired resistance does not disappear, even if the organism is grown in a Hg-free medium, as it is hereditary (Webb, 1966). Tonomura et al. (1968a, b) isolated, from a PMA-contaminated rice field, Hg-resistant Pseudomonas. which required for growth inhibition the following concentrations of Hg-compounds in the medium; 450 ppm HgCl2, 120 ppm PMA or 20 ppm EMP. The resistance to HgCl2, PMA and EMP was about 1000 times higher than normal. Usually IS. coli, P. aeruginosa. B. megaterium and JB. subtilis cannot grow in media containing higher concentrations than

0. 1 ppm HgCl2 0. 1 - 0. 05 ppm PMA <0. 05 ppm EMP

Penicillium roqueforti Thom. can grow in pulp containing 25 to 35 ppm PMA (Russell, 1955). Appling et al. (1949) even noticed that 1. 5 to 7 ppm PMA stimulated the growth of this mould. However, 8 ppm was already inhibitory. Webb (1966) also mentions observations of the stimulation of fungus growth by mercury. The mechanism of the resistance is not known in detail, but the microbes seem to bind the mercurial from the medium into an inactive

13 form. Penicillium bound 75% of the PMA in pulp (25 to 35 ppm) (Russel, 1955).

A mercury-resistant E^ coli bound about 35% of the HgCl2 in the medium (5 ppm) (Stutzenberger and Bennett, 1965); it further inactivated an addi- tional 40% of the HgCl2 in the medium by excreting and H2S. Mercury was so well inactivated that mercury-sensitive Staphylococcus aureus could grow in this medium. Mercury-resistant yeast cells produce more H2S than normal cells (Webb, 1966). Wr&bel (1963) noticed that methyl-Hg (in the form of Panogen) prevented the formation of symbiotic root nodules in the roots of the lupine Lupinus luteus and the pea Pisum sativum (symbiosis between the plant and Rhizobia), thus retarding fixation and growth of the plant. In general, it can be stated that the effects of organomercurials on the soil microflora have been little studied.

2.3.2. Plants a. Uptake from soil and from seed dressing and leaf spraying

The uptake of inorganic mercury from the soil by plants is small (Smart, 1968), but Yamada (1968) found that rice accumulates about 1% of the 203Hg-labelled PMA mixed with the soil. When the soil contained 13. 7 ppm PMA, the rice contained 31. 4 ppm Hg in the roots, 0. 38- 3. 51 ppm Hg in the leaves, and 0. 61 ppm Hg in the unpolished grain (on the basis of dry weight). The mercury concentration of wheat grain grown in CH3Hg- contaminated soil is in direct correlation with the CHgHg-concentration of the soil (Saha et al., 1970). In Japan, the mercury content of rice (even of untreated plants) has steadily grown since 1952. A well-drained rice field which contained 0. 33 ppm Hg in the dry soil produced grain with 0. 3 ppm Hg, while a poorly-drained rice field containing 1. 36 ppm Hg in the soil produced grain containing 0. 8 ppm Hg (on the basis of dry wt) (Furutani and Osajima, 1965, 1967). Rice takes up phenyl mercury acetate from solution with high efficiency (28 to 80%; Yamada, 1968). When the solution contained 5. 8 ppm PMA, the root tips contained 2150 ppm Hg and the stem, above the solution, 820 ppm Hg (on the basis of dry wt). When 203Hg-labelled PMA was used in the seed dressing, Tomizawa (1966) studied its uptake byrice seedlings. A small part (5 to 20%) was found in the roots and in the green portions of the seedling. Seed dressing with methyl mercury compounds increased the Hg content of the resulting grain by two- fold (0. 030 ppm), the control grain grown from undressed seed containing only 0. 014 ppm (Lbfroth and Duffy, 1969). However, Saha et al. (1970) found no significant difference in the mercury content of wheat and barley grain whether they were grown from alkyl mercury dressed seed or not. Mercury fungicides sprayed on plants are translocated into growing tissue, but there is little translocation into mature tissue (Ross and Stewart, 1962). Tomizawa (1966) studied the accumulation in rice grain of 203Hg- labelled phenyl-, ethyl- and methoxyethyl-Hg when sprayed onto the plant at either the tillering or the flowering stage. An fungicide containing 50 ppm Hg, which was sprayed during the tillering stage, resulted in only 0. 04 to 0. 1 ppm Hg in polished grain, but 0. 15 to 0. 58 ppm when sprayed onto the flowering plants. The mercury concentration in the raw rice was about 2 times, and in the husk even 3 to 4 times, higher than in the polished rice. A Japanese scientific committee reports the following

14 observations on the fate of PMA sprayed onto rice (Fukunaga and Tsukano, 1969): About half of the mercury sprayed as phenyl mercury onto the leaves is absorbed and about 5% of this is translocated into grain. About 30 to 50% of the mercury in the raw rice can be removed by polishing. The raw grain of untreated plants contained 0. 05 ppm Hg, while that of plants sprayed twice with PMA contained about 0. 2 ppm Hg. Higher concentrations are found if the treatment is carried out at a later stage of growth. b. Effect of Hg fungicides on plant growth

The ideal fungicide kills the pest but does not affect the plant. In practice, most fungicides affect the plant if over-applied. They can cause growth retardation, abnormal development or chromosomal damage. Excessively high concentrations of methoxyethyl-, ethylene-, phenyl- or octyl-Hg cause retardation or inhibition of the growth of the seedling, while methyl-, ethyl-, butyl- or cyclohexyl-Hg cause swelling of the cotyle- dons and roots (Gassner, 1951). With sufficiently high concentrations, the leaves become ball-formed and the roots tuber-like. Treatment of onion roots by methyl-, phenyl- or methoxyethyl-Hg causes hook-like malforma- tions (Ramel, 1969) and treatment of germinating wheat and rye grain with a 20 to 100 ppm solution of ethyl-HgCl causes swelling of the seedling and its root tips (Kostoff, 1939). According to Kostoff (1940), Bruhin (1955) and Ramel (1969), mal- formations like those described above are due to the c-mitotic effect of organic mercury compounds. In metaphase, the chromosomes are not organized at the equatorial plate but are random in the cell. Chromosomes are split and the sections are drawn apart, but they are not conveyed to the poles, evidently due to the lack of achromatic figures. The chromosomes are divided but the cell is not. The methyl and phenyl derivatives are especially c-mitotic. Addition of 2, 3-dimercaptopropanol (BAL) eliminates nearly completely the c-mitotic effect of phenyl-Hg. The compounds may cause their c-mitotic effect through a direct action on the sulphhydryl groups of those proteins actually involved in the formation of the spindle and in this way prevent them from polymerizing. Possibly, the mercurials may also react with disulphide groups already formed. In the light of this, the protective effect of BAL is logical (Ramel, 1969). Methyl-Hg and phenyl-Hg derivatives are the most effective c-mitotic compounds known. They cause a significant increase of c-mitoses in the roots of Allium cepa after 72 h at a concentration of 2 x 10_7M. The less effective methoxyethyl-Hg requires a concentration of 30 x 10"7M, while the concentration of inorganic Hg needed is 10'5M. Only slightly higher concentrations than the above will cause cell death.

2.3.3. Animals

In this connection are discussed the uptake, distribution and, to some extent, effects of only those mercury fungicides which are agriculturally important and in only those species which can be exposed to them, namely seed-eating birds and mammals, predators living on these animals and man. Since the toxicity of mercury compounds to animals and man is dealt with in Chapter 6 of this book, only a brief discussion is given here.

15 a. Birds

Seed dressed with mercury fungicides, especially methyl mercury, constitutes a great danger to seed-eating birds and birds of prey that feed on them. This was indicated by deaths of seed-eating birds in agricultural areas of Sweden during the 1950s. Borg (1958) was the first to suspect alkyl mercury fungicides as the cause of these deaths. Later, this was proved to be true (Borg et al., 1965). In Canada, birds from regions where the use of mercury seed dressing is widespread contain 8-10 times higher concentra- tions of mercury than birds from areas where seed treatment is much less common (Fimreite et al., 1970). About 0. 9% of seeds remain uncovered in the spring and are available to seed-eating wild birds. Sometimes farmers have ignorantly given seeds treated with Hg fungicides to hens (Tejning and Vesterberg, 1964), even though such seeds are always marked with the red fluorescent Rhodamine B as a warning. Some farmers have attempted to remove mercury from the dressed seed by washing them with water, however, this procedure removes only 3% of the mercury in Panogen-treated seed and 53% in dust-treated seed. In Sweden in the early 1960s, attention began to be focused upon birds of prey that were unable to fly. Predators consuming seed-eating birds seem to have been more exposed than those which prey largely on mice and other small mammals (Borg et al., 1969; Fimreite et al., 1970). Only methyl mercury is effectively absorbed in birds — when given per os, only about 11% is excreted within 3 days (Tejning, 1967a). The excretion of different mercury fungicides was studied by Swensson and Ulfvarsson (1968). Ten days after their administration by a single injection, 20% of methyl-Hg, 60% of Hg(N03)2, 80% of phenyl-Hg and 90% of methoxyethyl-Hg had been excreted. The biological half-time of methyl-Hg in the hen is reported to be 23 - 27 d, while that of methoxyethyl-Hg is 2 - 5 d (Swensson, 1967). These values may be too low, since the excretion was followed for only a short time. The distribution of methyl, phenyl, methoxyethyl and inorganic mercury in quail was studied by Backstr&m (1969) using whole-body autoradiography. Methyl-Hg is distributed rather evenly throughout all tissues. High con- centrations are found in the , , spleen, oviduct, albumin, blood, brain, muscle and epithelium of the mouth. Differences in the distribution of mercury in quail after injection of the above four mercury compounds are shown in Figs 1 to 4. The blood: brain ratio is 1: 1 and slowly increases with time (Swensson and Ulfvarsson, 1968). The other three compounds (phenyl-, methoxyethyl- and inorganic Hg) are effectively accumulated only in the kidney, liver, growing follicles, germinal disk and intestinal mucosa. The chronic intake by hens of seeds dressed with mercury fungicides showed that the ingestion of methyl mercury lead to the highest concentration of mercury in eggs. In decreasing order of mercury concentration were phenyl, methoxyethyl and inorganic mercury. An intake of 0. 3 mg Hg/day (as CHgHg) results in 0. 3 mg Hg/egg; ingestion of 1. 6 mg Hg/ day gives 1. 3 mg Hg/egg (Tejning, 1967a; Kivimae et al. , 1969, 1970). The methyl- Hg is found mostly in the egg-white, the ratio yolk/white being 0. 1 - 0. 2 (Smart and Lloyd, 1963; Kivimae et al., 1970) and about 80 - 90% of the total mercury in the egg-white is in the form of methyl-Hg, when this is the compound ingested (Kivimae et al., 1969). Other mercury fungicides are located mainly in the yolk, e. g, 95 - 97% of methoxyethyl-Hg appears in the

16 1234 5 6 7 8 9

10 11 11

FIG. 1. Distribution of mercury (white areas) in a quail 4 days after an i.v. injection of methyl mercuric nitrate. An isotope staircase has been placed below the autoradiogram. 1 brain; 2 thymus; 3 heart blood; 4 lung; 5 liver; 6 follicular yolk; 7 kidney: 8 yolk in magnum of oviduct; 9 uterine part of oviduct; 10 crop; 11 pancreas; 12 intestines. (BackstrBm, 1969).

12 3 4

o

FIG.2. Distribution of mercury in a hen quail 1 day after an i.v. injection of phenyl mercuric nitrate. 1 thyroid; 2 lung; 3 follicle, accumulating yolk: 4 kidney: 5 pancreas. (BSckstrQm, 1969).

17 1 2 3 4

FIG.3. Distribution of mercury in a hen quail 4 days after an i.v. injection of methoxyethyl mercuric hydroxide. 1 brain; 2 adrenal; 3 kidney; 4 yolk in oviduct; 5 heart; 6 liver; 7 spleen; 8 oviduct; 9 pancreas. (Backs trOm, 1969).

BRAIN KIDNEY UROPYGIAL GLAND

EGG WHITE EGG YOLK

FIG.4. Distribution of mercury (light areas) in a hen quail 4 days after an i.v. injection of mercuric nitrate. Note the accumulation of mercury in kidney, yolk and pituitary. A ruptured follicle has spread mercury- containing yolk in the abdominal cavity. (BackstrSm, 1969).

18 * * MORE THAN 2mg OF MERCURY PRO kg LIVER o o MORE THAN 5mg OF MERCURY PRO kg LIVER •/. D • MORE THAN 10 Tig OF MERCURY PRO kg LIVER i10(

NUMBER OF BIROS

FIG. 5. Seasonal variation of mercury levels in seed-eating birds found dead in 1964. (After Borg et al., 1969).

yolk (Helminen et al., 1966; Tammisto et al., 1968), the ratio yolk/white being about 1. 5 with low (0. 4 mg Hg/day) intake and 2-3 with high (1. 6 mg Hg/day) intake. The ratio for phenyl-Hg is 2 and 10 at the same respective feeding levels (Kivimae et al., 1970). Eggs and feathers are important routes of methyl-Hg elimination, the latter especially in cocks (Tejning, 1967a). Hens fed methyl-Hg at the rate of 1. 6 mg Hg/day die within 32 - 56 days, while methoxyethyl-Hg, phenyl-Hg and inorganic Hg can be fed at this rate for at least 140 days without symptoms (Helminen et al., 1966; Karppanen et al., 1968; Swensson and Ulfvarsson, 1969; Kivimae et al., 1970). Feeding methyl-Hg at the rate of about 8 mg Hg/day produces symptoms in 2 weeks and death in 3 weeks. A dose of 0. 4 mg Hg/day does not produce symptoms within 140 days. The range from non-symptomatic to is thus very narrow, 0.4 to 1.6 mg Hg/day or four-fold, which is typical of methyl mercury. Chicks are more sensitive to methyl-Hg than hens, 0. 1 - 0. 2 mg Hg/day retarding growth and increasing the death rate five-fold (Fimreite, 1970b). The first symptoms of methyl mercury in birds are reduced food consumption and weakness of the extremities. The co-ordination of muscle function is poor, is evident, and the birds cannot walk or fly. At the end stage, the birds may lay on their chest in opisthotonus or on their back in a comatose state. At , a reduction in the amount of fatty tissue and atrophy of the muscles are noticed. Histological study reveals focal demyelinization of the cerebellar medulla and medulla oblongata, as well as degeneration of nerve cells (Borg et al., 1965, 1969, 1970). Mercury fungicides affect bird reproduction by reducing egg production. Eggs are also more frequently laid outside the nest (Tejning, 1965, 1967a; Karppanen et al., 1968; Kivimae et al., 1970). A methyl mercury concentration in hen's egg of 0. 3 mg Hg/egg may allow the development of a live chicken, but 0. 6 mg Hg/egg kills the embryo within 10 days (Tejning, 1967a). Borg et al. (1965, 1969) found a reduced hatchability of eggs containing 1. 3 - 2. 0 mg Hg/kg, after feeding pheasants with methyl-mercury-treated grains. Only methyl mercury has been proved to have an effect on egg hatchability.

19 Of mercury compounds that have been used in agriculture, only the alkyl-Hg compounds seem to cause a substantial risk to birds. Study of bird feathers obtained from museums shows that, in Sweden, Hg-concentrations began to increase in 1940 - 1950 along with the increased use of methyl- mercury fungicides (Berg et al., 1966). In Sweden (1964) half of the seed- eating wild birds and 2/3 of the birds of prey contained more than 2 ppm of Hg, mostly as methyl-Hg, in their liver (Borg et al., 1969). Seasonal peaks in seed-eating bird deaths occurred in spring and autumn, a few weeks after sowing (Fig. 5). Increased Hg levels in birds have been found in Norway (Holt, 1969), Canada (Fimreite et al., 1970), and Finland (Karppanen et al., 1970). In Sweden, a marked decrease in the numbers of several bird species was noticed in the 1950s and 1960s (Larsson, 1970). The use of methyl mercury was restricted in Sweden in 1965 and forbidden in 1966, and a marked reduction of the Hg-level in birds was soon observed (Wanntorp et al. , 1967; Tejning, 1967b). The ecological risks connected with the agricultural use of methyl mercury are considerable. Methyl mercury is very persistent in nature and has a tendency to accumulate in food-chains. Much lower levels than those found experimentally toxic may be fatal in nature. When chronic accumulation occurs, it may reduce the ability of birds to gather food and to defend themselves. In addition to these somatic effects, methyl mercury reduces their reproduction.

b. Mammals other than man

Mammals can ingest mercury fungicides directly either by consuming the treated seeds (e. g. small rodents) or possibly by eating spray-treated stems and leaves, especially in tropical countries. Predators can contract indirectly by eating contaminated animals (birds or rodents). The elevated mercury levels found in small rodents living in Swedish fields and grain storage areas have been due to fungicide-treated seeds (Lihnell and Stenmark, 1967a). Increased mercury levels and symptoms of methyl mercurialism have been reported in some predators as well (Borg et al. , 1965, 1969). The feeding of domestic animals with left-over treated seed has resulted in intoxication and death of the animals (Curley et al., 1971). The absorption, distribution and elimination of a mercury fungicide depends primarily on the structure of the compound. Alkyl-Hg compounds (especially methyl-Hg) differ from aryl and alkoxyalkyl derivatives in that they are rather easily absorbed and evenly distributed in many tissues, without decomposition, whereas aryl and alkoxyalkyl derivatives are less easily absorbed, are decomposed largely to inorganic mercury and are accumulated (as such or as Hg) mainly in the kidneys and the liver (Swensson, 1967). The elimination of alkyl-Hg usually follows an exponential function and occurs rather slowly. By measuring the 203Hg content of excreta, Ulfvarsson (1962) determined the biological half-time of methyl-Hg in the rat to be 15- 20 days. Berlung (1969) obtained by whole-body counting a half-time of 40 days; however, about a third of the body burden was "eliminated" in the . Ostlund (1969) measured the half-time of CHjHg in mice "correcting for the hair", and obtained values which increased with the dose level: a

20 TABLE VI. METHYL MERCURY IN LIVER OF SMALL RODENTS IN SWEDEN (1964- 65)

Number of small rodents analysed Liver ' (mg Hg/kg) Borg et al. (1965) '<" Lihnell and Stenmark (1967b)

0-2.0 101 172

2.1-5.0 8 6

5.1-10.0 3 3

10.1 -20.0 2 2

20.1 -40.0 1 0

>40 1 1

Total 116 184

dose of 0. 03 mg Hg/kg body weight resulted in a half-time of 3. 7 days, 0. 3 mg Hg/kg in 5. 3 days, 1. 0 mg Hg/kg in 8. 4 days and 5. 0 mg Hg/kg in 12. 6 days. In the rat, 85 - 95% of the elimination takes place via the faeces (Ulfvarsson, 1969), and in piglets this value is about 80% (Platonov, 1968b). Aryl-Hg, alkoxyalkyl-Hg and inorganic Hg are eliminated quicker than alkyl-Hg. The elimination mechanisms of the two former types of compound are complicated by their simultaneous decomposition and, therefore, the elimination functions are not clearly exponential. In the rats, Ulfvarsson (1962) found "half-times" of 4 - 10 days for these compounds. A small portion of inorganic mercury is eliminated through the lungs and skin of rats (Clarkson and Rothstein, 1963). When the intake is chronic, the alkyl compounds are the most dangerous due to their high resorption and slow elimination. Methyl-Hg injected into the rat chronically at the rate of 0. 3 mg Hg per kg and per day does not produce any symptoms, while 0. 9 mg Hg per kg and per day produces typical symptoms within a few weeks (Berglung, 1969). The symptoms of methyl mercurialism in mammals are approximately the same as those in birds (Hanko et al., 1970; Piper et al., 1971). In the case of methyl-Hg, a level of 30 ppm Hg in the brain is always lethal. Such high levels are seldom found in nature (Borg et al., 1969). A polecat with cramps had 32 ppm Hg in its liver and 34 ppm Hg in its kidneys; a staggering fox that was running in circles, half-blind and without functioning olfactories, had 30 ppm Hg in its liver. Of 22 foxes studied in Sweden in 1964 - 65, 11 had more than 2 ppm Hg and 3 had over 20 ppm Hg in the liver. Swedish results on the alkyl mercury content of small rodents during these years, when the use of methyl mercury was maximal, are presented in Table VI. Of mammals, only small rodents can act as a significant link in the food-chain that transfers methyl mercury to predators. Since the mercury contents of small rodents have been rather low, it is easily understandable that birds of prey catching only rodents have lower body burdens of mercury than those catching only or mostly seed-eating birds.

21 c. Man

Man can absorb mercury fungicides via the lungs, skin or stomach. Personnel of fungicide factories, persons dressing seed and farmers handling treated seeds, sprays or dusts are subjected to the first two routes of intake. The adsorption of mercury fungicides through handling can be kept to a minimum by carefully following instructions and occupational safety regulations. The accumulation of mercury fungicides into foodstuffs is quite low if the normal precautions are observed. The mercury content of cereal grown from dressed seed is very low. Neither spraying nor dusting plants significantly increases the Hg content of the edible parts, provided that the treatment is not carried out when that part of the plant is developing. However, heavy treatment of rice plants with PMA in Japan has increased the Hg level of the Japanese people to about three-fold over the average of other populations (Fukunaga and Tsukano, 1969). Consumption of game bird is not common. Mercury fungicides are absorbed, distributed and excreted by man in the same manner as in animals. The half-time of the elimination of methyl mercury in man is 75 days (Aberg et al., 1969; Miettinen et al., 1971); that of inorganic mercury about 42 days (Rahola et al., 1971). It is thus evident that the use of mercury fungicides in agriculture does not constitute any serious health risk to man, so long as instructions and safety regulations are closely observed. In earlier decades, when organomercurials first came into common use, several severe accidents occurred due to the manufacturing or handling of these compounds. Kurland et al, (1960) give a list of such accidents, some of which caused death and some permanent severe disability. With the improvement of dressing techniques and safety measures, such accidents have disappeared. However, the mercury level in the blood of persons working in seed-dressing factories is higher than the average (Tejning, 1967c). The symptoms of methyl mercury poisoning and the resulting are described accurately by Hunter et al. (1940) and Hunter and Russel (1954). The symptoms of methoxyethyl mercury poisoning have been reported by Zeyer (1952). Aryl mercury have occurred only rarely, but some of the symptoms involved are described by Swensson and Ulfvarsson (1963). All commercial mercury fungicides, as well as sacked seeds dressed with Hg-compounds, are labelled to prevent their misuse as livestock feed. The mercury formulations even contain a red pigment, which makes it easier to distinguish between the treated and the untreated portions of seed. However, it has been verified that farmers occasionally may use left- over treated seed for animal feed, thus causing increased mercury levels in the meat of the animals, and also in the eggs of hens. Only alkyl mercury compounds administered in high concentrations have caused poisoning in chickens and hogs. Mercury poisoning of human beings as a result of eating contaminated chicken meat or eggs has not been reported. However, in New Mexico, four members of a family were seriously poisoned by eating the meat of a healthy looking hog fed with alkyl-mercury-treated seed (Curley et al., 1971). One of the victims was a newly-born delivered of an apparently healthy mother (Snyder, 1971).

22 Secondary alkyl mercury poisoning has for the present been restricted to narrow family circles, but in the New Mexico case, there was the possibility of an epidemic because some contaminated hogs had already been slaughtered and mixed with uncontaminated animals. In this and other similar cases, Federal action has prevented meat containing mercury residue from reaching the general public (USDA, Jan. 23, 1970). Hundreds of deaths have resulted due to the ignorant human ingestion of treated cereal seed. The precise number of fatalities, as well as permanent injuries, that have resulted can only be estimated because most of these accidents have occurred in developing countries, where relatively few of the patients received medical attention. In Sweden, three persons were poisoned by eating porridge made from treated cereal seed; one of those victims was the infant of a healthy appearing mother (Engleson and Herner, 1952). In the northern part of in 1956, many hundreds of persons were inflicted by eating bread home-made from ethyl mercury-treated cereal seed; 14 of the 100 patients hospitalized died. There was a similar incident in central Iraq in 1960; again, hundreds of persons became ill and many died (Jalili and Abbasi, 1961). In West Pakistan (Haq, .1963), more than one hundred persons were poisoned by the ingestion of bread made from wheat seed treated with ethyl and phenyl mercury compounds; many of these victims also died. From 1963 to 1965, many farmers and their families living in Guatemala were similarly poisoned by methyl mercury (Eyl, 1971). In the above-mentioned cases, the persons involved had been warned not to eat the treated cereal seed. However, these persons, most of whom were undernourished, ignored the warning. In some cases, attempts had been made to decontaminate the seed by washing. In other cases, the treated seed was fed to chickens in order to see what effect it had on them; when no apparent illness occurred after a few days feeding, some of the farmers themselves began to eat it. When these farmers remained apparently healthy after a few weeks consumption of the treated cereal seed, even the people who had been initially cautious began to ingest it (Jalili and Abbasi, 1961). The long latency of alkyl mercury poisoning proved fateful for these people.

2.4. ADSORPTION OF MERCURY FUNGICIDES BY SOIL AND THE NATURAL Hg LEVELS

A small portion of the mercury compounds used in treating the seed or plant is always accumulated in the soil, either directly or by the decom- position of plant material. Since there is a wide variation in the natural mercury content of soil, it is difficult to estimate the often small amount of mercury added to the soil by agricultural treatments. Not only the natural geological level of mercury in the soil must be considered (Warren and Delavault, 1969), but also the amount added by air contamination from nearby towns and cities (Stock and Cucuel, 1934). The increased mercury content of arable land is not always due to agricul- tural treatment alone; the influence of variations in pH (addition of lime), humus content and hydrological factors (draining) (Andersson, 1967a) also have some effect. In Germany (ca. 1930), the mercury content of cultivated land was 30 - 140 ng Hg/g, while that of uncultivated land far from population centres was 30 - 81 ng Hg/g and near to these centres 100 - 290 ng Hg/g (Stock and

23 Cucuel, 1934). Andersson (1967a) determined the mercury content of African soils to be 11-41 ng Hg/g (avg. 23 ng Hg/g), while the corresponding value for Swedish topsoil was 4- 922 ng Hg/g (avg. 60. 1 ng Hg/g). Cultivated Swedish soil contained an average of 64. 2 ng Hg/g, while the uncultivated soil averaged 56. 4 ng Hg/g. The use of alkyl mercury compounds in agriculture has been estimated to contribute about 5 - 10 ng Hg/g to Swedish soil since 1940. Soil suspected of being polluted by mercury must always be analysed for humus content, because only soils rich in humus may naturally contain more than 150 ng Hg/ g. The average mercury level of Swedish topsoil, 60. 1 ng Hg/g, corresponds to about 150 g Hg/ha (Andersson, 1967b). It has been calculated that Swedish soil receives about 1. 2 g Hg/ha annually due to rainfall; this amount is approximately the same as that contributed by the use of mercury in agriculture. The mercury level of rain-water is quite high: 200 ng Hg/litre (Stock and Cucuel, 1934). The use of 5 - 10 tons/ha (dry wt) of animal manure as fertilizer adds about 1 - 2 g Hg/ha. Conversely, the use of equal amounts of sludge from treatment plants would add 30 - 290 g Hg/ha, a level far exceeding that contributed to the soil via rainfall, the use of mercury fungicides and manure (Andersson, 1967b). The natural distribution of mercury is the same in profiles of both cultivated and uncultivated soils. The mercury content of subsoil is usually very low (2- 10 ng Hg/g), while topsoil contains 5-10 times this amount. The concentration of mercury in the topsoil, seemingly in the humus fraction, is probably due to rain water, plant matter and ground-water (Andersson, 1967a). The chemical properties of the soil particles are of great significance. Mercury residues resulting from the use of fungicides are normally 203 bound in the upper 10-cm soil layer (Ross and Stewart, 1962). When HgCl2 was placed at a depth of 5 cm in a pot of topsoil, which was then exposed to the environment and had some vegetative growth on the surface, after one year most of the radio-mercury was found over a depth range of 2. 5 to 12. 5 cm. Only 0. 01 - 0. 02% of the added radio-mercury was eluted by rain-water (Andersson, 1967a). CHgHg compounds bound in soil are removed slowly by rain-water, plants and evaporation. After 5 months, 51 - 53% of the original amount remains bound in the soil (Saha et al., 1970). Methyl-, ethyl- and phenyl-Hg compounds in water solution are effectively adsorbed by soil. When the pH of the solution is lowered to 1.5-2.8, or increased to 8. 8 - 10. 5, the adsorption is decreased (Miller et al., 1957). The distribution of inorganic Hg between the organic and the mineral material of soil is a function of pH. The lower the pH, the more Hg is bound in the organic material (Andersson, 1967a). In Japanese rice fields that have received extensive treatment with phenyl mercury fungicides, significant amounts of mercury residue have been found, not only in the topsoil, but also at greater depths, or not at all, indicating the movement of mercury to deeper strata (Aomine et al., 1967). This fluctuation in Japanese results has been explained by variations in the clay content of the soil and the minerological composition of the clay fraction. The chemical form of the mercury compound also effects the amount of mercury adsorbed into the clay. PMA is adsorbed well into montmorillonitic clay, but poorly into allophanic and kaolinitic clays; on

the other hand, HgCl2 is adsorbed well into allophanic clay but poorly into montmorillonitic clay.

24 The desorption of the respective mercury compound is opposite to the magnitude of adsorption (Aomine and Inoue, 1967; Inoue and Aomine, 1969). The adsorption maximum of PMA in clay occurs at pH 6 and the adsorption follows the law of cationic exchange.

2. 5. DECOMPOSITION REACTIONS OF MERCURY COMPOUNDS

The alkyl derivatives are the most chemically stable of the mercury compounds used in agriculture. Aryl and alkoxyalkyl compounds are more easily decomposed, both chemically and enzymatically. This decomposition, which takes place in soils and in plant and animal tissues, often liberates ionic or metallic mercury.

2. 5. 1. Microbial decomposition

In this section, microbial decomposition occurring in soils is of the greatest interest. Booer (1944) noticed that all mercury compounds de- compose in soil and "volatilize", either as such (ethyl mercury) or as metallic mercury. He did not measure the disappearance of mercury chemically but by the growth inhibition of wheat seedlings, i. e. he measured the inactivation ability rather than the true volatilization. Spanis et al. (1962) isolated Penicillium and Aspergillus spp. from soils and conditioned the isolates to withstand normally lethal concentrations of 2-chloro-4-(hydroxymercury). The isolates were able to "inactivate" the fungicide. Bacillus spp. were similarly able to withstand and inactivate cyano-()guanidine. The authors assumed that the organisms produce intra- or extracellular enzymes which decompose the mercury compounds and then use the chains for their own growth. The grew in a deficient nutrient media better with methyl mercury than without it. Kimura and Miller (1964) studied the decomposition and volatilization of methyl-, ethyl- and phenyl-Hg compounds in soil and published chemical data on the formation of metallic mercury in non-sterilized soil. Phenyl mercury acetate was 14- 16% volatilized as metallic mercury vapour in 28 days; ethyl mercury acetate was 21 - 32% volatilized (as ethyl mercury and metallic mercury vapour) in 53 days. Methyl derivatives were not decomposed but 6 - 14% were volatilized as CH3Hg in 35 days. Several Japanese authors (Tonomura et al., 1968a, c; Tonomura and Kanzaki, 1969; Furukawa et al. , 1969) have studied the action on mercury compounds of a Hg-resistant Pseudomonas. which was isolated from a PMA- contaminated rice-field. The bacterium volatilized metallic mercury from solutions containing methyl, ethyl or phenyl mercury. Methane, ethane and , respectively, were also liberated and identified by means of chromatography. Cleavage of the C-Hg bond in phenyl and ethyl mercury was catalysed by enzymes present in the cell-free extract which required both a sulphhydryl compound and NADH (Tonomura and Kanzaki, 1969).

2. 5. 2. Decomposition by plants

Sumino (1968) found that PMA sprayed onto the leaves of rice plants was rapidly decomposed; during the first 2 weeks, the phenyl-mercury content

25 was only 5 - 20% that of the total mercury. Growing wheat root can also decompose PMA (Isobe et al., 1967).

2. 5. 3. Decomposition by animals

a. Birds The decomposition of methyl mercury in birds has not thus far been observed (Borg et al., 1970); ethyl, methoxyethyl and phenyl mercury, however, are known to be partially decomposed to inorganic mercury in the liver and kidneys of the hen (Miller et al., 1960, 1961). BSckstrBm (1969) confirmed the decomposition of PMA by using molecules labelled with 14C or 203Hg.

b. Mammals Ostlund (1969) showed that even methyl mercury is slightly decomposed 14 in mice. About 4. 2% of the "C activity of injected CH3HgOH is exhaled within 24 hours. 203Hg-labelling of this compound did not result in exhaled 203Hg activity because the main excretion route of mercury is via the faeces. After exposure of the rat to methyl mercury chloride, Norseth (1969) found that about 40% of the total mercury excretion, 50% of the faecal excretion and 5 - 20% of the urinary excretion (which increased with time) occurred as inorganic mercury. Decomposition takes place primarily in the intestinal lumen of the rat coecum. Secondary sources of the inorganic mercury appearing in the faeces are the liver, pancreas, intestinal epithelium and secretion from above the pylorus. Ethyl mercury is also decomposed to inorganic mercury in both the rat and the pig, evidently by the kidneys (Platonow, 1968a; Miller et al., 1961). Phenyl mercury is detoxified very rapidly in the rat by decomposition to inorganic mercury; only 10% of the total mercury excreted in the within the first 48 hours was in the form of phenyl mercury (Miller et al., 1960).

Hg(N03)2 is eliminated from the rat in volatile form by an unknown mechanism (possibly through the lungs or skin) (Clarkson and Rothstein, 1964). This elimination may occur as metallic mercury.

2.6. CONCENTRATION BY FOOD-CHAINS

Since methyl mercury is eliminated from most organisms rather slowly, chronic exposure to this compound results in its accumulation at the end of food-chains, especially in predatory birds and mammals. Possible ter- restrial food-chains are presented in Fig. 6. The aquatic food-chains which lead to the accumulation of mercury in the seal and in man are not discussed here, as they are not likely to originate from mercury used in agriculture but rather from mercury of industrial origin. The number of terrestrial food-chains ending in man is somewhat mis- leading since only the food-chain, dressed seed game-bird man, can normally lead to the accumulation in man of mercury compounds used in agriculture. Crops grown from methyl-mercury-dressed seed may well contain slightly higher amounts of mercury than those grown from undressed

26 SEED-EATING •r BIRD - • EGG

SEED

SEED-EATING ^MAMMAL _

FIG. 6, Possible terrestrial food-chains. or methoxyethyl-mercury-dressed seed, but this increase is negligible (West&S, 1969). The misuse of agricultural mercury compounds may, however, lead to highly increased concentrations of mercury in man. The serious problem is that these mercury compounds are carried along the food-chains and concentrated in predatory birds and mammals. Little experimental food-chain research has been performed so far (and none using radioisotopes) in which actual food consumptions have been controlled. Borg et al. (1970), who studied the food-chain dressed seed -» seed-eating bird -> predatory bird, and Hanko et al. (1970), who investigated the food- chain dressed seed -» seed-eating bird - predatory mammal, have carried out controlled-feeding experiments. In both studies, secondary intoxication was much faster than primary. Chickens fed seed containing 8 ppm Hg (as methyl mercury) were still seemingly healthy after 40 - 44 days, at which time the mercury levels of the muscle and liver were 10 and 40 ppm Hg, respectively. Goshawks fed a of these tissues (avg. Hg content: 10-13 ppm) died after 30 - 47 days and high concentrations of mercury were found in the muscle (40 - 50 ppm Hg), brain (30 - 40 ppm Hg) and especially in the testicles (up to 280 ppm Hg) (Borg et al., 1970). Ferrets fed a diet containing chicken tissue (avg. Hg content of diet: 5 or 7 ppm) died after 58 or 35 days, at which time the mercury level of the muscle was 30 or 40 ppm, respectively. About 30 ppm Hg was found in the brain (Hanko et al., 1970). Concentrations are given on the basis of fresh weight. The concentration factors observed in these experiments were:

dressed seed to chicken = ca. 2, chicken to goshawk = ca. 4-5. chicken to ferret = ca. 6.

It was found that methyl mercury is able to pass along a food-chain without splitting of the carbon-mercury bond. Muscle tissue plays the central role in food-chain transformations. Usually, 60 - 80% of the total body burden of mercury is found in muscle tissue; the entrails are not commonly eaten, at least not by most predatory birds (Tejning, 1967a). Food-chain transformations are also emphasized by the fact that most predators prey on the weakest individuals and as methyl mercury poisoning first affects the extremities, the ability of the animal to escape is curtailed. Such disabled individuals may be caught even by predators which do not normally prey on that species.

27 2.7. RESIDUES IN VICTUALS

All plant and animal tissues contain traces of mercury, usually of the order of nanograms of Hg per gram of tissue. As the natural variations are great, it is not always easy to distinguish contaminated, levels from the natural ones. Furthermore, the analytical methods used in earlier times were not always sufficiently sensitive to determine the natural levels.

2.7.1. Residues in plant tissues

Mercury residue levels resulting from the fungicidal use of sprays or dusts on fruits and vegetables are extensively described by Smart (1968). Generally, the values are quite low compared with natural levels, provided that the treatment was not carried out during the growing season. Some natural levels (as ppm Hg) reported are: apples < 0. 02; pears 0. 04; tomatoes 0. 012 - 0. 044 and potatoes 0. 003. Approximately equal levels were reported by Stock and Cucuel in 1934. The mercury content (as ng Hg/ g) of wheat grown in different countries varied greatly in 1965 (Kim and Silverman, 1968): Brazil 75; Canada 34; France 250; Germany 81; India 24; Japan 150; Pakistan 35; the UAR (Egypt) 37; the United Kingdom 84 and the USA 20 - 68 (range for 12 wheat-growing states). Japanese rice is the only commercial plant product in the world that has had a clearly elevated Hg level. This resulted from the accumulation of Hg residues in soil and from the spray-treatment of growing plants. The mercury level of rice from treated fields has been about 100% higher than that from non- treated fields (Furutani and Osajima, 1967). Treated plants contained 0. 24-2.4 ppm Hg in the raw rice and 0.23- 0.8 ppm Hg in the polished rice, while non-treated plants contained, respectively, 0. 16- 0. 32 and 0. 23- 0.24 ppm Hg (Tomizawa et al., 1966). Ingestion of rice probably explains the fact that the average level of Hg in the hair of Japanese is three times higher than that of other populations. Rice from other countries has had a much lower Hg content, of the order of 0. 02 ppm Hg (Smart and Hill, 1968).

2.7.2. Residues in animals

Cereal grown from methyl mercury-treated seed, when fed to the hen, results in 0. 027 ppm Hg'in eggs, while cereal grown from methoxyethyl - mercury-treated or non-treated seed gives only 0. 010 - 0. 012 ppm Hg in eggs (WestbO, 1969). This explains why, in 1964, hen eggs in Sweden and Norway (where methyl-mercury-dressed seed was extensively used) contained on the average 0. 029 and 0. 021 ppm Hg, respectively, whereas eggs in other European countries contained only 0.007 ppm Hg (Underdal, 1968; WestaQ, 1969). After the use of methyl mercury in seed-dressing was forbidden in Sweden (1966), the Hg content of eggs rapidly decreased to 0.009 ppm Hg (1967). The feeding of domestic animals with Hg-treated grain is strictly forbidden in all countries. If misuse occurs, however, then the mercury content of victuals may become quite high, especially in the case of alkyl mercury (Curley et al., 1971). Even the normal use of alkyl mercury fungicides causes elevated levels of Hg in game birds (Sweden: Borg et al., 1965; Ireland: Eades, 1966; Norway: Holt, 1969; Finland: Karppanen et al., 1970; Canada: Fimreite et al., 1970). Alkoxyalkyl derivatives may

28 also cause mercury levels in game-birds that slightly exceed the food- hygiene standard of 0. 05 mg Hg/kg (Helminen et al., 1966) but the risks to man are likely to be low. High levels of methyl have recently caused concern in many countries. This is usually of industrial origin but even natural levels of 0. 3 - 1. 0 ppm Hg have been found in pelagic species. Although mercury levels in fish are not discussed in this chapter, an exception is the high levels of ethyl mercury found in some river fish in Japan (Ui, 1970; Aoki, 1970). This may be partially of agricultural origin since large amounts of ethyl mercury were used in Japan for the seed-dressing of rice. As river water is extensively used in the irrigation of rice fields and as excess water returns into the river, some agricultural ethyl mercury may have entered the river system. However, industry is not excluded as a source of this ethyl mercury.

2.8. CONCLUSIONS The agricultural use of mercury fungicides is relatively harmless for man if the proper safety measures are followed. Mercury fungicides are also the most effective seed-dressing agents and their use increased rapidly after the Second World War, when a shortage of food existed in most countries. After 10- 15 years of continued heavy use, however, severe pollution in agricultural regions became evident where alkyl mercury compounds had been used. In Sweden, for instance, the use of methyl mercury began about 1940 and the first bird deaths due to mercury poisoning were observed approximately 10 years later. During the ensuing decade (1950s) these deaths became more common. Methyl mercury was first suspected as their cause in 1958 (Borg, 1958), but this view was not officially accepted until 1965. At the beginning of 1966, Sweden, closely followed by the other Scandinavian countries, forbade all agricultural use of methyl mercury. In 1970 it was also forbidden in the USA and in Canada, even though the Hg level in seed-eating birds was relatively low. The following reason for the suspension was given by the USDA in a news release (USDA, March 9, 1970): "Directions for proper use and caution statements in product labels have failed to prevent misuse of the treated seed as livestock feed". This occurred after the New Mexico poisonings (Curley et al., 1971). The consumption of pheasants was also forbidden in some states (Saha et al., 1970). Thus, it seems likely that the agricultural use of alkyl-mercury compounds will be discouraged in many countries. Two risks .are involved, especially with the use of methyl mercury: the poisoning of natural food- chains and the misuse of treated seed as livestock feed. Much less risk is involved with the use of alkoxyalkyl and aryl-mercury compounds. The use of these compounds for seed dressing is therefore allowed in all countries except Japan where their use has been forbidden in rice-disease control since 1968. The Japanese situation is exceptional, however, because the heavy use of phenyl mercury in particular has increased the mercury levels of rice fields and rice grain to such an extent that the average mercury body burden of the Japanese is about three times greater than in other populations (Fukunaga and Tsukano, 1969). The tragic accidents at Minamata and Niigata, caused by the consumption of fish

29 contaminated by methyl mercury of industrial origin, have raised fear to the extent that worried people have repeated the phrase: "All those who live on rice are sure to contract the " (Sumino, 1968). Attempts have been made to replace even the alkoxyalkyl- and aryl- mercury compounds by mercury-free fungicides, but no compound with an equally wide fungicidal spectrum has been found so far. The suspension of seed dressing with alkoxyalkyl-mercury compounds is neither possible nor necessary. Even slight neglect of seed dressing causes the occurrence of fungus disease in cereal, at least in the Scandin- avian climate. This was demonstrated in Sweden and Finland in 1968, when farmers left even contaminated seed partially undressed because of the wide- spread publicity concerning mercury-polluted fish (Lihnell, 1969). A portion of the harvest was so diseased that it could not be accepted in mills or fed to domestic animals. A marked increase in cereal yield is obtained through seed dressing with methoxyethyl-mercury fungicides. In Finland, the winter rye yield has been increased 19. 4%, winter wheat 6. 1%, spring wheat 6. 6%, barley 7.9% and oats 12. 5% (Jamalainen, 19 54). Seed dressing has substantially reduced the disease frequency due to the snow mould Fusarium nivale, as well as that of other fungi for which preventive agents have been developed. Alkoxyalkyl-mercury compounds are not significantly concentrated within food-chains. Bird deaths caused by their use have never been detected, although a slight increase in the mercury level of birds has been observed. Apparently, this increase is insignificant relative to the great benefits which are obtained from their present use in agriculture.

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WR<3BEL, T., Influence of fungicides on symbiosis of Rhizobium with pea and lupine. Acta Microbiol, Polon. 12 (1963) 203.

YAMADA, T., Uptake of phenylmercuric acetate through the root of rice and distribution of the mercury in rice plants, Nippon Nogeikaguku kaishi 42 (1968) 435 (in Japanese).

ZEYER, H.G., Methoxyathylquecksilberoxalatvergiftung, Zentbl. ArbMed. ArbSchutz 2 (1952) 68.

34 3. USE OF MERCURY AND ITS COMPOUNDS IN INDUSTRY AND MEDICINE

N. SAITO

3.1. INTRODUCTION

Apart from its use in agriculture, mercury is extensively used in industry, medicine and in the laboratory. It is used in the electrolytic production of chlorine and caustic soda, in catalysts, electrical appliances, instruments, pharmaceuticals, paints, amalgams, dental preparations, the pulp and paper industry and in general laboratory use. However, certain uses of mercury have recently been abandoned since increasing consumption of this element brought about the risk of accumulation of mercury in man and his environment beyond the permissible levels. For this reason, up-to-date statistics on the consumption of mercury on a world-wide basis is difficult to compile, but some estimates may be made from the figures [ 1] in Table VII on the uses of mercury in industrialized countries.

TABLE VII. CONSUMPTION OF MERCURY IN THE USA IN 1969 IN THOUSANDS OF POUNDS

Electrolytic chlorine 1572

Electrical apparatus 1382

Paints 739

Instruments 391

Catalysts 221

Dental preparations- 209

Agriculture 204

General laboratory use 126

Pharmaceuticals 52

Pulp and paper making 42

Amalgams 15

Other 1082

Total 6035

Note: Consumption based on 76-pound flasks. Source: US Department of Interior, Bureau of Mines.

35 3.2. USES IN CHEMICAL INDUSTRY

Several chemical processes use mercury and its compounds on a large scale. The importance of the use of mercury in mercury-cell chlorine- alkali production is shown by the fact that, in 1969, more than 25% of the 6 million pounds of mercury consumed in the USA went into the mercury process [ 1] . In this process, chlorine and caustic soda are produced by the following reactions:

NaClaq • Cl2 ()

NaClaq »- NaHgx (mercury ) electrolysis

NaHgx - NaOH + xHg + |H2

Figure 7 shows a typical flow sheet [ 2] of the process in a Swedish plant. Mercury is employed as a mobile cathode in a and the formed by electrolysis is decomposed by water in a secondary cell to give caustic soda and hydrogen. In such a chlorine-alkali production plant, a small part of the mercury used in the electrolytic cell is lost from the plant with the chlorine, hydrogen, caustic soda, brine, hydrogen condensates, ventilation systems and effluents. The loss of mercury quoted in the literature is of the order of 150 to 200 g per ton of chlorine produced. According to the Federal Administration

FIG. 7. Plant for the production of chlorine and caustic soda by the mercury process.

36 FIG.8. Modified chlorine plant.

in the USA, American producers of chlorine and caustic soda purchased 1. 3 million pounds of mercury in 1968 in order to just maintain inventory [ 1 ]. Very recently the discharge of mercury from chlorine plants into lakes and rivers has become an object of public concern [ 1, 3-5]. For instance, the St. Clair River-Lake Erie water system was polluted with the mercury discharged from chlorine plants operating in this area, and fish and bottom sediments in this water system were found to contain unusually high levels of mercury. Consequently, Canadian and state governments have banned fishing or warned against eating fish from these waters. The loss of mercury from chlorine plants can be minimized by the modification of the plants. As an example, Swedish industry, in collabo- ration with the Swedish Water and Research Laboratory, has taken the necessary measures to prevent the discharge of mercury to the environment. Figure 8 is a flow sheet of the process in a modified Swedish plant. The loss of mercury from the modified plant [ 2] is shown in Table VIII. As may be seen from this table, the loss of mercury from the modified plant is much less than the amount quoted in the literature. Installation of settling basins and systems in existing plants is effective in reducing the discharge of mercury into effluents from these plants [ 1 ]. A plant in Michigan, USA, which adopted these systems is reported to be in operation without any mercury discharges in waste waters.

37 TABLE VIII. LOSS OF MERCURY FROM THE MODIFIED CHLORINE PLANT

Source Grams of Hg/ton of Cl2 Remarks

Chlorine <0.5

Hydrogen 1 Cooling to < +5°C

Caustic soda 1 After filtering

Brine, precipitation <2 Vacuum salt used sludge

Brine, Nil sludge

Hydrogen condensate 0 Returned to denuder

Other effluents <5

Ventilation system Variable

Mercury sulphate is an excellent catalyst for the hydration of to give :

0^2+HzO CHgCHO HgS04 catalyst

Hg2+Hg Hg2+ (regeneration of Fes+ the catalyst)

Mercury chloride loaded on active charcoal is a good catalyst for the production of vinyl chloride by the reaction:

CH = CH + HC1 — CH2= CHC1

HgCl2

Several years ago, the discharge of mercury from a chemical plant in Minamata, Japan, caused a serious social problem [6], This plant is one of the large plants in Japan producing acetaldehyde and vinyl chloride. The amount of the mercury catalysts used in this plant was estimated to be about 200 tons (as Hg) during the period 1960 to 1964. The mercury discharged into from the plant was accumulated in fish and the bottom sediment in the bay. Some of the inhabitants in this area who had eaten fish from the bay were found to suffer from a strange disease which is now called "Minamata disease". Careful investigations by a Japanese medical group revealed that the patients of the Minamata disease show typical symptoms of poisoning with methyl-mercury compounds. The producer operating the plant has already taken the necessary steps to modify the facilities of the plant in order to minimize the release of mercury into the bay. Figure 9 shows a flow sheet of the process used in the modified plant. As shown in this flow sheet, the drain from the first is not released to the effluents but recycled to the reactor so that no mercury is discharged into the environment. A recent survey indicates

38 1 REACTOR 2 EVAPORATOR 3 FIRST RECTIFIER i DRAIN TANK 5 COOLING UNIT E SECOND RECTIFIER

FIG. 9. Modified plant for the production of acetaldehyde.

that the average mercury content of the effluents from the modified plant is of the order of 0.01 ppm at the highest. However, the mercury levels in the bottom sediment in the bay still range from several tens to several hundred ppm. Although methyl mercury was not detectable in the sediment, the levels of methyl mercury in fish caught from the bay in August 1969 ranged from 0. 01 to 0.3 ppm (calculated as Hg) t 7]. In the dyestuff industry, mercury sulphate is used as a catalyst for introducing sulphonic acid groups into the anthraquinone nucleus by the reaction:

iSOoOH oleum

HgSQ4

Acetyl mercury sulphate is used as a catalyst to synthesize vinyl acetate.

39 3.3. USES IN ELECTRICAL APPLIANCES

Mercury has a variety of uses in electrical appliances. Metallic mercury is used in low-pressure mercury lamps, high-pressure mercury arc lamps, ultra-high pressure mercury lamps, mercury switches, mercury-arc rectifiers, hot-cathode mercury-vapour tubes (, phanotron) and pool-cathode mercury-arc tubes. In the manufacture of dry cells a small amount of mercury chloride is added to an consis- ting of chloride and zinc chloride. The is basically a dry-cell-type battery consisting of a cathode of mercuric mixed with graphite, a zinc anode, and a solution saturated with . The overall electrochemical reaction is: Zn + HgO • ZnO + Hg

3.4. AMALGAMATION AND AMALGAMS

Mercury forms amalgams with a variety of metals. The amalgamation process used to be a method of extracting gold and from their , but it has been largely replaced by the cyanide process. In chemical laboratories, some amalgams, such as sodium and zinc amalgam, are used as reducing agents in chemical analysis and organic syntheses. The amalgams of a few metals can be used to make . An consisting of Hg (4 - 8%), (3 - 6%) and lead melts at about 250°C and can be used as a solder.

3.5. USES IN PULP AND PAPER INDUSTRY

In paper mills, such mercury compounds as ethyl mercury phosphate, phenyl mercury acetate and pyridyl mercury acetate are employed as slimicides. In some countries, the use of mercury compounds in paper mills was banned, as such use brought about the risk of accumulation of mercury in the biosphere. In Sweden, for instance, the use of phenyl mercury compounds was discontinued in 196 7 [8]. Phenyl mercury • compounds can also be mixed with paraffin for the production of paraffin paper which is used to protect fruit from germs.

3.6. USES IN PAINTS

Several mercury compounds are added to some antifouling paints for coating the bottom of ships. The most important is mercury oxide, either red or yellow. Phenyl mercury acetate is added to mildew-proofing paints as a . The latter compound can also, be added to latex-emulsion paints to prevent its deterioration during storage in the can.

3.7. GENERAL LABORATORY USES

Mercury is used in mercury diffusion pumps, barometers, manometers, thermometers, mercury-pool , mercury air pumps, mercury

40 porosimeters, mercury jet , dropping mercury electro'des, mercury coulometers, mercury-sealed stirrers, Toepler pumps and in McLeod gauges. The element is suited also for the production of standard cells. The , one of the important standard cells, contains mercury, amalgam and a layer of mercury (I) sulphate. A number of mercury compounds are frequently used as reagents in chemical experiments. The can also be employed as a vibration damper, a and a radiation shield. The hazard of mercury poisoning in laboratories is well known. A recent investigation in the UK [ 9] has indicated that mercury decontami- nation and changes in working procedures in a small industrial laboratory have a striking effect in reducing the mercury concentration in the head hair, body hair, finger nails and urine of laboratory workers.

3.8. DENTAL PREPARATIONS

Some amalgams (tin and silver amalgam, etc.) are used as dental fil- lings. Dentists and their assistants are subjected to a risk of mercury poisoning. A survey in the UK [ 9] by means of radioactivation analysis showed that the uptake of mercury in a group of 20 female dental-surgery assistants was higher than in a control group, although no clinical symptoms of poisoning were found.

3.9. USES IN MEDICINE

Mercuric chloride has been used as an for many years but has been largely replaced by less toxic chemicals. Mercuric oxycyanide is also used as an antiseptic. A number of organic mercury compounds are antiseptics. The popular compounds are (thimerosal; merthiolate; sodium ethylmercuri- thiosalicylate), and mercurochrome (; disodium 2, 7-dibromo- 4-hydroxymercurifluorescein). The important mercurials employed as are (sodium o- {[ 3-hydroxymercuri-2-methoxypropyl] carbamyl} phenoxyacetate) and chlormerodrin (a derivative of y-substituted /3-alkoxypropyl-mercuric acetate). The latter compound is prepared, as an example, by refluxing a solution of allylurea and mercuric acetate. Mercuric salicylate, mercuric succinimide and such inorganic com- pounds as mercury oxycyanide have been used as antisyphilitics, but most of these compounds have now been replaced by antibiotics. The ointments containing mercury include mercury oleate (yellow mercury oxide + oleic acid), mercurial ointment (mercury oleate + lanolin + white wax + petrolatum), ammoniated mercury ointment (amidomercury(II) chloride + lanolin) and mercuric oxide ointment (yellow mercury oxide + white ointment ). A compound called disodium mercury haematopor- phyrin was synthesized in Japan [ 10], It has been found effective as an antineoplastic agent. Phenyl mercury acetate finds its use as a spermicidal agent. Some soaps contain a minute amount of this compound as a skin antiseptic. Acetyloxymercuryhydroxyundecylate can be employed in the treatment of water-eczema.

41 However, the pharmaceutical industry in some countries has recently begun to make every effort to replace the mercurials by other chemicals which are less toxic than the mercury compounds.

3.10. OTHER USES

An interesting use of mercury in large amounts in the USA has been in mercury boilers in which the mercury vapour drives turbines in power plants. The metal is also used in the frozen mercury casting process [ 11]. Mercury is used as a primer for . Mercury sulphide is a red pigment called . Mercuric chloride is used in to intensify negatives. In the so-called mercury method, metallic mercury is employed to measure the softening point of -tar pitch.

REFERENCES TO CHAPTER 3

[1] Mercury stirs more pollution concern, Chem. Engng News 48 26 (1970) 36. [2] BOUVENG. H.O., ULIMAN, P., Reduction of Mercury in Waste Water from Chlorine Plants, Report of the Swedish Water and Air Pollution Research Laboratory, April 1969. [3] Mercury: widespread spillage, Chem, Engng News 48 29 (1970) 15. [4] Mercury: blast at industry, Chem. Engng News 48 32 (1970) 14. [5] Pollution: flurry of moves, Chem. Engng News 48 30 (1970) 18. [6] FUJII, M., "Mercury and environmental contamination", Chap. 6, Japan Public Health Association, (1970) 14. [7] Survey Report on Environmental Contamination with Mercury, Japan Public Health Association, Tokyo (1970), [8] WESTERMARK, T., LJUNGGREN, K., "Problems of analysis of individual mercury compounds", Table I (presented at IAEA Expert Meeting on Mercury Contamination in Man and His Environment, Amsterdam, 1967). [9] LEN1HAN, J.M.A., SMITH, H., "Activation analysis and public health", Nuclear Activation Techniques in the Life Sciences (Proc. Symp. Amsterdam, 1967), IAEA, Vienna (1967) 601, and NIXON, G.S., SMITH, H., J. Oral Ther. Pharmac. 1 (1965) 512. [10] 1IJ1MA, N., et al, Studies on chemotherapy (1) - Studies on the anti-neoplastic action of MH (haematoporphyrin-Hg) upon Ehrlich-carcinoma, Chemotherapy (Tokyo) 6 2 (1958) 121. [11] Frozen Mercury Casting Process, Am. Metal Market 61 185 (1954) 1,5.

General reference

Kirk-Othmer, Encyclopedia of Chemical Technology, 2nd edn, Interscience Publishers, New York (1963 - 1969).

42 4. BEHAVIOUR OF MERCURY IN THE ENVIRONMENT

S. JENSEN and A. JERNELOV

Metallic mercury and mercury compounds occur naturally in the environment but they are normally present at low levels. Besides these natural sources, increasing amounts are released from industrial wastes and from the use of organic mercurials as seed-dressing preparations and as slimicides in the paper and pulp industries. The most important discharges of mercury into the environment fall into two groups.

1. Inorganic mercury. Metallic mercury (Hg°) and inorganic mercury (Hg2+), including mercury sulphide (HgS).

2. Organic-bound mercury. Alkylmercury (RHg+), including methyl- + mercury (CH3Hg ) used as a seed-dressing preparation in agriculture. + + Aromatic mercury compounds (ArHg ), including phenylmercury (C6H5Hg ) used as a slimicide in the paper and pulp industries, as well as alkoxyalkyl- + + mercury (R-0-R'-Hg ), including methoxyethylmercury (CH3OCH2 CH2 Hg ) used as a seed dressing which is less harmful to seed-eating birds. Depending on the biological, chemical and physical conditions in the environment,these mercury compounds can be converted. In this chapter, these conversions are treated separately according to their inorganic or organic nature, even when they are only steps in a pathway leading to the most stable form under the existing circumstances.

Conversions between inorganic forms

2+ Hg „ Hg y HgS

> HgS: Mercury sulphide is a compound with an extremely low solubility product. If sulphide are present, mercury sulphide will be formed. Even if the theoretical solubility product of 10 "53 in water does not apply to a system where and competing complexing agents are present, the concentration of mercury ions (Hg2+) will be extremely low. Under anaerobic conditions mercuric sulphide seems to be stable, or it will be converted to the complex HgS2" if an excess of sulphide ions are present.

HgS > Hg2+: A direct biological effect on mercury sulphide — probably as an enzymatic oxidation — has been indicated (Fagerstrom and Jernelov, 1971). Normally the sulphur originally present as sulphide is slowly oxidized to sulphite and sulphate under aerobic conditions. This conversion of mercury sulphide to divalent ions (complexed to other com- pounds) makes the mercury available for further conversion.

43 Hg2* >Hg°: Reduction of divalent mercury may occur in nature under reducing conditions where the potential is in favour of this transformation. On the other hand, it has been shown that bacteria in the genus Pseudomonas can convert divalent mercury to metallic mercury under aerobic conditions (Yoshida, 1967, and Furukawa etal., 1969) as a method for detoxification. Under ideal conditions in cultures this conversion may be very rapid (close to 100% in a few hours). The extent to which this conversion occurs naturally and its significance for the turnover of mercury in the environment is not known.

Hg° > Hg2*: This is an oxidation reaction and, as such, dependent upon the redox-potential in the medium. The potential necessary for the oxidation of metallic mercury to inorganic divalent mercury can be calculated from the formula:

H 2+ tQtal E = 850 + 30 log S

where the coefficient a is a measure of the binding strength of the complexes between divalent mercury and the available complexing agents. For mercury complexes with organic mud the coefficient a has been calculated to be > 1021 (Werner, 1967). This means that oxidation of metallic mercury to inorganic divalent mercury will take place in an aquatic environment if organic substances and oxygen are present.

+ C2H3Hg

2+ + ArHg • Hg CH3 Hg .. CH3HgCH3 t CH30-CH2CH2-Hg

Conversions between organic and inorganic forms

+ 2+ CH3Q-CH2-CH2-Hg —> Hg : The decomposition of all alkoxyalkylmercury compounds can be understood by studying the decomposition of methoxyethyl- mercury. The compounds are all very unstable in acid media:

+ + 2+ CH3 -0-CH2-CH2 -Hg + H -» CH3OH + CH2 = CH2+Hg

At pHO the half-life of this reaction is about 10 min, and at pH2 more than 20% of the compound is broken down after 1 hour. This instability in acid media gives rise to the difference in accumulation between these types of organic mercurials and methylmercury. In humid soil (pH5) methoxyethyl- mercury has a half-life of 3 days.

44 + + + 2+ ArHg , CH3Hg , C2HgHg Hg : All known organo-mercury compounds can be degraded to inorganic mercury biologically, chemically and physically. (It is assumed here that under the influence of, for example, u. v. -light the degradation of organic mercury to elementary mercury occurs in a two- step reaction with inorganic divalent mercury as an intermediate product. ) The stability of the organo-mercurials, however, is very different. In general, it can be said that the short-chain alkylmercury compounds are the most stable and that the stability increases as the carbon chain decreases.

2+ + Hg *-C2H3-Hg : The biological formation of small amounts of has been reported by Kitamura et al. , 1969. However, the possibility of this being an artefact due to transalkylation activity of tetraethyl lead during the process of extraction has been discussed.

2+ + + (C2H5)4Pb + Hg »(C2H5)3Pb + C2H3 -Hg

2+ + Hg >CH3Hg : A biological methylation of mercury has been shown to occur in organic sediments from aquaria (Jensen and Jernelov, 1967 and 1969), fresh-water and coastal water of Sweden (Jernelov, 1968). Mercury- resistant bacteria with the ability to synthesize methylmercury have been isolated (Kitamura et al. , 1969, and Jernelov, 1969). Two different pathways for methylation of mercury have been described. Cell-free extracts of Methanobacterium smeliansky in the presence of methyl- cobalamin (B12-CH3) and ATP in a mild reducing environment have been shown to convert inorganic divalent mercury to methylmercury (Wood et al. , 1968). This methylation process is described as a non-enzymatic transport of the methyl group from methylcobalamin to the mercuric and an enzymatic regeneration of methylcobalamin. This process is a strictly anaerobic one. The other methylation process strongly indicated from in-vivo experi- ments with Neurospora crassa is an enzymatic methylation of mercury bound to homocysteine in the cell (Landner, 1971). The end-product of this process is methylmercury homocysteine or methylmercury , the mercury in both compounds being more or less inactivated (detoxificated) with respect to these organisms as one of the valences of mercury is bound to the sulphur in the amino acid and the other to the carbon in the methyl group. The ability of Neurospora to methylate mercury at a high rate is correlated with a resistance to high concentrations of inorganic divalent mercury in the substrate. Studies of the formation and release of methylmercury from sediment have indicated a negative correlation between the methylation and release rates and the oxygen content in the water (Jernelov and Lord). In natural waters, however, when the oxygen is exhausted, hydrogen sulphide starts to be formed and the divalent mercury is bound as mercuric sulphide. In this form mercury is not available for methylation under anaerobic condi- tions (Jernelov, 1968, and Ris sanen 1969), and even under (later occurring) aerobic conditions the methylation rate is low (Fagerstrom and Jernelov, 1971).

45 The ability to methylate mercury does not seem to be restricted to one or a few types of micro-organisms but seems to be a widespread process. Thus, the activity in the environment is enhanced by the same conditions as the general microbiological activity. This also means that the highest methylation rate in the aquatic environment occurs in the very upper part of the organic sediment and on suspended organic material in the water (Jernelov, 1971). Micro-organisms in the intestine and in the slime on the fish have been shown to methylate mercury, but the fish itself does not seem to do so. It has been reported that live hens and isolated liver from calf should have the ability to convert organo- mercurials to methylmercury. It is, however, still uncertain if impurities are responsible for the presence of methylmercury in the tissue.

Conversions between organic forms

+ -CH3Hg > (CH3)2Hg: has been shown to be formed from monomethylmercury in decomposing fish (Jensen and Jernelov, 1968) and from (originally) inorganic mercury in sediments. Here, the formation of dimethylmercury from inorganic mercury is regarded as a two-step process with monomethylmercury as an intermediate product. Of the two described biochemical processes for the methylation of mercury, the methylcobalamin process (Wood et al. , 1968) forms dimethylmercury as an end-product if there is an excess of methyl groups. The formation of dimethylmercury in aquatic sediments seems to be favoured by high pH values (Olsson, 1969, and Jernelov, 1969) and by inorganic nitrogen.

+ (CH3)2Hg •—^CH3Hg : Dimethylmercury is unstable at low pH values (< 5. 6 ?). During its breakdown, monomethylmercury is believed to be formed as an end-product or as an intermediate metabolite. Under the influence of u. v. -light, metallic mercury is formed.

Summary

The "mercury problem", as it is recognized today, is mainly based on the occurrence of mercury in fresh-water and marine fish. In fish muscle mercury exists mainly as methylmercury (Norfen and Westoo, 1967). This is a result of the difference in the accumulation rates of inorganic mercury and methylmercury. The decrease in the concentration of methyl- mercury in fish seems to be brought about more by growth of the fish than by excretion. As fish have not been shown to methylate mercury, the methylmercury found in the fish must be assumed to have been formed outside the body. Thus, the methylation rate is one of the key factors in determining the methylmercury content in fish. Some of the factors influencing this process are discussed above. However, as dimethylmercury is only very slightly soluble in water and is volatile, it will, when formed in an aquatic environ- ment, be lost by evaporation. This means that it will no longer be available for accumulation in organisms in that body of water. Thus, the ultimate net result of the methylation process (mono- or dimethylmercury) is im- portant for the resulting levels of methylmercury in fish. After evaporation,

46 dimethylmercury may be broken down in the air. If the end-product is monomethylmercury it will be deposited with any precipitation and be a possible source for mercury contamination of organisms in a new area. Mercury thus has two cycles. One well-known global cycle in which there is an atmospheric circulation of elementary mercury, and one more local cycle in which there is an atmospheric circulation of dimethylmercury. For the local contamination with organomercurials the latter cycle seems to be the more important, sometimes giving rise to high levels in the organisms.

REFERENCES TO CHAPTER 4

FAGERSTROM, JERNELOV, A., Water Research 5 (1971) 121.

FURUKAWA, K., SUZUKI, T., TONOMURA, K., Agr. Biol. Chem. 33 (1969) 128.

JENSEN, S., JERNELOV, A., Biocidinformation No. 10 (1967) 4.

JENSEN, S., JERNELOV, A., Biocidinformation No. 14 (1968) 3 (in Swedish).

JENSEN, S., JERNELBV, A., Nature, Lond. 223 No. 5207 (1969) 753.

JERNELOV, A., Vatten 24 (1968) 456.

JERNELOV, A., Vatten 25 (1969) 304.

JERNELOV, A., Limnology and Oceanography J15 6 (1971) 950.

JERNELOV, A., LORD, in press.

KITAMURA, M., SUMINO, K., TAINA, M., Synthesis and decomposition of organic mercury compounds by bacteria, Jap. J. Hyg. _24 (1969) 132.

LANDNER, L., Nature, Lond. 230 No. 5294 (1971) 452.

NORiN, K., WESTOO, G., VSr foda No. 2 (1967) 13.

OLSSON, M. , Nord. hyg. Tidskr. 50 2 (1969) 179.

RISSANEN, Swedish-FinnishSymposiumonMercury, Helsinki, Nov. 1969. Reprint series in Radiochemistry, Dept. of Radiochemistry, University of Helsinki.

WERNER, Analytiska aspekter p£ utslapp till vattendrag av metalliskt och oorganiskt kvicksilver, Analysdagar, Lund (1967).

WOOD, M., SCOTT, K.F. , ROSEN, C.-G., Nature, Lond. 220 (1968) 173.

YOSHIDA, Transference Mechanism of Mercury in Marine Environment, Tokyo, Japan (1967).

47

5. MECHANISMS FOR METHYLATION OF MERCURY IN THE ENVIRONMENT

J.M. WOOD, M.W. PENLEYand R.E. DeSIMONE

5.1. INTRODUCTION

Oxidative addition reactions involving metals are well documented in the chemical literature. In 1964 Halpern and Maher [l] were able to demonstrate that methylpentacyanocobaltate would react with mercuric ions to give methylmercury as the product. Since pentacyanocobaltate was regarded as a model compound for vitamin B12) ^ seemed feasible that B12-containing enzyme systems should be capable of this synthesis in biological systems. A survey of the methylating agents which are available for methyl- transfer reactions in biological systems reveals that there are three major coenzymes which are known to be involved in this reaction.

(1) S-adenosylmethionine (2) SN -methyltetrahydrofolate derivatives (3) Methylcorrinoid derivatives

S-adenosylmethionine and 5N-methyltetrahydrofolate derivatives are not capable of transferring methyl groups to mercuric salts because, for both these coenzymes, the methyl group is transferred as a carbonium ion(CHg). Therefore, the only known methylating agents capable of methyl transfer to inorganic mercury salts are methylcorrinoids since they are theoretically capable of transferring methyl groups as (CH+), carbonium ions (CHg), or radicals (CHj) (Scheme I).

Scheme I

CH|

CH3

CH®

B = base (usually 5, 6 dimethylbenzimidazole, but a variety of bases can co-ordinate to the atom as an axial ligand).

Theoretically, methylcorrinoids have the capacity to methylate inorganic mercury salts with CH®.

49 Wood et al. (1968) [ 2] demonstrated that methylcobalamin was capable of the synthesis of both monomethylmercury and dimethylmercury in both enzymatic and non-enzymatic systems. The relative proportions of the monoalkyl to the dialkyl derivative were shown to be dependent on the concentration of Hg2+added to reaction mixtures. Although this research proved that the biosynthesis of methylmercury compounds could occur under anaerobic conditions, it was not possible to determine details of how the methyl group is transferred from Co+3 to inorganic mercury. More recent studies in our laboratory have confirmed the non-enzymatic and enzymatic methylation of mercury but, furthermore, we have ob- tained experimental data which allows us to propose mechanisms for these reactions.

5. 2. NON-ENZYMATIC METHYLMERCURY SYNTHESIS

Hill et al. (1970) [3] studied the reaction of various alkylcobalamins with mercuric ion and concluded that this alkylation reaction proceeds by electrophilic attack on a species which is stabilized by the cobalt atom. Schrauzer et al. (1971) [4] determined rate constants for methyl transfer to concentrated solutions of mercuric diacetate for methylcobalamin, methylcobinamide and a number of methylcobaloxim.es with different axial bases. U.V.-visible spectral methods were used in this study of the overall demethylation reactions, and although it was ob- served that the displacement of benzimidazole from methylcobalamin occurred as a prerequisite to methyl transfer, no attention was paid to this reaction in the kinetic interpretation of the experiments reported. Furthermore, the variation in rate constants for the different methyl- cobaloximes are difficult to reconcile since the different axial bases are not co-ordinated to the cobalt under the reaction conditions employed, therefore, the same rate constant should be obtained for all the methyl- cobaloxime species tested (Halpern, personal communication (1971)). Adin and Espenson (1971) [5] have shown that cobaloximes are demethyl- ated by a bimolecular mechanism of electrophilic substitution. No kinetic or spectrophotometric evidence was found for complexation or association of mercuric ions with cobaloximes. Therefore, in this respect cobaloximes are poor models for the methyl-cobalamin reaction according to our studies. The mechanism for methyl transfer from methylcobalamin to mer- curic ion became further complicated by conflicting reports from Imura et al. (1971) [6] and Bertilsson and Neujahr (1971) [7], Imura et al. (1971) [6] claimed that dimethylmercury was the first product of the reaction, and that this compound then reacts with mercuric ions to give monomethylmercury. Bertilsson and Neujahr [7] claimed that methyl- mercury was the product, but the rate and stoichoimetry depends upon + the anion co-ordinated to the CH3Hg cation. When methylcobalamin is mixed with an excess of mercuric diacetate in acetate buffer at pH5. 0, then a rapid colour change from red to yellow is observed followed by a slower reappearance of the red colour. This red-to-yellow conversion has been postulated to involve the dis- placement of benzimidazole from the sixth co-ordination site of the co- balt with the possible replacement by attacking mercuric species

50 -1 ^ 400 500 600 nm FIG.10. Changes in the visible spectrum of methylcobalamin(6.95 x 10_5M) upon the addition of 5 15. 0 x 10" M Hg (OAC)2 . Methylcobalamin (—). Solid lines are scans in ascending order at 525 nm beginning at 5 sec, and at 2,4, 6 and 8 min after mixing.

(Ridsdale (1970) [8]). The spectrum of the yellow intermediate is similar to that of "base off" methylcobalamin. In the reaction of ethylcobalamin with an excess of mercuric diacetate, a sufficient quantity of the yellow intermediate accumulates because this reaction is much slower than that with methylcobalamin. Hill et al. (1970) [3] and Ridsdale (1970) [8] have shown that in the case of ethylcobalamin, the yellow intermediate has an identical u. v. -visible spectrum to "base off" ethylcobalamin. Closer examination of the reaction between methylcobalamin and mer- curic diacetate shows a characteristic change in the spectrum which is identical to the spectral change observed when benzimidazole is pro- tonated and displaced from methylcobalamin (Fig. 10). (Scheme II.)

Scheme II

CH3 CH3 3 \ I t F, ^J J3 6 Co-^ + Hg(OAc)2 —1- ^rcd^ + OAc

LBZ LB ®HgOAc

where = Kj/K.j and Bz = 5, 6 dimethylbenzimidazole.

51 ORDER OF INITIAL "FAST" REACTION 1.2

-32 -30 -2.8 -2.6 -2.4

log [Hg]

FIG. 11. Plot of log Hg(OAc)2 concentration versus the log of the observed rate constant for the "fast reaction". The order of the reaction in Hg(OAc)2 is given by the slope of the line. [Methylcobalamin] = 5 x 10_SM.

Once this intermediate is formed, then electrophilic displacement of CH® can occur by the mechanism presented in Scheme III.

Scheme III H H CH3

+ + + (HgOAc) Co + CH3HgOAc + (HgOAc) It® H2° t l L Bz - HgOAc B

The initial reaction with mercuric monoacetate and benzimidazole (Scheme II) is a "fast reaction" which is followed by a "slow reaction" for the methyl-transfer step. (Scheme III.) Figure 11 contains the rate data for the "fast reaction" plotted as log k

observed versus log [ Hg(OAc)2]. In these experiments we work under pseudo first-order conditions with Hg(OAc)2 always in at least ten-fold excess over methylcobalamin. Therefore:

Rate = k-J CH3B12 ] [ Hg(OAc)2 ]" (1)

= kobs[CH3B12] (la) where log k^ = log k1 + n log[Hg(OAc)2] (lb)

The slope of the straight line of the plot in Fig. 11 gives the order of the reaction in Hg(OAc)2. Since n = 0.99, then we can conclude that the fast

52 DETERMINATION OF EQUILIBRIUM CONSTANT

FOR CH3B,£-Hg ADDUCT FORMATION

(a)

(b)

L _L J_ 270 290 310 330 350 370 Wavelcnght, nm

FIG. 12. Representative oscilloscope traces of the 270-370 nm region showing: (a) initial and final products (methyl and aquocobalamin), and (b) buildup of the intermediate from methylcobalamin and its conversion to aquocobalamin.

53 [HQ]

FIG. 13. Plot of the observed rate constant versus the concentration of Hg(OAc)2 over a thousand-fold concentration range. [Methylcobalamin] = 5x 10"5M.

reaction is first order in Hg(OAc)2. The order of the reaction obtained in this manner is correct; however, log kj^ obtained from the intercept on the vertical axis must be corrected for the equilibrium constant K1.

The equilibrium constant Kx for the "fast reaction" was determined by scanning the 270 to 370 nm range rapidly (30 times/sec) and recording the change in absorbance at 306 nm as a function of Hg(OAc)2 concentra- tion. An experimental oscilloscope trace for this type of reaction is recorded in Fig. 12. Similar data were obtained at several Hg{OAc)2 concentrations allowing us to calculate the equilibrium constant for reac- tion 1 as (Ki = 70 ± 20). Analysis of the slow reaction is more complicated since the observed rate constant is a function of the equilibrium constant for the "fast reaction. " Data for the Hg(OAc)2 and methylcobalamin dependence of the observed rate constants are presented in Figs 13 and 14, respectively. We would like to emphasize the unusual shape of the curve in Fig. 14 which is indicative of an induction period at high methylcobalamin concentrations. There is a rapid increase in the reaction rate as the methylcobalamin concentration drops below 10"° M. The effect of increasing concentra- tions of methylcobalamin on the kinetics for methylmercury synthesis is clearly shown in the representative oscilloscope traces of the different

54 FIG. 14. Plot of the observed rate constant versus the methylcobalamin concentration over a hundred- 3 fold range. [Hg(OAc)2] = 5 x 1CT M.

reactions shown in Fig. 15. This decrease in the reaction rate with in- creasing methylcobalamin concentrations is indicative of an active mer- curic species being used up rapidly. Under the reaction conditions which we have employed, this species could only be mercuric monoacetate. Proof for the stoichiometry of the methyl transfer from cobalt to mercuric ion is readily obtained by 220 MHz NMR. NMR provides an elegant means of confirming that methylmercury is the pro- duct of this reaction. Furthermore, NMR has been used to indicate that mercurous'salts are not reactive in this reaction. The reaction of methyl- cobalamin with mercuric diacetate shows the rapid displacement of the methyl group from the cobalt at +0. 3 ppm and its transfer to Hg2* to = give methylmercuric acetate at -1. 05 ppm. (JHg.H 240 Hz). (Fig. 16c). When ascorbic acid is added to the reaction mixture, no reaction is ob- 9 2 + served to occur (Fig. 16b); since ascorbic acid reduces Hg to Hg2. The NMR spectrum presented in Fig. 16 c shows that no mercuric species is co-ordinated to benzimidazole at the end of the reaction since the C2o methyl resonance at -0. 55 ppm indicates that benzimidazole is co-ordinated to the cobalt, and the remainder of the spectrum is identical to that of aquocobalamin at the same pH (Penley et al. (1970) [9], Wood and Brown (1971) [ 10]). The pH dependence of methyl transfer from methylcobalamin to mercuric ion is presented in Fig. 17. In this experiment the protona- tion and displacement of benzimidazole, together with the changing

55 OSCILLOSCOPE TRACES : % TRANSMITTANCE vs TIME

(a)

(b) (c)

FIG. 15. Representative oscilloscope traces during the course of the demethylation reaction (slow reaction).

(a) Two repetitive scans showing the excellent reproducibility at 306 nm with a large excess of Hg(OAc)2. (0.5 seconds/division on the horizontal scale). (b) High methylcobalamin concentrations at 520 nm showing the lag at the beginning of the reaction. (2.0 seconds/division on the horizontal scale). (c) Low methylcobalamin concentrations at 520 nm showing the first order behaviour over the entire course of the reaction. (1.0 seconds/division on the horizontal scale).

56 (PPM)

FIG. 16. 220 MHz NMR spectra of the high field portion of the methylcobalamin spectrum in D2 O. (a) Methylcobalamin.

(b) Methylcobalamin + Hg(OAc)z + ascorbic acid.

(c) Methylcobalamin + Hg(OAc)2. (The products observed are aquocobalamin and methylmetcuric acetate). 3 4 5 6 PH

FIG. 17. The pH dependence of the observed rate constant for the demethylation reaction (slow reaction).

HgOAc+ concentration (as the pH changes), combine to complicate the situation and together determine the position of the maximum of the pH profile. The reaction with "base off" methylcobalamin, when the base has been displaced by protonation and not mercuration, is very slow. These data are in accord with those of Schrauzer et al. (1971) [4] who showed that reactions between methylcobinamide and mercuric diacetate are much slower than the reaction with methylcobalamin. We also ob- served that the rate of reaction is slowed down appreciably as the OAc0 concentration increases. For methylcobalamin the interaction of HgOAc+ with benzimidazole increases the electron density on the cobalt atom to

facilitate CH3 displacement from the Co atom, but for cobinamide a direct interaction between mercury and cobalt stabilizes the Co-C bond. One point of minor importance is the fact that methylmercury it- self reacts with methylcobalamin to give dimethylmercury as product. However, the reaction with methylmercury is several orders of magni- tude slower than the reaction with mercuric acetate. In summary, we have shown that the chemical transfer of methyl groups from methylcobalamin to mercuric ion involves heterolytic cleavage of the Co-C a bond which facilitates electrophilic attack by mercuric ion on the carbanion generated by heterolysis. Stopped flow

58 kinetic experiments are consistent with an initial reaction involving the displacement of benzimidazole by a mercuric species which then facili- tates methyl displacement from the. cobalt atom by a second mercury. These two reactions can be separated kinetically into a "fast reaction" (displacement of benzimidazole) and a "slow reaction" (displacement of CH-f). The "fast reaction" involves an equilibrium which is first order in mercuric species. The "slow reaction" is kinetically complex, but the pH optimum for together with variations for the observed rate constants with different concentrations of mercuric diacetate and with different concentrations of methylcobalamin, have been interpreted in terms of a reaction with low concentrations of mercuric monoacetate with the methyl group on the cobalt atom. To our knowledge this reaction provides the only well-documented example of a Grignard reaction which occurs smoothly in aqueous solution.

5.3. ENZYMATIC SYNTHESIS OF METHYLMERCURY COMPOUNDS

Methylcorrinoids are implicated in methyl-transfer reactions in three enzyme systems.

(1) Cobalamin-dependent TNI-methyltetrahydrofolate-homocysteine transmethylase ( synthetase). (2) Acetate synthetase. (3) Methane synthetase.

A number of micro-organisms, including a mutant of the foecal organ- ism Escherichia coli, use the cobalamin-dependent methionine synthetase for the synthesis of the essential amino acid methionine. This enzyme is . present in some aerobes, facultative anaerobes and strict anaerobes; in addition to mammalian liver, Guest et al. (1960) [11] and Brodie (1970) [12]. In this enzyme system methylcobalamin binds to the methionine synthe- tase apoenzyme to give the active enzyme-substrate complex. (Scheme IV.)

Scheme IV

CH3 SH S-CH3 SAM

Enzyme mni/nu Enzyme e Methionine

Methionii synthetas

®NCH3-THF THF THF = tetrahydrofolate

SAM = catalytic amounts of S-adenosylmethionine

59 For this enzyme reaction catalytic amounts of SAM are required, 5 and NCH3-tetrahydrofolate regenerates the methionine synthetase enzyme- substrate complex. Aerobic micro-organisms and facultative anaerobes which utilize the cobalamin-dependent methionine synthetase enzyme are capable of the synthesis of methylmercury. Also, mammalian liver will catalyse this reaction slowly, Westoo (1967) [13]. (Scheme V. )

Scheme V

CH3 H H \ / (

(a) 3 2+ ^3 + ^C< + Hg + CH3Hg Enzyme Enzyme

H

•J .+3 v.. +i (b) FADH„ + FAD Enzyme Enzyme

CH,

^1 +3 (c) + NCH3-THF + THF Enzyme Enzyme

+ CH3Hg + SH CH^-Hg

d ( ) (CH2)2 S

e CH ) + H+ NH3-CH-COO ( 2 2

NH+-CH-COO®

In this mechanism (Scheme V), electrophilic attack on CH® of the methylcobalamin-methionine synthetase complex yields methylmercury and the aquocobalamin enzyme complex. Reduction of the resulting Co+3 +1 (by two electrons) (FADH2) gives the Co species which is remethylated + by CHj transfer from ^ CH3-THF. The CH3Hg which is formed re- moves the substrate homocysteine from the active site of the enzyme. However, some methyl-transfer to the methyl-Hg-homocysteine product may occur to give methyl-Hg-thiomethyl (CHgHg-SCHg) as a second product. Clostridium thermoaceticium and CI. sticklandii are anaerobic micro- organisms which synthesize from . Ljungdahl et al. (1965) [ 14]. In this reaction methylcobalamin is the substrate for the C2 carbon, and CO2 forms the substrate for the Cj carbon of acetic acid. In this reaction methylcobalamin is continuously synthesized from

60 +1 B12s (CO ) and ^NfCH3- THF. Carboxymethylcobalamin may be an inter- mediate in this reaction, but a Grignard reaction between C02 and methylcobalamin has some experimental support [15], (Scheme VI.)

Scheme VI

Methane synthetase is a very common enzyme system in any anaerobic ecosystem (e. g., sediments of lakes or rivers and in the rumen). Methanobacillus omelianskii was isolated as a symbiotic culture of two organisms from canal mud in Delft, Holland in 1940 by Barker [16], In 1967 Bryant et al. [ 17] were able to separate this culture into two or- ganisms.

(1) S organism

CH3CH^OH + HzO • CHgCOOH + 2H2 (2) M. O. H. organism

4H2+ C02 ^ CH4+2H20

The S organisms provided the H2 required by M. O. H. for the reduction of CO2 to CH4. Symbiotic relationships between evolvers and H2 utilizers are abundant in anaerobic ecosystems.

Cell-extracts of M. O. H. grown with 80% H2 and 20% C02 catalyse the formation of CH4 from a variety of substrates which include, in addition to C02, C3 of serine (Wood et al., 1965 [18]), Ci of pyruvate (Wolin et al., 1964 [19]), SN methyl-THF (Wood and Wolfe, 1965 [20]) and a variety of methyl-corrinoids including methylcobalamin (Wolin et al., 1964 [21]), methyl-cobinamide (Wood et al., 1966 [22]), and methyl Factor III (Wood and Wolfe, 1966 [23]). Methyl Factor III was isolated as the natural cobalamin in this methane synthetase system, and it was

61 this corrinoid which was isolated from anaerobic sewage sludge by Bernhauer and Friedrich (1953) [24]. The methane synthetase reaction is presented in Scheme VII. ATP is required in catalytic amounts, and the role of ATP in this reaction is not yet understood. In this methane synthetase system experimental evidence is avail- able that the methyl group is transferred to a second coenzyme, of un- known structure (X), before reductive cleavage to CH4 occurs (McBride and Wolfe, 1970 [25]). (Scheme VII. )

Scheme VII

CH3

\ I +3 x +2 jco^ + x + X'CH; t

X H + CH4 X - CH3 ATP— "

B = H20, 5, 6 dimethylbenzimidazole or 5 OH benzimidazole

Good evidence is available to support the concept that the methyl group is transferred to X as a radical (CH3). When methylcorrinoids are photol- ysed under anaerobic conditions homolysis of the Co-C bond occurs to give Co+2 and methyl radicals. (Scheme VIII.)

Scheme VIII H H \/ ?H3

B (rate limiting step) B B

The use of isotopes together with photolysis control experiments has been effective in proving that methyl transfer in methane synthetase occurs by a radical mechanism.

The rate of methane formation from CD3 cobalamin and CH3 cobalamin were compared for the enzymatic reaction and for the photolysis reaction. Identical isotope effects were demonstrated for both these reactions with kH/kD = 1.32 j 0.06. Examination of tritiated and deuterated methanes formed from CT3 cobalamin and CD3 cobalamin by scintillation counting and mass spectro- metry, respectively, showed that all three of the methyl group were conserved to give CT3H and CD3H as products. Reactions with methylcobalamin in full deuterated buffer systems yielded CH3D, indicat- ing that the H added to the methyl group is exchangeable with .

62 These experimental data provide a basis for CHj transfer, and since the redox potential required for growth of these strict anaerobes is less than - 400 m volts, then any inorganic mercury added to the methane synthetase enzyme system should be reduced to Hg°. The synthesis of dimethylmercury by this anaerobic system (Wood et al,, 1968 [2]) is easily explained in terms of methyl-radical addition to mercury metal, (Scheme IX.)

Scheme IX

CH„

+3 ^CcC Hg (CH3)2 Hg t B

It seems feasible that Hg may be transported across the cell membrane 2+ by these anaerobes, but once inside the cell, Hg would be reduced to Hg° and methylated as indicated in Scheme IX. Dimethylmercury would readily diffuse from the microbial cell, and if the pH of the sediments were on the alkaline side, this compound would be released into the water. If the pH of the sediments were on the acid side, then the dimethyl- mercury would be converted to monomethylmercury and methane. (Scheme X.)

Scheme X

CHg-Hg-CH, + H CH3 Hg + CH4

5.4. DISCUSSION

Although methylcorrinoids provide the first example of a clearly de- fined mechanism for the synthesis of methylmercury in the environment, it would be dangerous to assume that alternative methylating agents do not exist. Recently DeSimone (1972) [26] has shown that methylsilicon derivatives react with Hg in aqueous systems to yield methylmercury. (Scheme XI.)

Scheme XI

(CH3)3Si(CH2)3 SOsNa + Hg(OAc)2

(CH^a-Si-(CH2)3 SOgNa + CH3Hg OAc

OAc

However, there is increasing evidence to support that methylcorrinoids are extremely important in methylmercury biosynthesis in sediments. Wood et al. (1968) [27] and Penley et al. (1970) [9] have shown that chlorinated hydrocarbons irreversibly inhibit methane synthetase and methionine synthetase by the general reaction presented in Scheme XII.

63 Scheme XII

R-C-C12 +1 \ I +3 ^CcT + R-C-CI3 + CI t B B

It has been observed in a number of locations in the USA that when sediments are polluted with both inorganic mercury compounds and chlorinated hydrocarbons that very little methylmercury accumulates in fishes which live in this type of polluted environment. However, chlorinated hydrocarbons do accumulate in fish under these conditions (Wood, 1971 [ 10, 28, 29]). In conclusion, it seems to us that methylcorrinoids are present in a sufficient number of micro-organisms to permit the synthesis of methylmercury compounds in both aerobic and anaerobic ecosystems (Scheme XIII). Therefore, the rate of synthesis of methylmercury com- pounds will be dependent on the population of micro-organisms in these ecosystems. This population of micro-organisms will in turn depend on the quantities of , nitrogen compounds, and phos- phate added to our aqueous systems. In more affluent societies, we have had a tendency to convert our inland and coastal waterways into ideal bacteriological culture media which has lead to a situation where methyl- mercury synthesis is rapid and hazardous to public health.

Scheme XIII 2+ TT 0 . TT 2 + Hg2 Hg + Hg

e ; CH [CHa L or 7 + :CH 3-ln CHgHg CH3Hg (CH^ Hg 2 Chemical and aerobic] [ Enzymatic and anaerobic]

The research conducted for this chapter was subsidized by grants from the National Science Foundation GB 26593X and GP 13329, The United States Public Health Service USPH AM 12599, and the Dow Chemical Company.

ACKNOWLEDGEMENTS

We wish to thank Prof. S. G. Smith and Dr. L. Charbonneau for their help with the kinetic experiments reported in this manuscript.

64 REFERENCES TO CHAPTER 5

[1] HALPERN, J., MAHER, J.P., J. Am. chem. Soc. 86(1964) 2311. [2] WOOD, J. M., KENNEDY, F.S., ROSEN, C.G., Nature, Lond. 220 (1968) 173. [3] HILL, H.A.O., PRATT, J. M., RIDSDALE, S., WILLIAMS, F.R., WILLIAMS, R.J.P., Chem. Commun. (1970) 341. [4] SCHRAUZER, G.N., WEBER, J. H., BECKHAM, T. M., HO, R. K. Y., Tetrahedron Lett. 3 (1971) 275. [5] ADIN, A., ESPENSON, J.H., CHEM. Commun. 13 (1971) 653. [6] IMURA, N., SUKEGAWA, E., PAN, S.K., NAGAO, K., KIM, J.Y., KWAN, T., UKITA, T., Science 172 (1971) 1248. [7] BERTILSSON, L., NEUJAHR, H.Y., Biochem. 10 (1971) 2805. [8] RIDSDALE, S., Thesis in , Part II, Oxford University, Oxford, England (1971). [9] PENLEY, M.W., BROWN, D.G., WOOD, J.M., Biochem. 9 (1970) 4302. [10] WOOD, J.M., BROWN, D.G., Structure and Bonding, Vol.11, Springer Verlag (in press) 47-105. [11] GUEST, J. R., HELLEINER, C. W., CROSS, M., WOODS, D.D., Biochem. J. 76 (1960) 396. [12] BROD1E, J., Biochem. 9 (1970) 4295. [13] WESTOO, G., Vlr Foda 19 (1967) 121. [14] LJUNGDAHL, L., IRION, E., WOOD, H.G., Biochem. 4(1965) 2771. [15] PARKER, D.J., WOOD, H.G., GHAMBEER, R.K., LJUNGDAHL, L.G., Biochem. (in press). [16] BARKER, H.A., Antonie van Leeuwenhoek, 6 (1940 ) 20. [17] BRYANT, M. P., WOLIN, E.A., WOLIN, M.J., WOLFE, R.S., Arch. Mikrobiol. 59 (1967) 20. [18] WOOD, J.M., ALLAM, A M., BRILL, W.J., WOLFE, R.S., J. biol. Chem. 240(1965) 4564. [19] WOLIN, E.A., WOLIN, M.J., WOLFE, R.S., J. biol. Chem. 238 (1963) 2882. [20] WOOD, J.M., WOLFE, R.S., Biochem. biophys. Res. Commun. 19 (1965) 306. [21] WOLIN, M.J., WOLIN, E.A., WOLFE, R.S., Biochem. biophys. Res. Commun. 12(1964) 464. [22] WOOD, J.M., WOLIN, M.J., WOLFE, R.S., Biochem. 5 (1966) 2381. [23] WOOD, J.M., WOLFE, R.S., Biochem. 5 (1966) 3598. [24] BERNHAUER, K., FRIED RICH, W., Angew. Chem. 65(1953) 627. [25] McBRIDE, B.C., WOLFE, R.C., Fed. Proc. 29, 344 abs. (1970). [26] DeSIMONE, R. E. , Chem. Commun. (in press). [27] WOOD, J.M., KENNEDY, F.S., WOLFE, R.S., Biochem. 7 (1968) 1707. [28] WOOD, J.M., Advances in Environmental Sciences, Vol. II, Wiley Interscience(1971) 39-55. [29] WOOD, J.M., Enzymes and the Environment, Bogden and Quigley, N.Y. (1971).

65

6. THE TOXICITY OF MERCURY IN MAN AND ANIMALS

F.C. LU, P.E. BERTEAU and D.J. CLEGG

6.1. INTRODUCTION

The presence of contaminants in the food of man is one of the most serious aspects of the overall problem of environmental pollution. In recent years the fact that mercury has been detected in food in many parts of the world has given rise to considerable concern. The existence of this element in food was recognized at least as'early as the 1930s (Stock and Cucuel, 1934). However, during the last two decades there has been a considerable increase in the use of mercury in industry (Anon., 1966). In addition, mercurial fungicides have proved to be of great value in agriculture. For these reasons there is an increasing potential for contamination of food by mercury. In certain parts of the world, high mercury levels have been found in fish and birds (Westermark et al. , 1965; Anon., 19 70 and Borg, 1958), and signifi- cant levels have also been found in fruit, vegetables and cereals (Novick, 1969). These observations have led to much concern about the potential for intoxication to humans due to mercury in foodstuffs. In addition, much of the mercury found in food, particularly that in fish, is in the form of methyl- mercury (WestOO, 1966), and it is this form which is generally considered to present the most serious toxic potential to man and animals. In this chapter, the information available on the toxicity of various types of mercury compounds toman and animals is reviewed. The various chemi- cal forms of mercury are considered separately, and some comparative studies are also summarized. However, in view of the environmental pro- blem and potential hazard existing from the presence of methylmercury in food, special emphasis has been given to methylmercury.

6.2. HISTORY

Although the first unmistakable reference to mercury toxicity appears to be by Dioscorides in the first century A.D. (Dioscorides, cited in "The Greek Herbal of Dioscorides" trans. 1934) it is known that mercury in the form of cinnabar was used by the ancient Egyptians as early as 1600 B.C., although probably not for medicinal purposes. Arab physicians in the early middle ages made extensive use of mercurial ointments. The ability of mercury compounds to produce salivation also became known at this time and later led to its extensive use in the treatment of . It was not, however, until 1861 that Overbeck attempted a systematic clinical and experimental study of mercury which resulted in a clear description of the toxic effects of mercury in man and animals (Almkvist, 1929). The presence of mercury intoxication among workers using mercurial compounds in the -hat industry has also been long recognized. The bizarre symptoms observed in these persons have been implicated in the

67 origin of the term "mad hatter" although there may be other explanations for this expression (Turk, 1963). Toxicity from organomercury compounds was reported as early as the mid-19th century (Edwards, 1965). The classical description of methyl- mercury poisoning is, however, attributable to the studies of Hunter et al. (1940).

6.3. INORGANIC MERCURY

6.3.1. Metallic mercury

The major route of human exposure to metallic mercury is by inhalation of mercury vapour. Studies have shown that mercury vapour diffuses rapidly across the alveolar membrane (Kudsk, 1966) and that following absorption, a proportion of the mercury is slowly oxidized to give mercuric ions (Magos, 1968a). However, inhalation of metallic mercury results in higher mercury levels in the brain and myocardium than those which result from the intra- venous injection of mercuric ions (Berlin et al., 1966), presumably because of the slow conversion of metallic mercury to mercuric ions in the blood (Magos, 1967). Diffusion of this elemental mercury within the body is facili- tated by its lipid solubility, and its lack of charge, thus resulting in the different distribution pattern compared to that found after direct adminis- tration of mercuric ions (Magos, 1968a; Berlin et al., 1969). Following intravenous injection of 0. 1 jug of radioactive mercury as a finely divided (possibly colloidal) suspension into rats, nearly 20% of the dose was exhaled. The amount of mercury which penetrated the lung, brain and heart was larger than that obtained by injecting mercuric ions, whereas the amount remaining in the blood was considerably smaller (6% of 45% of the dose given). These observations provide further indication of facilitated transportation of metallic mercury within the body (Magos, 1968b). The biochemical activity and toxicity of metallic mercury are generally attributed to the action of the mercuric ion. There is a marked affinity for mercury to bind primarily to -groups and to a lesser extent to the amino- carbonyl- and hydroxyl-groups of enzymes (Battigelli, 1960; Webb, 1966). In man, acute poisoning from mercury vapour is now rare, although occasional instances have been reported (Bidstrup, 1964). The major symptom is that of acute pneumonitis ("metal fume fever"), but gastrointestinal dis- turbances and effects on the central are also,evident. In cases of chronic exposure to mercury vapour, the principal signs are , psychological disturbances (erethismius mercurialis), and occa- sionally proteinuria. The latter may progress to a nephrotic syndrome (Kazantzis et al. , 1962; Joselow and Goldwater, 1967). Oral signs such as gingitivis, stomatitis and excessive salivation may occur, as well as occa- sionally dermatitis. Mercuralentis (a yellow discolouration of the lens) is often seen following long-term exposure to mercury. In addition, some less specific symptoms such as fatigue, weight-loss, , pallor, etc. may be evident (Bidstrup, 1964). Hypersensitivity and allergic reactions to mercury have also been reported (Stock, 1937). A number of attempts have been made to try to correlate mercury vapour concentrations with the degree of mercury intoxication. Neal et al. (1937; 1941) reportedly found no cases of poisoning when air levels were

68 below 0. 1 mg/m3 air although their erratic results, correlating symptoms of poisoning with urinary levels of mercury, have been subject to question (Goldwater, 1964). Exposure to higher air concentrations have usually been associated with some incidence of intoxication (Bidstrup et al. , 1951; Bruusgaard, 1958; Williams et al., 1947; Benning, 1958). There have been numerous attempts to try to correlate levels of exposure to mercury, evidence of toxic symptoms and levels of mercury present in the plasma and/or excreted in the urine. In general, very inconsistent results have been obtained (Goldwater et al., 1964; Jacob et al., 1964; Goldwater and Nicolau, 1966; Lundgren et al. , 1967; West and Lim, 1968). Levels in the urine appear to be higher in workers exposed to mercury for the first time, and to be lower in workers who had been exposed to similar levels over a long period even though symptoms may be evident in this latter group (Armeli and Cavagna, 1966). Exposure to metallic mercury via routes other than inhalation is infre- quent. Oral doses of 100 - 500 g have been given to man with little effect because of poor absorption, although they occasionally resulted in diarrhoea (Battigelli, 1960). Entry of mercury into the blood stream in man has been reported to produce serious pulmonary lesions (Popper, 1967). Death within one month of injection of between 1 and 2 ml of metallic mercury into the forearm has been reported. Autopsy revealed extensive necrosis of the kidney and myocardium in addition to degeneration of anterior horn cells of the spinal cord (Johnson and Koumides, 196 7). On the other hand recovery has been reported following the injection of 20-40 g into the thigh muscle (Hill, 1967).

6.3.2. Inorganic mercury salts

The comparative toxicity of inorganic salts of mercury is related to their absorption. Thus, insoluble mercurous salts such as calomal (mercu- rous chloride) are relatively non-toxic (Battigelli, 1960). In man, some data are available following accidental or intentional oral overdosage with mercuric chloride. The immediate effects of acute poisoning are due to irritation, coagulation and superficial of tissues which are exposed. Chronic effects include kidney damage as well as intestinal haemorrhage and ulceration (Bidstrup, 1964). Inhalation exposure may also occur in man. An investigation of labora- tory technicians exposed to 0.2 - 0.3 mg mercuric chloride/m3 air showed high levels of urinary protein. This finding was considered suggestive of early kidney tubule dysfunction (Taylor, 1968). The distribution of mercury in the body of mice was studied following single intravenous injections of 203Hg-labelled mercuric chloride to 24 mice. The animals were sacrificed at various intervals from 5 min to 16 days, and the mercury in the organs was determined by densitometric comparison in autoradiograms. Mercury accumulated mainly in the kidney, liver, myocardium, intestinal mucosa, upper respiratory tract, mouth and skin. Uptake by the brain was much slower than by the other organs. Slow elimina- tion and considerable retention was found in some parts of the brain, in the testes, skin, buccal mucosa and in the kidney (Berlin and Ullberg, 1963a). The mechanism of transport of mercuric ions has been further investi- gated following intravenous injection of 0. 12 to 1.2 mg/kg 203Hg-labelled mercuric chloride in rats. At the lower dose the mercury in the serum is

69 bound to cr-globulin. With higher doses, association with albumin occurs, once the a-globulins are saturated (Cember et al., 1968). In another study, rats received intravenous injections of from 0.1 to 0.3 mg/kg of mercury as 203Hg-labelled mercuric chloride. Tissue analysis revealed three different types of mercury binding: in all media except urine the element formed complexes of high molecular weight (proteins); in kidney and liver and occasionally in urine it occurred as compounds of medium molecular weight (11 000), whereas in the urine it was found mainly as low molecular weight compounds (100 - 300), traces of such compounds also being found in the kidney (Jakubowski et al., 1970). The of inorganic mercury salts is summarized in Table IX. In man, Kaye (1968) states that the minimum lethal dose for mercuric chloride is approximately 500 mg for a 70-kg man (i.e. about 7 mg/kg body-weight). Mercuric chloride injected into the yolk of the fertile incubated hen egg caused 100% mortality at a dose of 0.5 mg/egg, and 80% reduction in hatch- ability at 0. 25 mg/egg (McLaughlin et al., 1963). Further data on inorganic mercury toxicity are included in the section on comparative studies.

TABLE IX. ACUTE TOXICITY OF INORGANIC MERCURY SALTS

LD50 Compound Species Route Reference (mg/kg body-weight)

Mercurous iodide Mouse oral 110 Gothe and Sundell, 1964

Mercurous iodide Rat oral >310 Gothe and Sundell, 1964

Mercuric acetate Rat oral 104 Ikeda, 1968a

Mercuric iodide Mouse oral 80 Gothe and Sundell, 1964

Mercuric iodide Rat oral 40 Gothe and Sundell, 1964

Mercuric chloride Rat s. c. 3 Clarkson, 1968

Mercuric chloride Mouse s. c. 6 Clarkson, 1968

6.4. ORGANIC MERCURY COMPOUNDS

As stated in Section 6.1, concern has been expressed relating to the environmental contamination with mercury. Although much of the mer- cury released into the environment is probably in the inorganic form, a considerable part of the mercury found in animal tissues appears to be in the form of methylmercury. In fact it has been reported that regardless of the nature of the mercury pollutant only methylmercury has been found in fish (West66, 1969). The possible mechanisms for converting inorganic mercury to methylmercury in nature have been reviewed by JernelOv (1969). In addition, it has been suggested that the use of phenylmercury compounds as seed-dressing agents may result in their being converted to methyl- mercury compounds, following the initial conversion to inorganic mercury (Miller et al., 1960; WestOO, 1967).

70 6.4.1. Arylmercury compounds

Immediately after single intravenous injections of phenylmercuric hydroxide in cockerels the concentrations of mercury in the organs was fairly consistent. However, relatively slow elimination of mercury from the kidney, as compared to other organs, was observed (Swensson and Ulfvarson, 1968a). In mice the phenylmercuric acetate content of the whole body was determined after a single intraperitoneal injection of 5 mg/kg. One hour after injection, 4.5 ppm phenylmercuric acetate was present. By 24 hours, the level had decreased to 0. 1 ppm. However, total mercury at this time was 3.5 ppm, indicating that there was conversion to another form of mercury (Sumino, 1968). Similar studies in chicks, rats and dogs after oral, intramuscular or intravenous administration, indicated that phenyl- mercuric acetate was absorbed unchanged and disappeared within 96 hours. However, the levels of inorganic mercury increased in the liver and kidneys (Miller et al., 1960). The accumulation of mercury in these organs was also observed in mice (Berlin and Ullberg, 1963b). Urinary excretion of mercury in rabbits administered phenylmercuric acetate is in the form of inorganic mercury (Berlin, 1963). The acute toxicity of arylmercury compounds is summarized in Table X.

TABLE X. ACUTE TOXICITY OF ARYLMERCURY COMPOUNDS

Compound Species Route LDso Reference (mg/kg body-weight)

Phenylmercuric acetate Mouse oral 13 Piechocka and Krauze, 1967

Rat oral 60 Piechocka and Krauze, 1967

Mouse oral 70 Goldberg et al., 1950

Rat oral 46 Ikeda, 1968a

Chicken oral ca 60 Miller etal., 1960

Rat i.p. ca 10 Swensson, 1952

Phenylmercuric Mouse i.v. 27 Wein, 1939 nitrate

In a two-year study in rats fed phenylmercuric acetate at 0, 0.1, 0.5, 2.5, 10, 40, 160 ppm of the diet, growth retardation was observed at 40 ppm in the females, and at 10 ppm in the males. Longevity was reduced at 160 ppm, but mortality rates were unaffected at other dose levels. Gross pathological examination revealed enlargement and granularity of the kidney and moderate paleness of the viscera (suggestive of anaemia) at 0.5 ppm and above. Histopathological examination demonstrated severe renal tubule damage at 10 ppm in the females and slight to moderate damage in the males at 40 ppm. Other pathological manifestations included caecal wall necrosis in two animals and normoblastic hyperplasia in the bone marrow in some

71 animals fed high doses, but these changes were not clearly established to be due to mercury. Mercury accumulation in liver and kidney was apparent even at 0. 1 ppm (Fitzhugh et al. 1950). Rabbits were fed levels of phenylmercuric acetate up to 50 ppm for 10 months and monkeys for 2 years. The major toxic manifestation in the rabbit was renal tubular degeneration. Such degenerative change was less marked in the monkeys. Proteinuria was observed in both species (Ichikawa, 1970). Long-term (670) days) dministration of 0.03 mg phenylmercuric acetate/day to a guinea-pig resulted in no apparent ill effects. The level of mercury in the kidney was increased from 0.3 ppm (control animal) to 4.8 ppm (Kluge et al., 1938). Reproduction studies in rats fed mercury (as phenylmercuric acetate) prior to, and during, at 0, 1, 2, 4 and 8 ppm resulted in a reduction in size (44%) at the 8 ppm exposure level (Piechocka, 1968). In a carcinogenic study, groups of 18 mice of each sex from two hybrid strains were given 10 mg/kg each day of phenylmercuric acetate by gavage from 7 to 28 days of age and, thereafter, fed a diet containing 24 ppm phenyl- mercuric acetate until they were 18 months old. There was no increase in the incidence of tumours when compared with untreated controls (Innes et al., 1969). Human chronic exposure resulting from inhalation of phenylmercury compounds at air levels up to 5.1 mg mercury/m3 air have not resulted in evidence of clinical injury (Massman, 1957); Ladd et al., 1964; Goldwater et al., 1964).

6.4.2. Alkylmercury compounds

As already pointed out, the current problem concerning contamination of the environment by mercury is mainly related to'the presence of methyl- mercury in foodstuffs. For this reason the methylmercury compounds are considered in some detail.

6.4.2.1. Biochemistry

The methylmercury compounds such as the hydroxide, chloride, di- cyandiamide and propanediolmercaptide, some of which have been widely used as seed-dressing agents, have been shown to be similar as regards absorption, distribution and excretion after oral administration. Distribu- tion and elimination have also been shown to be similar for the hydroxide and dicyandiamide when injected subcutaneously (Ulfvarson, 1962). It seems likely that the compounds dissociate in the body; the methylmercury group attaches to other ligands (probably proteins) and is then similarly metabolized. Methylmercury compounds are absorbed via the skin, the respiratory tract (Hunter et al. , 1940; Swenson, 1952) and there is almost complete absorption from the digestive tract (Berglund, 1969; Ekman et al., 1968). In cockerels and rats methylmercury distribution after intravenous injection is fairly even throughout the body, as determined 24 hours after injection. However, the rate of elimination of methylmercury in both species was much slower than for other forms of mercury (Swensson and Ulfvarson, 1968a, 1968b). Mercury distribution after intravenous administration in mice has been studied using autoradiographic techniques (Berlin and Ullberg,

72 1963c). Results were similar to those found in rats. However, methyl- mercury resulted in much higher mercury levels in the brain than did other forms of mercury. Levels in the brain reach a maximum several days later than in other organs. Thus, the blood-brain barrier may provide some hinderance to methylmercury penetration. This speculation is supported by work in mice by Suzuki et al. (1963). A number of studies in the rat (Gage, 1964; Ulfvarson, 1962), mouse (Suzuki, 1969) and hen (Smart and Lloyd, 1963) indicate that a progressive increase in tissue concentration of mercury occurs when methylmercury compounds are administered daily in experiments lasting up to 6 weeks. In another study, rats were fed 0, 0.2, 1 or 5 ppm of 203Hg-labelled methylmercury in their diet for 9 months. At all three test-dose levels an equilibrium was reached after 6 months. At this point, excretion was found to be about 2% of the total body burden (Berglund and Berlin, 1969; Berglund, 1969). Other studies have indicated that tissue levels of mercury decrease after continued feeding of ethylmercuric chloride to mice. Rats were fed dietary levels of this compound of from 0.1, 2 or 40 ppm for up to 18 months. After 7 months, all the males fed 40 ppm and eight out of 13 of the females had died. Mortality was not adversely affected at the 0. 1 or 2 ppm levels. The maximum level in the tissues of most organs was reached after 2 months. After this time a steady decrease of mercury level was evident in brain, lung, liver, kidney, spleen, muscle and blood. Except for the kidney, levels in all these organs were less than 1 ppm in all surviving animals after 18 months (Ikeda, 1970a). In the dog brain, radioactive techniques using labelled and unlabelled methylmercuric thioacetamide have demonstrated maximum mercury levels in the hippocampus; and frequently, but not always, high levels in the cerebellum. The mercury was mainly in the protein fraction of the brain, little being found in the lipid, or nucleic-acid fractions. Mercury can be released by hydrolysis of this protein. Immediately after injection, mercury levels were highest in the mitochondrial, and lowest in the supernatant fraction, but 24 hours after injection mercury levels were similar in mitochondrial, microsomal and supernatant fractions, and remained similar until termination of the experiment 16 days later (Yoshino et al., 1966a). The rate of excretion of methylmercury has been studied in man, after oral ingestion of 203Hg-labelled methylmercuric nitrate. The half-life was estimated to be about 73 days (Aberg et al., 1969). The highly volatile compound, dimethylmercury has also been found to occur in animal tissues following exposure of the animal to various forms of mercury (WestOO, 1969). When dimethylmercury was administered to mice, either by inhalation or intravenous injection, there was rapid elimination by exhalation, the compound behaving as a chemically and physically inert substance. However, a portion was retained in the tissues where it was metabolized to non-volatile mono-methylmercury and distributed as already described for these compounds (Ostlund, 1969). Biotransformation of methylmercury compounds in the rat was studied, using detection of the cleavage of the carbon-mercury bond as an indicator. After exposure to methylmercuric chloride, inorganic mercury accounted for 40% of the total excretion, 50% of the faecal excretion and 5 - 20% of the urinary excretion. Most of the faecal inorganic mercury originated from biotransformation in the lower part of the small intestine or in the caecum.

73 Of the mercury from the original dose of methylmercuric chloride, 10% was secreted in the bile within 24 hours, falling to 5% on day 10. Only 3% of the dose was excreted daily in the faeces over the same period. Methylmercury cysteine is the major biliary metabolite together with mercury proteinate. Methylmercury cysteine is rapidly and nearly completely re-absorbed in the small gut and is accumulated to a greater extent than the originally injected methylmercuric chloride in the kidney. This accumulation of methvlmercury cysteine could be inhibited by bile-duct ligation despite increased blood and plasma levels or mercury (Norseth, 1969). After a single intraperitoneal dose of 75 mg/kg body-weight of methyl- mercury thioacetamide was administered to rats, neurological symptoms appeared within five days with death about 1 week later. At various intervals after administration of the dose, determinations were made of the rate of glycolysis in brain cortex slices and ability to synthesize protein. The activity of various sulphhydryl-containing enzymes, such as succinic de- hydrogenase, were also measured in various parts of the brain. The only parameter affected before the onset of the neurological signs was protein synthesis which was decreased. Later, oxygen consumption and succinic dehydrogenase activity were also decreased (Yoshino et al. 1966b).

6.4.2.2. Acute toxicity of alkylmercury compounds

The acute toxicity of alkylmercury compounds is summarized in Table XI. TABLE XI. ACUTE TOXICITY OF ALKYLMERCURY COMPOUNDS

LD50 Compound Species Route Reference (mg/kg body-weight)

Methylmercuric Mouse i.p. 47 Nose, 1969 chloride

Methylmercuric Rat oral 58 Ikeda, 1968a chloride

Methylmercuric Rat oral 26 Swensson and Ulfvarson, 1963 dicyandiamide

Ethylmercuric Mouse i.p. 28 Nose, 1969 chloride

Ethylmercuric Rat oral 50 Ikeda, 1968a chloride

n-Propylmercuric Mouse i.p. 18 Nose, 1969 chloride

n-Butylmercuric Mouse i.p. 15 Nose, 1969 chloride

6.4.2.3. Chronic toxicity studies in experimental animals

Pheasants fed wheat treated with 15 - 20 mg/kg methylmercuric di- cyandiamide for 9 days laid eggs with a reduced hatchability. Longer feeding (29 - 61 days) resulted in death of the birds. Histopathological examination revealed degeneration of central and peripheral nerves (Borg et al., 1966).

74 Oral administration to 10 mice of 14.7 mg mercury/kg as methylmercuric chloride for seven days resulted in 100% mortality within 5 days after the last dose. Only one of 10 mice died at a dose of 10 mg mercury/kg per day. Neurological signs were observed at 10 mg mercury/kg per day, but not at 4.6 or 6.8 mgmercury/kg per day. Analysis of mercury in brain indicated that there were neurological signs in those animals having a mercury content exceeding 20 ppm on awet-tissue basis (Suzuki, 1969). In rats, 0.3 mg dimethylmercuric sulphide administered by mouth, daily for 90 days, resulted in the appearance of neurological signs from the 50th day of the experiment. Histological examination of the brain showed lesions in the cerebellar granular layer (Miyakawa et al., 1969). Intubation of 0. 76 or 3. 78 mg/kg of ethylmercuric chloride daily to rats for up to 91 days resulted in 100% mortality at the higher dose. At the lower dose, ataxia, tremor and occurred. No effects on body-weight, food intake or haematological parameters were apparent (Ikeda, 1968b). Groups of 10 rats of each sex were fed either methyl- or ethylmercuric chloride at dietary levels of 0. 1, 2 or 40 ppm for up to 3 months. Mortality was 100% within 1 month in the group fed 40 ppm of methylmercuric chloride but there were no deaths in any other group. Signs of toxicity were evident after 1 week in the group fed 40 ppm of the methyl compound and after 2 weeks in the group fed 40 ppm of the ethyl compound. The only abnormality at lower dose levels were moderately pale kidneys in the group fed 2 ppm of methylmercuric chloride (Ikeda, 1970b). Dietary administration of 0.2, 1 or 5 ppm of methylmercuric dicyandia- mide to rats for 9 months did not cause any weight loss, behavioural changes, or pathological damage (Berglund and Berlin, 1969; Berglund, 1969). Cats treated orally, with 1 mg mercury/kg each day as methylmercuric chloride showed neurological signs after several weeks of administration. At autopsy, pathological degeneration and loss of granular cells in the cerebellum, and loss of other nerve cells in the cerebral cortices were observed. In severe cases, Purkinje cells in the cerebellum were damaged (Irukayama, 1966). In a second study in cats, 1 - 2. 5 mg/kg of mercury/day of methylmercuric chloride caused extreme convulsions in 20 - 30 days. Mercury concentration was highest in liver, hair , kidneys and brain, in that order (Tokutomi, 1968). Comparative studies on the toxicity of the various alkylmercury deriva- tives indicate that the time of onset of toxic signs, following daily oral ad- ministration of 5 mg mercury/kg to rats, increases with increasing length of the alkyl chain. In these experiments tissue analysis failed to indicate any relationship between mercury levels in the brain and manifestations of intoxication (Nose, 1969).

6.4.2.4. Effect of alkylmercury compounds on reproduction

Reproduction studies with methylmercuric dicyandiamide administered intraperitoneally at 0. 1 mg/mouse before mating reduced the incidence of but had no effect on litter size in those animals which mated successfully. A similar dose administered on the 10th day of pregnancy resulted in significantly increased embryonic mortality (Frolen and Ramel, 1969). In the rat, daily administration of 0. 1 mg mercury/kg (as methyl- mercury) throughout pregnancy resulted in reduced birth-weight, and postnatal neurological disorders (Fujita, 1969). Matsumoto et al. (1967) reported on

75 the histological examinations of embryonic rat brains, following maternal administration of single doses of 2 or 20 mg methylmercuric chloride/kg on days 9, 10 and 11 of pregnancy. In the foetal brain the morphological changes were more pronounced in the cerebellum as distinct from the cerebral cortex. Abnormalities were evident at the low dose but at the higher dose there was distortion of the whole shape of the cerebellum as well as enlarged ventricular cavities, and nerve cell degeneration in the mid- brain. A similar experiment at 2 mg of methylmercuric chloride/kg during the first 20 days of gestation also resulted in histopathological damage in the brain comprising disappearance of nucleoli, reduction in chromatin granules and decrease of ribosomes in granular cells of the cerebral cortex. Purkinje cells were vacuolated or electron dense (Nonaka, 1969). Reproduction studies with ethylmercuric phosphate injected subcutane- ously at 40 mg/kg on the 10th day of pregnancy in mice did not affect litter size. However, foetal body-weight was reduced, and a 31. 6% incidence of cleft palate was observed. In a separate reproduction study mice were sacrificed on days 5 and 19 after receiving 40 mg/kg body-weight of ethyl- mercuric phosphate. The incidence of abnormal chromosomes was 19.3% on day 5, and 16.6% on day 9 in the treated mice compared with 3.2% for the controls (Oharazawa, 1968).

6.4.2.5. Toxicity of alkylmercury compounds to man

Hunter et al. (1940) observed four cases of poisoning in workers using methylmercury compounds in the manufacture of fungicidal dusts. The symptoms were severe generalized ataxia, dysarthria and gross constriction of the visual fields, but and intelligence were unaffected. Similar symptoms have been reported following the use of methyl- mercuric thioacetamide dermally in the treatment of fungal diseases (Okinaka et al. , 1964). Two major outbreaks of methylmercury intoxication have occurred in recent years in Japan. Duringthe period from 1953 to 1960 a total of 111 cases of a special syndrome occurred in the Minamata Bay area of Japan. The voluminous literature which arose from this incident is largely cited in the report of the Study Group of Minamata disease, Kumamoto University, Japan (1968). In the Minamata outbreak, 29 of 59 adults, 10 of 30 children and 2 of 22 "congenital" cases had died up to the time of writing of the report (Takeuchi, 1968). Again in 1964 to 1965 a second outbreak occurred in Niigata, in which 26 diagnosed cases were reported (Irukayama, 1966). In both outbreaks, industrial pollution of water (Minamata Bay and River, and the Agano River) and subsequent contamination of fish and shellfish by mercury as methylmercury were shown to be the cause of the reported in- toxications (Nomura, 1968; Kosaka and Takizawa, 196 7). The symptoms and signs in both outbreaks were similar to those de- scribed by Hunter et al. (1940). The disease started with sensory distur- bances of the extremities, the lips and tongue followed by tremor, ataxia, concentric constriction of the visual fields and impaired hearing. In Minamata, the frequency of the occurrence of the various symptoms and signs have been tabulated for 34 individuals in the report of the study group mentioned above. Often there is a long latent period between exposure and appearance of symptoms and signs which are irreversible, although the

76 motor functions did improve somewhat after medical rehabilitation (Kitagawa, 1968). The of methylmercury intoxication in man (fatal cases of Minamata disease) has been reported in detail by Takeuchi (1968). It ap- peared to affect almost all parts of the . However, as would be expected from the clinical syndrome, the greatest effects were observed in the cerebellar cortex and the cerebral hemispheres, especially the gyrus precentralis and area striata. These effects included various degrees of degenerative changes up to complete disappearance of the neu- rones, glial proliferation, hydrocephalus, perivascular oedema and demy- elination. The peripheral symptoms of paraesthesia and numbness have been explained after examination of the peripheral nerves from patients with Minamata disease, when demyelination and destruction of nerve fibres were revealed (Takeuchi, 1970). In Minamata, the prenatal intoxication usually appeared as non-specific , characterized by mental retardation and motor disturbances (Harada, 1968). The high incidence of the reported cases of cerebral palsy in the Minamata Bay area during the epidemic tends to indicate that the foetus may be at greater risk than the adult. This view is further supported by the fact that the mothers of affected offspring did not normally show any signs of methylmercury intoxication (Kakita, 1961). It is of interest to note that animal experiments have indicated that alkylmercury compounds cross the in the mouse (Berlin and Ullberg, 1963a; Ukita et al., 1967; Suzuki et al., 1967) and the rat (Fujita, 1969), and that once across the placenta the mercury may have a greater affinity for the foetal central nervous system than for that of the adult (Matsumoto et al. , 1967). Chromosome analysis of cultured lymphocytes from normal controls, and individuals with high mercury levels in erythrocytes, due to ingestion of contaminated fish, indicated that increased chromosome breakages could be correlated with increased mercury levels. The significance of these results is not known (Skerfving et al., 1970). There are many reports of poisoning following accidental ingestion of methylmercury compounds. Such intoxication has resulted both from the ingestion of treated seed grain (Englesson and Herner, 1952) or indirectly after consuming meat from animals which had been fed grain treated with alkylmercury compounds (Storrs et al., 1970). In Pakistan, in 1961, several families became ill after eating wheat-seed treated with phenylmercuric acetate and ethylmercuric chloride. Five out of 34 patients hospitalized were known to have died, but the total mortality may have been higher. Symptoms were typical of alkylmercury poisoning (Haq, 1963). A similar outbreak occurred in Guatemala in 1965 when there were 20 deaths among 45 people who displayed typical symptoms of poisoning after eating wheat-seed treated with methylmercury dicyandiamide (Ordonex et al., 1966). After ingestion of 200 - 500 g of peas treated with 95 mg ethylmercuric chloride (expressed as mercury)/kg by a 13-year-old boy, nausea and headache appeared at 2 - 3 hours followed by loss of orientation, visual hallucinations, hyperemia and hyperkinesia. Recovery was incomplete (Slatov and Zimnikova, 1968). In Iraq, 331 cases of poisoning with 36 fatali- ties resulted between 1956 - 60, due to ingestion of seed treated with ethylme rcuric-p-tolyl sulphanilide. Symptoms were slightly different to those encountered with methylmercury intoxication. Distinct electrocardiographic

77 effects (Dahhan and Orfalay, 1964), optical atrophy and reduced vision, skeletal and muscular spasm, polyuria and skin changes, ataxia and difficulties in walking were reported (Jalili and Abbasi, 1961). In Ghana, in 1967, a total of 17 out of 65 persons died in Keta Government Hospital following ingestion of stolen maize which had been treated with Merkuran, a product containing 2 per cent ethylmercuric chloride. The maize was reported to contain the equivalent of 15 - 20 ppm of mercury (Anon., 1967; Sandi, 1970). Levels of mercury in hair, as well as in blood, have been used to determine the intake of mercury. In Sweden a relationship between the average daily quantity of fish eaten and the level of mercury in hair and blood is evident (Birke et al., 1967). Similar comparisons between levels of mercury in hair and frequency of eating fish have been made in Japan. Analysis of long hair cut into segments enabled a determination of the peak period of mercury intake (Irukayama, 1966; Aoki, 1970).

6.4.3. Alkoxyalkyl mercury salts

In the rat, methoxyethylmercuric chloride has been shown to break down at the carbon-mercury bond to yield ethylene and inorganic mercury (Daniel and Gage, 1969). Achievement of a balance between intake and excretion, principal route of excretion, and general toxicology are similar to those en- countered with inorganic .and arylmercury salts. The half-life in the rat is approximately 10 days (Ulfvarson, 1962). The repeated oral administration of methoxyethylmercuric hydroxide at 50 ppm in the diet of cockerels did not elicit any adverse reactions. As determined at termination of dosing, the methoxyethylmercuric hydroxide was excreted more rapidly than other mercury compounds (Swensson and Ulfvarson, 1969). Intraperitoneal administration (2 mg/kg) to rats for 50 days resulted in an increased latency period of reaction to conditioned and situational reflexes, and a reduction of discriminating ability. At 0.2 mg/kg administered for 80 days, increased reaction time was noted in the latter part of the study, after the appearance of somatic signs (Lehotzky and Bordas, 1968).

6.5. COMPARATIVE STUDIES

There have been several studies reported in which various mercurial compounds have been compared with each other. Several of these have been reported under the sections on individual types of mercury. The following data provide additional information on the toxicity and distribution of various mercury compounds. In the long-term study in rats, already described, in which the animals were fed phenylmercuric acetate in their diet for two years, companion groups of rats were fed comparable levels of mercuric acetate instead. The storage in the liver and kidney was 10 to 20 times higher with phenylmercuric acetate. Renal damage occurred with both compounds, but was more severe with phenylmercuric acetate and occurred at much iower dose levels (Fitzhugh et al. , 1950). Injections of methylmercuric dicyandiamide or phenylmercuric acetate were given three times a week for up to 6 weeks to groups of female rats. Each dose injected was equivalent to 0.15 mg of mercury per rat. Tissue

78 levels of mercury were determined' after 1, 3 or 6 weeks using a dithizone method of analysis accurate to 1 ppm. No toxic effects were observed. In the animals given the methylmercury compound there was a progressive increase in mercury in kidney, liver, spleen and brain and cord and only about 25% of the dose appeared in the excreta. With the rats given phenyl- mercuric acetate a steady state in these tissues was reached by week 3 and excretion was then fairly constant (Gage, 1964). Following subcutaneous administration of mercuric chloride (3 mg 203Hg/kg), phenylmercuric chloride, ethylmercuric chloride, ethyl- mercuric cysteine or n-butylmercuric chloride (10 mg 203Hg/kg) to rats, the alkylmercury compounds were found to be more slowly excreted than the aryl or inorganic forms. Excretion via the faeces was highest for the phenyl- and inorganic mercury compounds although the faeces appeared to be the major route of excretion for all compounds. Mercury levels in the brain were highest for ethylmercuric cysteine, and then ethylmercuric chloride, butylmercuric chloride, mercuric chloride and phenylmercuric chloride, in descending order. In the early period after administration, the distribu- tion of the arylmercury was imilar to that of alkylmercury, but later it became similar to that of the inorganic mercury. Ratios of the levels of mercury in the brain to the levels in the plasma were about 2-3 for the ethylmercury compounds, about 1 for butylmercuric chloride and less than 0.6 for the phenyl- and inorganic mercury compounds (Takeda et al., 1968). Comparative studies following 18 months administration of 0, 0.1, 2 or 40 ppm mercuric chloride, ethylmercuric chloride or phenylmercuric acetate confirm that the brain concentrations attained were higher with ethylmercury than with the inorganic or arylmercury. However, brain content decreases with all compounds with increasing time, although again, alkylmercury seems to decrease more slowly. The only organs in which mercury levels remained high were the kidney and, to a lesser extent, the liver (Ikeda, 1970a). Suzuki et al. (1967) have reported the ratios of maternal:placental;foetal blood levels of mercury in mice, as 1: 19:0.4 for mercuric chloride, 1: 1.2: 2.1 for methylmercuric acetate and 1:4. 5:0. 3 for phenylmercuric acetate. The placenta appears to act as a barrier to the passage of mercuric chloride and phenylmercuric acetate but not to methylmercuric acetate. Cytotoxicity in HeLa cells for various mercury salts has also been investigated. Growth of cells was slightly inhibited by concentrations in the medium of 3. 2 ug mercuric chloride/ml and by 0.32 /ug/ml of phenyl-, ethyl- and butylmercuric chlo ride. Lethality to cells occurred at 32 jug/ml with mercuric chloride, and between 1 and 3.2 /ug/ml for the other compounds; the most toxic material was the ethylmercuric chloride (Umeda et al., 1969). The compounds methylmercuric dicyandiamide, methylmercuric hydro- xide, phenylmercuric hydroxide and methoxyethylmercuric chloride have been screened to determine their effect on Allium root cells. Treatment was from 4 to 72 hours. At concentrations below those which caused fixation or extensive death of the cells, typical c-mitosis and polyploidy were found. The lowest concentration necessary for a cytologically observable effect was 2.5 X 10"7 mol/litre (0.05 ppm of mercury) for the methyl- and phenyl- compounds. In another experiment with Drosophila melanogaster, larvae treated with mercury compounds displayed a highly significant increase in frequency of XXY females, but there was no difference in the frequency of XO males compared to controls (Ramel, 1967).

79 6.6. CONCLUSIONS

Exposure to mercury or its compounds may result in serious intoxication. It may occur among workers engaged in certain occupations or among indivi- duals accidentally consuming grossly contaminated food. This type of health problem is more readily identifiable and usually involves relatively small numbers of individuals. On the other hand, the potential health hazards of low levels of mercury in food is of greater concern, because the symptoms and signs of poisoning might not be readily diagnosed and because a very large number of people may be affected. It is well documented that mercury is present in different foodstuffs, although its levels vary according to the type of food, background level of mercury in the environment, etc. The health hazards involved in the consumption of such foods depend not only on the levels of mercury but also on the chemical nature of the mercury com- pounds because they possess different . Since it is impossible to completely eliminate mercury from our food, and because its levels vary greatly, it is imperative that appropriate tolerance levels of mercury compounds in foods be established for use by regulatory agencies in order to protect the health of the consumer. Such tolerances must be based on proper evaluation of relevant toxicological and related data. Although a considerable literature exists on mercury to date, there do not appear to be sufficient studies which permit the assessment of an (ADI) for man. This conclusion has been reached in the toxicolo- gical evaluations of organomercury compounds as assessed by the FAO/WHO Joint Meetings on Pesticide Residues in 1965, 1966 and 1967 (FAO/WHO, 1965, 1967a, 1968) and by the Joint FAO/WHO Expert Committee on Food Additives in 1966 and 1970 (FAO/WHO, 1967b, 1970). The latter, at its 19 70 meeting, noted the various points of toxicological concern (epidemics of poisoning, the higher sensitivity of the foetus, the severity and persistence of central nervous-system damage, and the relation between mercury expo- sure and chromosome breaks in lymphocytes) and urged that further work be initiated before an ADI could be established. Meanwhile, it recommended that mercury contamination from all sources should be reduced to a minimum.

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85

7. DETERMINATION OF MERCURY BY INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS

V.P. GUINN

7.1. INTRODUCTION

Mercury is a toxic element, even when present in foodstuffs at levels of the order of some parts-per-million. In environmental science studies, it is important to have reliable, rapid, inexpensive methods available for the measurement of mercury in samples of many different kinds (sea-water, fresh-water, marine sediments, plankton, shellfish, fish, flesh from birds and mammals, hair, blood, rocks, etc.), in some cases down to levels as low as 10"5 ppm. As will be developed below, high-flux thermal-neutron activation analysis (NAA) - essentially the only analytical method capable of accurately measuring mercury levels as low as 10"5 ppm — can be used in its purely instrumental, non-destructive form to measure mercury levels in many kinds of important matrices down to levels as low as 0. 01 ppm. Since mercury "tolerance" levels in some foodstuffs are currently set at 0. 5 or 1.0 ppm, this instrumental limit of detection is already more than adequate for large-scale screening of foodstuffs for mercury.

7.2. STABLE NUCLIDES OF MERCURY AND THEIR (n, y)

Mercury in nature consists of a mixture of seven stable nuclides. Upon exposure to a flux of thermal neutrons, these form five radionuclides, by (n, 7) reaction. Since all five of these radionuclides are gamma-ray emitters, each offers the possibility of being used in the instrumental NAA determina- tion of mercury. These seven stable nuclides, their abundances, their isotopic thermal-neutron (n, 7) cross-sections, their (n, 7) products, and the half-lives of their five radioactive products are listed in Table XII (data from Refs [1] and [2]).

7. 3. DECAY SCHEMES OF THE FIVE (n, 7) PRODUCT MERCURY RADIONUCLIDES

The X-ray and gamma-ray emissions of the five (n, 7) product mercury radionuclides are summarized in Table XIII (detailed decay schemes, /B"Emax energies, and internal conversion coefficients may be found in the "Table of Isotopes" by Lederer, Hollander and Perlman [1]).

7.4. CALCULATED INTERFERENCE-FREE LIMITS OF DETECTION

From the abundance, cross-section, half-life, and decay-scheme data of Tables XII andXIII, the specific activity of each of the five (n, 7) product

87 TABLE XII. STABLE NUCLIDES OF MERCURY, AND THEIR (n, 7) PRODUCTS

Thermal-neutron Stable Abundance (n.y) Half-life (n,y) cross-section nuclide product of product3 (% (barns)

I97m 196Hg 0.146 125 Hg 24 h

2930 "'Hg 65 h

198Hg 10.02 0.018 '»mHg 43 min

'"Hg 16.84 2500 »»Hg Stable

!°°Hg 23.13 <60 2»lHg Stable

2°'Hg 13.22 <60 !MHg Stable

202Hg 29.80 4.5 203Hg 46.9 d

304Hg 6. 85 0,43 M5Hg 5.5 min

a Abbreviations: min = minutes, h = hours, d = days.

TABLE XIII. X-RAY AND GAMMA-RAY EMISSIONS OF THE FIVE (n, 7) PRODUCT RADIONUCLIDES OF MERCURY

°h decay Half-life — —^ —- ^ and y-ray energies (MeV) by ITd by ECa by 0 and 70 emissions (unconverted)

197mHg 24 h 94 6 0 HgX-raysb 0.134 (42% 0.279 (7%

7Hg 65 h 0 100 0 AuX-raysb 0.0773 (18% 0.191 (2<7o) 0.268 (0.15%)

'"mHg 43 min 100 0 0 HgX-raysb 0.158 (53%) 0.375 (15"?.)

Hg 46.9 d 0 0 100 0.279 (77%

Hg 5.5 min 0 0 100 0.205 0?)?)

a Abbreviations: IT = isomeric transition, EC = (orbital) . b X-ray energies, in keV, with relative intensities in parenthesis:

Ka2 Kal Kg^ • Kg.2 Fluorescent yield

Mercury: 68.9 (55) 70.8 (100) 80.2 (35) 82.5 (10) 0.95

Gold: 67.0 (55) 68.8 (100) 77.9 (35) 80.1 (9) 0.95

88 radionuclides of mercury may be calculated, for any selected values of thermal-neutron flux (

ip = 1 x 1013n/cm2 sec (thermal) tj = one hour irradiation

tc = one hour counting Cmin.det. =100 net photopeak counts.

TABLE XIV. CALCULATED LIMITS OF DETECTION FOR MERCURY BY THERMAL-NAA 13 2 (plx 10 n/cm sec, ti = 1 h, tc = 1 h)

Ey Calculated photopeak Calculated limit of detection Radionuclide Half-life (MeV) (cpm/g Hg) (in |jg of Hg)

197 mHg 24 h 0.134 9.5 x 108 0.002

"'Hg 65 h 0.077 4.0 x 109 0.0003

199 mHg 43 min 0.158 2.4 x 10 0.01

!03Hg 46.9 d 0.279 1.9 x 108 0.01

2°5Hg 5.5 min 0.205 5 x 108a 0.02

a Assumes 5<7° gamma-ray emission by 205Hg.

Under the defined conditions, it is evident that the most sensitive deter- mination of mercury is attained by detection of the induced 65-h197Hg activity. The limit of detection for mercury, when using the 65-h 197Hg activity, is actually closer to 0. 00004 /ug than to 0. 0003 ng since about 6. 3 Au K X-ray counts (at 67 - 80 keV) are obtained for each 77. 3 keV 197Hg gamma-ray count, and 67 - 69 keV and 77 - 80 keV peaks - both resulting from the EC decay of 197Hg - are not resolved from one another by a Nal(Tl) detector. With a Ge(Li) detector, they are resolved from one another, but the counts in the two peaks can be added together, if desired. With longer irradiations (e. g. 10 h) and/or higher thermal-neutron fluxes (e. g. 1014 n/cm2 sec), the limit of detection for mercury can be reduced to 4 x 10"6pg, or even 4 x 10"7 pg.

89 7.5. PRACTICAL INSTRUMENTAL LIMITS OF DETECTION IN SOME ILLUSTRATIVE MATRICES

Whereas the interference-free limit of detection for mercury, via detection of the 67 - 80 keV composite X-ray and gamma-ray photopeak of 6 5-h 197Hg, is calculated above to be approximately:

4 x 10," 5 g for cp = 101 3 , tjt j = 1 h or 4 x 10"6 ng for q> = 1013, ttjj = 10 h, or

A. Instrumental NAA determination of Hg in grain

Grain (wheat, rice, barley etc.) or grain products such as flour or bread can sometimes be contaminated with significant levels of mercurial fungicide-treated seed grain. Such treated seed grain may typically contain from 10 - 30 ppm Hg, and is quite toxic to animals, birds and man. Although forbidden by law to be used for other than planting, sometimes such grain is accidentally or deliberately introduced into foods, either undiluted, or diluted with untreated grain. The range of mercury concentrations of interest, in checking possibly suspect samples of grain or grain products for mercury, is thus from about 0. 001 - 0. 01 ppm Hg(the approximate range of Hg levels in "natural" or uncontaminated grain) on up to perhaps 30 ppm Hg (pure treated seed grain). Experiments in various laboratories, including the author's former laboratory (Gulf General Atomic), showed that purely instrumental NAA could determine mercury in a wheat-flour sample contaminated with about 5 ppm Hg very precisely and accurately, and could detect, but not measure very precisely, a level of about 0. 04 ppm Hg in an almost uncon- taminated sample of wheat flour. These inter-laboratory comparison flour samples were distributed to many laboratories by the IAEA. Details regarding the analysis of these samples, carried out in the author's former laboratory, are described below. These measurements showed that the instrumental NAA approach, using ~2-g (4-cm3 bulk volume) samples of flour and only a 30-min activation at a moderate thermal-neutron flux (1. 8 x 1012 n/cm2 sec), gave precise and accurate results for mercury at the ~5 ppm level either via Nal(Tl) or Ge(Li) gamma-ray spectrometry measurement of the X- and gamma-ray emissions of the induced 65-h197Hg activity. The pulse-height spectrum obtained in a 30-min count of an activated flour sample containing 5. 1 ppm Hg, on a 15-cm3 Ge(Li) detector, is shown in Fig. 18. It is seen: (1) that the 67 - 69 keV K X-ray peak of gold and the 77 - 80 keV X-ray and gamma-ray peak, both due to197Hg, are almost completely resolved from one another; and (2) that the heights of the two peaks are almost the same

90 FIG. 18. Ge(Li) gamma-ray spectrum of neutron-activated flour containing 5.1 ppm Hg.

(however, the X-ray peak, including both the K^and K^ X-ray energies of gold, 68. 8 keV and 67. 0 keV, respectively - in the ratio of 1. 00 to 0. 55 - is somewhat broader than the 77 - 80 keV X-ray and gamma-ray peak, and hence has a somewhat larger area than the 77- 80 keV peak, even though the latter also includes 77. 9 -keV and 80. 1-keV gold Kg X-ray pulses, in addition to the 77. 3 keV 197Hg gamma-ray pulses). Depending upon the counting geometry (which determines the number of 197Hg K + L X-ray and 7 + K X-ray coincidence pulses), the 67 - 69 keV peak is found to have an area from 1. 5 to 1. 9 times that of the 77 - 80 keV peak. Aqueous Hg standard solutions, similarly activated and counted, gave pulse-height spectra identical with the spectrum shown in Fig. 18, in this region of the spectra. The same 5. 1 ppm Hg activated flour samples were also counted on top of a 3-in. x 3-in. solid Nal(Tl) scintillation crystal detector, resulting in pulse-height spectra such as the one shown in Fig. 19 (in this case plotted on a semi-logarithmic scale). For comparison, Fig. 19 also shows the Nal(Tl) pulse-height spectrum of a similarly activated and counted aqueous Hg standard solution. In the spectrum of the Hg standard, the photopeaks that are evident are: (1) the large combined 67 - 69 keV/ 77 - 80 keV peak of 197Hg, (2) a small peak of 197mHg at 134 keV, (3) a small peak of 197Hg at 191 keV, and (4) a small combined peak due partly to 197Hg (268 keV) but mostly to 203Hg (279 keV). In the spectrum of the activated 5. 1 ppm Hg flour sample, only the combined 67 - 80 keV peak of 197Hg was clearly discerned, the other mercury peaks being too small to show up above the Compton continuum caused by higher-energy gamma rays from the various other significant induced activities present in the activated flour sample (e.g., 35. 34-h 82Br, 14. 96-h 24Na, and 12. 36-h 42K). The degree of precision attainable by means of instrumental NAA, for grain samples containing ~ 5 ppm Hg, may be seen in Table XV. In this table, the mercury values found in three aliquots (each ~ 2 g) of the IAEA laboratory intercomparison mercury-contaminated flour sample are shown.

91 100,000

-I W 10,000

< X o 1,000 oc. Ui a

tn 100

3 O o 50 100 150 200 CHANNEL NUMBER FIG. 19. Nal(Tl) gamma-ray spectra of neutron-activated flour containing 5.1 ppm Hg and of a mercury standard solution.

TABLE XV. INSTRUMENTAL NAA RESULTS ON A MERCURY- CONTAMINATED FLOUR SAMPLE

Solid Solid Well-type Detector: Ge(Li) Nal(Tl) Nal(Tl) Nal(Tl)

Decay time: 1 day 7 days

Sample transfer to fresh vial: No No Yes Yes

Aliquot No. ppm Hg found

1 5, .07 ± 0.17 4. 94 ± 0.07 4.94 ± 0.,0 4 4.90 ± 0.04

2 5.,0 3 ± 0.16 5. 09 ± 0.07 5.11 ± 0.,0 4 5.05 ± 0.04

3 5.,3 0 ± 0.16 5. 35 ± 0.07 5.35 ± 0.,0 4 5.12 ± 0.04

Mean: 5.13 ± 0.15 5.13 ± 0.21 5.13 ± 0.21 5.02 ± 0.11

All three aliquots were activated in the reactor at the same time (30 min at a thermal-neutron flux of 1. 8 x 1012 n/cm2 sec). Each activated sample, as shown, was counted four different ways: once with a 15-cm3 Ge(Li) detector, twice with a solid 3-in. x 3-in. Nal(Tl) detector (with, and without, transfer to a fresh polyvial), and once with a well-type 3-in. x 3-in. Nal(Tl) detector (at a longer decay time, after transfer to a fresh polyvial). The mercury values shown, where the activated samples were not transferred to fresh polyvials for counting, are those obtained after subtracting a small blank value, obtained by separate analyses of several empty polyethylene vials. The blank level was found to be 0. 026 ± 0. 006 ng Hg per 2-dram polyvial. The mercury values shown, where the activated samples were

92 transferred to fresh polyvials for counting, are those obtained after adding 0. 19 ± 0. 02 /ug Hg to each value found - to compensate for the 197Hg activity retained in the walls of the polyvials when the samples were transferred. The 0. 19 ± 0. 02 /ug Hg correction (above the 0. 026 ± 0. 006 /ug Hg blank) was measured experimentally on the six irradiated polyvials from which , the activated flour samples were transferred. The ± values shown in Table XV, after each of the twelve experimental ppm Hg values, represent one standard deviation (la)- based only on the counting statistics. However, the ± values shown in the table after each of the four mean ppm Hg values represent one standard deviation, based upon the deviations, in each case, of the three values from the mean value. It is seen that the four mean values agree to within their respective ± 1 a values. The overall mean value, for all twelve determinations, is 5. 10 ± 0. 16 ppm Hg, i. e. the relative standard deviation of the mean value is ±3. 1% of the value. Except for the Ge(Li) values, it is seen that the precision of the mean values is appreciably poorer than one would expect from the counting statistics. This discrepancy is believed to be mainly due to sample heterogeneity. Even though considerable care was taken to try to obtain representative aliquots of the flour, it is evident from the table that aliquot 3 actually contained a higher concentration of mercury (5. 28 ± 0. 11 ppm) than did aliquots 1 and 2 (which averaged 5. 02 ± 0. 08 ppm). Because of the very low energies of the197Hg radiations (67- 69 keV and 77 - 80 keV), it was feared that the degree of in-sample radiation absorption and scattering might be significantly different in the flour samples than in the aqueous Hg+2 standard solutions, thus leading to a consistent error in the results. To investigate this possibility, precisely 1-, 2-, 3- and 4-cm3 samples of the aqueous Hg+2 standard solution, and 1-, 2-, 3- and 4-cm3 samples (0. 550, 0. 893, 1. 44 and 2.28 g) of the mercury- contaminated flour were activated (each in a 2-dram polyvial, which has a capacity of 7 cm3) and counted, as before. .No significant trend in the specific counting rate (197Hg photopeak cpm per gram of sample) could be detected in either the standard solution or flour-sample values. Thus, the 1-, 2-, 3- and 4-cm3 standard solution samples gave specific photopeak counting rates of, respectively, 9. 50 x 104, 9. 82 x 104, 9. 90 x 104, and 9. 50 x 104 cpm/g solution, and the 1-, 2-, 3- and 4-cm3 flour samples gave specific photopeak counting rates of, respectively, 3. 13 x 103, 3. 19 x 10s, 3. 18 x 103 and 3. 16 x 103 cpm/g. The ratio of cpm/g of flour sample to cpm/g of standard solution, comparing the values for the same sample volumes, were thus 0. 0330, 0.0325, 0. 0321 and 0. 0333, giving a mean value of 0. 0327 ± 0. 00053 (i. e., a relative standard deviation of only ±1. 6% of the value). The overall mean value found (5. 10 ± 0. 16 ppm Hg) was for the sample on an "as-received", undried basis. The moisture content was then determined, via the loss in weight during 24 h at 110°C, resulting in a mean value of 13. 9% moisture. Thus, on a dry-weight basis, the contaminated flour contained 5. 93 ± 0. 19 ppm Hg. It should be noted that, in mercury determinations - by whatever analytical method - the moisture content should be determined after measurement of the mercury content (or on separate aliquots), not before mercury measurement. Most materials will lose a considerable fraction of their mercury content during such a conven- tional drying. For example, it was found that drying this flour sample for 24 h at 110°C caused a 2 7% loss of its mercury content.

93 When the essentially uncontaminated IAEA flour sample was similarly analysed for mercury, only a very slight indication of the 197Hg 67 - 69 keV and 77 - 80 keV peaks could be observed in the Ge(Li) pulse-height spectrum - as shown in Fig. 20. From the data, an upper limit of 0. 04 ppm Hg was established for this sample. It may be noted that several other workers, employing NAA with radiochemical separations, obtained a value of 0. 038 ppm Hg for this sample. Further details on these mercury studies and on other related NAA pesticide-residue studies have been published by Guinn [4], Graber and Guinn [5], and Guinn, Graber and Fleishman [6],

1000 2-gram sample

15 cm3 Ge(Li) 0.25 keV/channel 30-minute count

< > > x 2 jc o ff> h- <0 t>-

CHANNEL NUMBER 1024

FIG.20. Ge(Li) gamma-ray spectrum of flour containing <0.04 ppm Hg.

B. Instrumental NAA determination of Hg in ocean fish

In recent years, considerable attention has been focused on the deter- mination of mercury in various species of fresh-water and ocean fish. This interest stemmed from two major mercury poisoning incidents in Japan (involving high levels of mercury in river fish, resulting from large accidental discharges of waste mercury effluents from chemical plants into rivers), and studies of mercury contamination of fresh-water fish in Swedish lakes and rivers (the contamination coming from pulp and paper-plant effluents, and from mercurial fungicide run-off into the lakes and rivers), and in Canadian and U.S. lakes and rivers (in this case, the major source of pollution being effluents from alkali-chlorine plants). Appreciable mercury levels have also been found in certain species of Swedish birds (both fish- eating and grain-eating), and in certain species of Canadian and U.S. grain- eating birds. The sources of mercury in these cases appear to be mainly mercury-contaminated fish, in the case of fish-eating birds, and mercurial fungicide-treated seed grain, in the case of grain-eating birds.

94 Most of the mercury determinations currently being reported, for samples of various kinds of fish, are based upon flameless atomic absorption measurements, plus a sizeable number based upon NAA with radiochemical separations. Both of these techniques suffer from the disadvantage that they require a time-consuming wet ashing of each sample. In the case of the flameless atomic absorption method, there is evidence that consistently low values are obtained, probably mainly due to losses of some dimethyl mercury during the wet-ashing step, prior to reduction with titanous chloride and purging into the absorption cell. In the case of the radiochemical-NAA method, such losses do not invalidate the results - provided that the equilibration with Hg2+ carrier has proceeded rapidly enough. It appeared that, just as in the case of grain samples, it should be possible to obtain quite sensitive, precise, and accurate measurements of the mercury levels in fish samples also, down to levels of a few hundredths of a ppm Hg - by purely instrumental NAA. Such measurements, being purely instrumental, would take very little of a chemist's time, and would be free of any type of mercury loss or contamination errors (since samples and standards would be sealed in quartz ampoules). In view of the reasonably long half-life of 197Hg (65 h), and the large sample capacity of the rotary specimen rack of the author's TRIGA reactor, it was obvious that many samples could be activated simultaneously. The major initially unanswered question was the extent to which the very high level of induced 14. 96-h24Na activity in ocean fish samples would affect the instrumental NAA limit of detection for mercury and affect the optimum decay time for measurement of the 197Hg activity. Preliminary measurements, employing 1- to 3-h activations of 1- to 2-g samples of , in a thermal-neutron flux of 0. 7 x 1012 n/cm2 sec, showed that the optimum decay time for precise measurement of the induced 197Hg activity is typically in the range of 5 - 7 days. Subsequently, many specimens of marine biological species (e. g. swordfish, tuna, totuava, cod, shrimp, mussels, anchovies, squid etc.) have been analysed for mercury in

TABLE XVI. ILLUSTRATIVE MEASUREMENT PRECISIONS, AND AGREEMENT BETWEEN THE TWO MERCURY-197 PEAKS (Instrumental NAA measurements on various tuna samples)

Ratio ppm Hg found: Sample of From 67 - 69 keV peak From 77 - 80 keV peak values

Fresh 0.125 ± 0.013 0.124 ± 0.015 1.008

Canned (Brand 1) 0.473 ± 0.036 0.486 ± 0.042 0.9733

Canned (Brand 2) 0.303 t 0.015 0.287 ± 0.017 1.056

Canned (Brand 3) 0.384 ± 0.031 0.366 ± 0.035 1.049

Mean: 1.022

95 the author's laboratory — particularly by R. Kishore - by means of instru- mental NAA, normally using 2-g samples, a 3-h activation at a thermal- neutron flux of 0. 7 x 1012 n/ cm2 sec, and gamma-ray spectrometry with a 36-cm3 Ge(Li) detector and 4096-channel pulse-height analyser (one to a few hours of counting). Under these conditions, as is shown below, good precisions are attained at the 0. 1 - 1 and higher ppm Hg levels, and the limit of instrumental detection is about 0. 02 ppm Hg. The precisions attained, and the degree of agreement of the ppm Hg values obtained from the two different 197Hg peaks (67 - 69 keV and 77 - 80 keV), may be illustrated by the values shown in Table XVI for some fresh and canned tuna specimens. These results are shown only to illustrate these two points, i. e. the different tuna specimens are not expected to have the same mercury concentrations and, in fact, are seen to range from about 0. 1 to 1 ppm Hg (wet-weight basis). The ± values shown represent 1 a, based only upon the counting statistics. It is seen that the ± values are all in the range of ± 0. 01 to ± 0. 04 ppm Hg (averaging ± 0. 026) and that the mercury values found separately from the two197Hg peaks agree within about 2%. At higher mercury levels (~1 ppm and higher), or with longer counting periods, the relative standard deviations of the resulting ppm Hg values improve to ± only 2 - 3% of the value. Under the irradiation and Ge(Li) counting conditions used, the 67-69 keV and 77-80 keV197Hg net peak counting rates, at zero decay time, are found to be re- spectively, 187cpm/|Ug Hg and 129 cpm//ug Hg (using, however, only the centre portions of each peak). For a 1-h irradiation at a flux of IX 1013 (instead of a 3-h irradiation at 0. 7 x 1012), these values become, respectively, 891 cpm//ug Hg and 615 cpm/pg Hg. The 615 cpm//ug Hg value, obtained with the 36-cm Ge(Li) detector, is fairly close to the calculated value for a 3-in. x 3-in. Nal(Tl) detector, shown in TableXIV, i. e. 4000cpm/jug Hg - and in keeping with the somewhat poorer counting geometry of the smaller-area Ge(Li) detector, and the fact that only the centre portion of the Ge(Li) 77 - 80 keV peak was used. If samples and standards are each sealed in quartz, measurements show no detectable loss of mercury from either, in 3-h irradiations at a thermal-neutron flux of 0. 7 x 1012 n/cm2 sec. If samples and standards are instead heat-sealed in conventional 2-dram (7 cm3) polyethylene vials, the same irradiation is found to result in a 10 ± 2% loss of mercury (both of stable mercury and 197Hg, as established by a later re-irradiation) from the aqueous standard solution (containing ~5 /ug of Hg), but in no detectable loss from the fish samples. Apparently (it is hypothesized), radiolysis of the aqueous solution produces reducing agents that can reduce a good deal of the Hg+2 to give free mercury, some of which can diffuse into, and some through, the polyethylene — some mercury actually being detected in the outer plastic capsule in which the polyethylene vial is placed for reactor irradiation. In the case of the fish samples, the reduction of mercury is perhaps less, and/ or any reduced mercury is recaptured within the sample and hence does not escape.

C. Other matrices

Mercury can also be easily detected and measured in various other kinds of matrices by purely instrumental NAA. For example, it is readily measurable in samples of hair.

96 REFERENCES TO CHAPTER 7

[1] LEDERER, C.M., HOLLANDER, J.M., PERLMAN, I., Table of Isotopes, 6th edn, John Wiley and Sons, New York (1967). [2] HUGHES, D.V., SCHWARTZ, R.B., Neutron cross sections, BNL-325 (and Supplement 2). [3] HEATH, R.L., Scintillation Spectrometry Gamma-Ray Spectrum Catalogue, USAEC Rep. IDO-I688O-I and -2 (1964). [4] GUINN, V.P., Neutron activation analysis and its application to the analysis of food products, J. Am. Oil Chem. Soc. 45 (1968) 767. [5] GRABER, F.M., GUINN, V.P., Instrumental neutron activation analysis determination of mercury pesticide residues in food-stuffs. Gulf General Atomic Rep. GA-8208 (1969) 14 pp. [6] GUINN, V.P., GRABER, F.M., FLEISHMAN, D.M., Ge(Li) gamma-ray spectrometry as a pilot for Nal(Tl) gamma-ray spectrometry, Talanta 15 (1968) 1159.

97

8. THE DETERMINATION OF MERCURY AND ITS COMPOUNDS BY DESTRUCTIVE NEUTRON ACTIVATION ANALYSIS

T. WESTERMARK and K. LJUNGGREN

8.1. INTRODUCTION

Most samples that have a mercury (Hg) content which it is of interest to determine contain elements that interfere with the Hg determination by neutron activation analysis since they give rise to activation products with radiations overlapping that of the Hg nuclides. Therefore the destructive type of activation analysis is often necessary. When a destructive activation analysis method is available, experience has shown that it is expedient to apply it to all samples as a matter of routine simply because one cannot predict which samples could have been analysed satisfactorily using a non-destructive method.

8.2. TECHNICAL PROCEDURES AND AN ATTEMPT TO APPRAISAL

8.2.1. The principles

An important principle in activation analysis is that care should be exercised in the collection of samples and that treatment of samples before irradiation should be kept to a minimum in order to avoid con- tamination during the only stage when it is fatal also to this method of analysis. The glassware etc. must be checked as well as the chemicals used for these purposes to ensure that no significant contamination occurs. There are, in our experience, two instances in destructive activa- tion analysis of mercury when pretreatment of samples is desirable: one is the separation of coarse particles from water samples, e.g. by centrifugation, the other is the preservation of fresh biological samples, e.g. fish, before storage or transportation by the addition of . After irradiation, carrier Hg and some acid for wet combustion are added in most methods. These additions should be made simultaneously and in such a way that no losses of Hg could occur, even under the worst conditions. After wet combustion, the Hg (sample content plus carrier) is re- covered in a pure form separated from other elements which may give interfering activities. This separation often consists of more than one step to ensure sufficient purity of the final Hg gamma spectrum. The recovery of the carrier is determined, usually by weighing the final preparation made for counting, and correction is made for loss of activity. This principle of "radiometric correction", given by de Hevesy as one of several varieties of the isotope dilution method, is very important

99 TABLE XVII. METHODS FOR DESTRUCTIVE ACTIVATION ANALYSIS OF MERCURY

Wet Main method of Carrier yield Applied to Reference Principle of detection Nuclide measured combustion separation measurement

Hamaguchi et al.[2] Beta counting 203 Hg Yes Precipitation as Yes Marine the sulphide organisms and water

Sjostrand [3] Gamma spectrometry 197 Hg Yes Distillation and Yes Virtually all electrolysis sorts of samples

Kellershohn et al. [4] Gamma spectrometry 203 Hg Yes Precipitation of Yes Blood Hg and distillation

Kim et al. [ 5] Gamma spectrometry '"Hg Yes Exchange with No a Biological metallic Hg samples

Kosta et al. [6] Gamma spectrometry 197 Hg No Evaporation and Nob Biological (burning in sorption on samples and hot air and water oxygen)

Johansen et al. [7] Gamma spectrometry 197 Hg Yes Sulphide Nob Biological precipitation and some at pH 8-9 industrial samples

b Hasanen [8] Gamma spectrometry 1W Hg Yes Desposition No Biological on Cu powder samples

a Yield determined to be 90% in separate study; all samples corrected for this, b Assumed to be close to lOO^o. since it corrects for all losses after the moment when sample and carrier have been brought together into a homogeneous system, or at least to a state where all Hg atoms have had a chance to exchange (which they do in acid aqueous solutions). For example, losses resulting from interrupting a distillation, a precipitation or an electrolysis a little too early are corrected for. Thus, several types of errors ascribable to the apparatus or to the human factor are ruled out automatically. This feature is unique to activation analysis.

8.2.2. Survey of published varieties of the method

Many papers have been published on the destructive neutron activa- tion analysis of Hg. Since their object is the same, they are, in principle, varieties of a common theme which is to produce a sample for counting - a sample that is physically well-defined and chemically sufficiently free from other elements with nuclides which emit interfering radiations. The quality of the sample to be measured can be defined according to the degree of freedom from .interfering activities. Assuming equal sample quality, the various methods may then be appraised solely in terms of ease of performance and speed. Table XVII, which is not claimed to cover all published methods, gives a survey of methods for Hg analysis (multi-element methods where Hg is determined as one of 20 - 30 elements have been omitted). For a more complete survey the reader is referred to the NBS Bibliography on Activation Analysis [1],

8.2.3. Choice of nuclear reaction

Of the many alternatives which exist for the neutron activation of Hg, the 196Hg (n, y)197Hg reaction is preferred by most analysts. The alternative of using very short-lived Hg nuclides gives much lower sensitivity, but these products can be used if the neutron flux is very high or if ultimate sensitivity is not striven for. The reaction 202Hg(n, 7)203Hg is useful, but the cross-section referred to the natural element is lower than that producing 197Hg. Also, one has to irradiate for several weeks to get a reasonable degree of saturation. 203Hg is useful for an independent check of values obtained with 197Hg, which is of particular value if the 197Hg measurement has to be discarded for some reason. The presence of 203Hg also confirms the identification of Hg should it be questioned. There is a 24-hour isomer, 197m Hg, to be considered together with the 65-hour 197Hg. The cross-section for the formation of the former is much lower than that leading to 197Hg [9]. The 133-keV gamma line of 197mHg may be seen in the spectrum, particularly well in a Ge(Li) detector provided that the amount of Hg is ample. Thus 197mHg, as well as 203Hg, may add to the certainty of identification although it is not very useful for the quantitative analysis.

8.2.4. Choice of detector

A typical Nal(Tl) spectrum is shown in Fig.21. It is recorded by means of a well-type crystal having a very high geometric (in addition to a high intrinsic) efficiency. This type of detector gives prominence to the coincident sum peak of the 68-keV X-ray and the 77-keV gamma ray of 197Hg.

101 If the sample is located outside (e.g. on top of) the crystal, this peak is not so pronounced (cf. Fig.22). Nal(Tl) detectors and spectrometers are very widely used and the technique is very practical and successful. Numerous examples of the use of this technique can be found in the literature, one being the work by Kosta and Byrne [6]; cf. their Fig.2 of a grass sample containing 140 ng/g of Hg. The presence of 198Au [10], resulting from external contamination or from Au actually present in the sample, say in minerals, is some- times a nuisance. The 412-keV peak of 198Au is very pronounced in a Nal(Tl) detector. The corresponding X-ray peak (actually Hg K X-rays) must be subtracted from the composite 70-keV 197Hg-peak in quantitative work. This correction can easily be made by means of a 198Au Standard measured in the same detector set-up. The X-ray peak is of the order of 10% of the intensity of the 412-keV line. (The reason why Au may follow Hg is given below.) The modern Ge(Li) detector has a much higher energy resolution than Nal(Tl), providing half-widths of a few keV. It resolves the 68- and 77-keV peaks, which is demonstrated in Fig.23. Here the peaks stand out on top of a continuous background. The 279-keV peak of 203Hg

102 is also seen. The Ge(Li) detector is likely to reveal the presence of contaminants emitting gamma or X-rays. Care has to be taken with heavy-element linings in detector shields since disturbing X-rays may be excited by sample beta or background radiation [10]. The Ge(Li) would, however, identify the X-rays and still make a correct analysis possible. Linings should preferably be made of relatively low atomic-number materials, e.g. Cd or Sn, see Ref.[10]. The disadvantages of the Ge(Li) detector equipment for routine work are the following: The detector has to be kept at a low temperature (about -190°C) all the time and should never be allowed to get warmer than this. Further, the efficiency is lower than for a well-type Nal(Tl) crystal. Finally, the price is considerably higher. On the other hand, well- or tube-types of Ge(Li) detectors are becoming available. Also, the cooling is simplified by liquid-air tanks which have to be refilled only every 10 days. So, as the price is also falling, time favours the Ge(Li) detector. To be quite explicit: for a large-scale Hg project of environmental or toxicological nature we would recommend the Nal(Tl) detector to-day, but in the near future the Ge(Li) detector may be recommended instead.

103 1,0-10* •

0.8 -

uj X 2 0.6 O s

2 O o O.i

0.2

___ I . 0.1 0.2 0.3 MeV

FIG. 23. A Ge(Li) detector gamma spectrum of the same sample. The 68-keV and 77-keV peaks are well separated and stand out from the continuous background. The sample is placed on the surface of the detector. As the scale in counts is the same as in Figs 21 and 22, the total efficiency may be assessed from the figures.

8.2.5. Chemical separation and purification

The purpose of the chemical procedures is to produce a mercury activity freed from other activities which may disturb the final spectro- metry. In our well-established procedure this is made, after wet combustion, by two different consecutive processes:

(a) Distillation under conditions which favour the formation of chlorides; (b) Electrolysis which also produces a physically well-defined precipitate for spectrometry.

Distillation is a very efficient decontamination step; besides Hg, only Au, the and perhaps some other volatile compounds of elements like Se, Te and As may be carried over. But the advantage of

104 the combined steps is that in the electrolysis of these possible elements only Hg and Au are deposited. Au can be corrected for in the gamma spectrometry, unless large amounts are present (cf. above). When large amounts of Au are found, the precipitate is again dissolved in (Au will not dissolve) and then a new electrodeposition is made [11]. It is worth noting that the interference from 198Au is due to the fact that the element gold and the actual nuclide 198Au have a number of properties which correspond to those of mercury and 197Hg: gold has volatile chlorides (probably complexes) and so does mercury, 198Au has nearly the same half-life as 197Hg (65 hours) and it emits X-rays of very nearly the same energy as the quanta from 197Hg. Kosta and Byrne (Ref.[6] p.1297) encounter difficulties in completely decontaminating the samples by means of electrolysis. It is not clear to us whether they use both distillation and electrolysis which we consider necessary. In our experience of more than 25 000 analysed samples (many of which were water containing as little as 0.03 ng/g) we have never had any remaining contamination serious enough to render the Hg determination unduly uncertain.

8.2.6. Further points

With regard to sample treatment after irradiation there are two main routes: either wet combustion or dry ashing in air or oxygen. The former procedure admittedly takes time (about 30 min), but as many as 12 samples can be handled simultaneously by one person. The introduction of the carrier and the almost immediate contact between sample and combusting liquid ensure that the Hg in the sample and/or carrier is treated identically. An important merit is that the sample may be solid, liquid (or jelly-like) or gaseous. The dry ashing or heating procedure - which is also the method of choice in many atomic ab- sorption procedures - is especially adapted for solid samples. can be treated according to the latter procedure but losses may occur, particularly of very volatile liquids. require special techniques. In the dry ashing or heating procedures, Hg is collected either on Au or Se. The Kosta-Byrne procedure [6] is probably the best developed one. However, the preparation of the selenium paper is critical according to Finnish experience 112]. It is advisable to follow the original procedure for precipitating Se, as described by Stitt and Tomimatsu [12a]. Further, it may be important that mercury is always brought completely to the atomic state. It should thus be proven that the procedure is really capable of giving complete recovery when

methylmercury, MeHg, and dimethylmercury, (Me)2Hg, are dominating. Kosta and Byrne [6] show that a certain amount of carrier, although a thousand times smaller than that used in the Sjostrand procedure [3], is important because the recovery is otherwise incomplete. Also this point should be investigated for all chemical states of Hg possible. With regard to the final form of the Hg preparation for spectrometry the Sjostrand procedure produces a uniform sample, essentially free from self-absorption, at no extra expense of work as opposed to filtering procedures. The liquid Hg drop which is measured in the Kim-Silverman procedure [5] gives rise to some self-absorption to be considered. Also Kosta and Byrne ascribe some lack of reproducibility

105 in their gamma spectrometry to variations in the position of their selenium paper and the uneven Hg deposition in it [6]. No corresponding difficulty exists in the Sjostrand procedure. Kosta and Byrne [6] state that determination of the recovery is not necessary in their procedure. Both Johansen and Steinnes [7] and Hasanen [8] make the same statement. This point might be questioned since the recovery of 98 - 99% or more has been reported only in special cases. In general, a burden of proof rests with any method which re- quires a very large variety of samples of different types to be tested for recovery before one dare omit the determination of carrier recovery in routine work. The Sjostrand procedure reports the recovery of each sample, which - as stated above - makes possible a correction for any differences in processing. With regard to time consumption, the Sjostrand electrolysis pro- cedure may seem to be lengthy - 15 hours - but the actual handling of the samples requires very little time in routine work (of the order of minutes) since the process can be left unwatched overnight and produces directly a Hg preparation suitable for spectrometry. Thus, the labour cost of this step is low.

8.2.7. Precision, accuracy and sensitivity

These questions have been dealt with by the authors at some length in Ref.[13], soonlythe main conclusions are given here. The precision, given as the coefficient of variation, in the Hg determination of the Sjostrand procedure is a few per cent as is shown by repeated analyses of kale powder [14], fish, minerals, oils, blood, water, etc. As our determination of Hg in the international standard kale sample agreed with the best interlaboratory average within the limits set by the precision [14], the accuracy can also be claimed to be as good as a few per cent, which is of great value when the results of these analyses are to be used as grounds for decisions on highest permissible concentrations in foods, in human tissue, etc. A normal 2-day irradiation in a 3 - 4 X 1012 n/cm2 sec thermal neutron flux in a reactor gives a limit of determination of about 0.1 - 0.3 ng. For the 0.3-g sample routinely used for biological samples containing water this yields a concentration limit of 0.3 - 1 ng/g, which is sufficient for all samples of medical or ecological interest that we have met. Water, however, may contain as little as 0.03 ng/g, as in unpolluted fresh-water in Sweden. In these cases, larger samples, 3 or 10 g, are used [13]. Air and other gas samples usually contain very little Hg, which suggests the use of large samples. The sensitivity increases with the neutron flux used for irradiation. Some problems arise, however, with high-flux irradiation. Experience shows that the quartz is embrittled after exposure to high integrated flux values and, furthermore, that there is a risk of explosion due to over-pressure from radiolytic gases. This can be overcome with the laborious procedure involving the use of solid carbon dioxide cooling suggested by Brune [15]. Up to 4 X 1012 n/cm2 sec there are very few losses due to breakage, fortunately.

106 8.3. APPLICABILITY

8.3.1. General

The determination of Hg by neutron activation analysis (NAA), particularly in the Sjostrand version [3], has a very wide applicability. In most cases, the sample is very small; 0.2 - 0.3 g is, as mentioned above, a standard amount. Of hair, nails, feathers, i.e. dry proteins, only 0.03 g is usually used. The sample may be in the form of a solid, a liquid or a gas; after sampling and sealing in the quartz vial, the procedure is identical. All kinds of samples can be accepted, although minerals have to be subjected to a special digestion procedure [13]. One advantage is that there is no need to know even the order of magnitude of the Hg content beforehand since the procedure is equally suited to high mercury contents.

8.3.2. The mineral kingdom

The following types of samples are typical examples of applications:

Minerals sulphide ores (high in Hg), ores (low in Hg), limestones (low) [16]; Coal 50 - 500 ng/g [13]; Mineral oil limited experience; 10 ng/g seems to be typical; Soils typical levels 20 - 800 ng/g; Water salt-water 0.3 ng/g [13]; brackish water 0.15 ng/g; fresh-water 0.03 - 0.1 ng/g; contaminated water is higher [13]; Snow as for water, see above; Air here, particulate matter collected on filters is of special interest; Hg content very variable.

8.3.3. The plant kingdom

Typical examples and levels from the vegetative realm of the biosphere are:

Algae collected from fresh-water with plankton nets, levels 10 - 200 ng/g, water content important; Lichens from trees or stones, up to 500 ng/g has been seen; Mosses 50 - 400 ng/g, submersed water mosses generally higher, cf. Riihling and Tyler's work [17] on other deposited on mosses; Fungi, ferns, moderate levels 5 - 100 ng/g; examples: needle mono- and trees, Chenopodium album, Chelidonium majus, dicotyledons Triticum sativum, Holosteum umbellatum [16],

These latter types also include the edible vegetables which may be of interest to check from the point of view of food hygiene.

107 8.3.4. The animal kingdom (including man)

This is one of the most important domains from the ecological and medical viewpoints:

Micro- from 10 ng/g up to extremely high levels, i.e. in organisms biochemical research; Earthworms 50 - 500 ng/g, very easy to deal with, but the intestine should be freed from soil [16]; Insects relatively low, 5 - 100 ng/g; Molluscs shells are low in Hg, 10 - 30 ng/g, but muscle tissue may be rather high; up to 1 000 ng/g has been recorded; Fish white muscle [18], which is the edible part, ranges from 10 ng/g in Ethiopia to 25 000 in some places in Japan. In Sweden, 17 000 ng/g is the highest value found [13]. Hg is, to a very large fraction, in the form of methylmercury which is very toxic. The liver, kidney etc. of fish under natural conditions contain less Hg than the quantities mentioned above. However, the Hg contents in these organs may increase if there is a constant supply of the element. Marine fish contain typically 60 - 100 ng/g; Birds very easy to analyse: muscle, blood, brain, liver, kidney, claws, feathers; Mammals very easy to analyse: blood and parts of blood like plasma, red corpuscles, cerebrospinal fluid (1 - 3 ng/g only), brain, kidney, bone (low), hair; Man from living persons blood, which should be separated into plasma and corpuscles, and hair are preferred samples [19]. Nails may also be of interest. Post- mortem investigations may cover all parts of the body (see mammals). Normal figures are 1000 ng/g in hair, and 10 ng/g in blood corpuscles. The Hg content of blood or blood corpuscles seems to be of great toxi- cologic al significance (cf. Ref.[24]), especially in relation to consumption of fish.

These sample types include important parts of man's food which makes it possible to assess intakes by analysis. From an ecological point of view, the range of interest may be from very high levels in contaminated fish down to 1 ng/g [17, 19, 20]. In order to encompass the whole range, destructive NAA is certainly preferable.

8.3.5. Industrial or other man-made products

A wide variety of samples of manufactured materials have been successfully analysed which is exemplified by:

Inorganic "heavy chemicals", like (up to chemicals 10 000 ng/g), sulphuric acid (up to 2000 ng/g). Products, e.g. fertilizer phosphates. Calcium hydroxide (a few ng/g, for ), sulphate (for water purification, 30 ng/g);

108 Metals aluminium; Products from tars, raw naphthalene (up to 3000 ng/g), , gas and ammonium sulphate, the gas itself; works Household 14 - 300 ng/g for solid materials ( is much detergents higher, or 200 - 4 000 ng/g); Pharma- some substances are preserved by means of Hg ceuticals * phenyl acetate or other Hg compounds; Plastics poly-propylene, 220 ng/g [10].

8.3.6. Conclusions

Destructive neutron activation analysis, as represented by the Sjostrand procedure [3], can deal with virtually all kinds of samples where Hg is of particular interest. The concentration need not be known in advance, it is immaterial whether it is 1 ng/g or 300 000 ng/g, the treatment will be the same. As is evident from other contributions to this book, Hg is a pollution problem in many countries. By means of activation analysis of environ- mental samples, sources of Hg emission to the environment can be revealed and the paths which the element follows in nature can be traced. For control of fish and mammals in ecological projects, destructive activation analysis is ideally suited. Also for checks on blood and hair of extreme fish-eaters or persons otherwise subjected to Hg uptake, the method is very convenient. The combination of NAA for total Hg determination and of gas chromatography according to the methods developed by Westod [22] or Kitamura [23] for methylmercury and ethylmercury determination is very important for assessing the risks of harmful exposures [24], A mere determination of total Hg content in biological samples and foodstuffs is not sufficient. NAA is, at present, meeting competition from some other methods of analysis, particularly from the atomic absorption method. Many versions of this analytical method have been developed for mercury [25-27]. The procedures used for mineral analysis are different from those for biological samples [28], To some extent, comparisons with these methods have been made in the preceding paragraphs. However, no objective and complete evaluation seems to have been made so far of the two methods in comparison with one another.

8.4. OUTLOOK

The future of NAA seems promising in spite of the competition mentioned. NAA has been developed to a high degree of automation. In the spectrometer, samples are changed automatically and the spectra stored on magnetic or punched tape. Computer programs are available for the processing of the spectra if desired. In the present author's experience, an annual volume of 7 000 - 8 000 samples analysed barely motivates complete computerization of the evaluation. The analysis of Hg can conveniently be combined with the determination of other trace elements. We have found that a combination with arsenic and cadmium is of particular interest for environmental samples [13].

109 In the near future, Hg analysis will be used in ecological and toxicological investigations and for testing the wholesomeness of food as well as for checking industrial products and industrial and other emissions to the air and water environment. Official restrictions on the Hg content of air, water and food will have to be based on extensive analytical data to be justified. Last, but not least, man1 s body burden of Hg and MeHg will have to be kept under observation by blood analysis.

REFERENCES TO CHAPTER 8

[1] Activation Analysis: A Bibliography, NBS Technical Note 467, Parti (Sep. 1968), Parti, Addendum 1 (Dec. 1969), Part 2, Revision 1 (Dec. 1969), US Department of Commerce, National Bureau of Standards, , D. C. [2] HAMAGUCHI, H., KURODA, R., HOSOHARA, K., J. atom. Energy Soc. Japan 2 (1960) 317; see also HOSOHARA, K. et al., J. chem. Soc. Japan 82 (1961) 163. [3] SJOSTRAND, B., Analyt. Chem. 36 (1964) 814. [4] KELLERSHOHN, C., COMAR, D., LE POEC, C., J. Lab. clin. Med. 66 (1965) 168. [ 5] KIM, C. K., SILVERMAN, J., Analyt. Chem. 37 (1965) 1616. [6] KOSTA, L., BYRNE, A. R., Talanta 16 (1969) 1297; see also BYRNE, A.R., DERMELJ. M., KOSTA, L., "A neutron activation study of environmental contamination and distribution of mercury in animals and fish", Nuclear Techniques in Environmental Pollution (Proc. Symp. Salzburg, 1970), IAEA, Vienna (1971) 415. [7] JOHANSEN, O., STEINNES, E., Int. J. appl. Radiat. Isotopes 20 (1969) 751. [8] HASANEN, E., Nord. hyg. Tidskr. 50 2 (1969) 78; see also Suomen Kemistilehti 43 (1970) 251. [9] LEDERER, C.M., HOLLANDER, J. M., PERLMAN, I., Table of Isotopes, 6th edn, Wiley & Sons, New York (1967). [10] CHRISTELL, R. et al., "Method of activation analysis for mercury in the biosphere and in foods", - Radioisotopes in the Detection of Pesticide Residues (Panel Proc. Vienna, 1965), IAEA, Vienna (1966) 90. [11] LJUNGGREN, K. et al., Nord. hyg. Tidskr. 50 2 (1969) 75. [12] MIETTINEN, J. K., Personal communication (1970). [12a] STITT, F., TOMIMATSU, Y., Analyt. Chem. 23 (1951) 1098. [13] LJUNGGREN, K. et al., "Activation analysis of mercury and other environmental in water and aquatic ecosystems", Nuclear Techniques in Environmental Pollution (Proc. Symp. Salzburg, 1970), IAEA, Vienna (1971) 373. [14] BOWEN, H.J. M., "Standard materials and intercomparisons". Advances in Activation Analysis, Vol. 1 (LENIHAN, J. M.A., THOMSON, S.J., Eds), Academic Press, London (1969) 101. [15] BRUNE, D., Aspects of low temperature irradiation in neutron activation analysis, Natn. Bur. • Stand., Spec. Publ. 312, Modem Trends in Activation Analysis, Vol. 1 (1969) 203. [16] WESTERMARK, T., SJOSTRAND, B., "Activation analysis of mercury and its applications", To be published in Advances in Activation Analysis, Vol.2 (LENIHAN, J. M.A., THOMSON, S.J., Eds), Academic Press, London (1971). [17] RUHLING, A., TYLER, G., Botaniska notiser (Lund, Sweden) 121 (1968) 321; ibid. 122 (1969) 248. See also Heavy metal contamination in the Oskarshamn area (in Swedish), University of Lund, Dept. of Ecological Botany Rep. Nov. (1969) and following reports. [18] JOHNELS, A. G. et al., Oikos 18 (1967) 323. [19] BIRKE, G, et al., Lakartidningen 64 (1967) 3628. [20] BERG, W. et al., Oikos 17 (1966) 71. [21] JOHNELS, A. G., OLSSON, M., WESTERMARK, T., Bull. Off. int. Epizoot. 69 (1968) 1439. [22] WESTOO, G., Acta chem. scand. 20 (1966) 2131; 21 (1967) 1790; 22 (1968) 2277. [23] KITAMURA, S., Medicine and Biology (Tokyo) 72 (1966) 274. [24] Methyl Mercury in Fish. A Toxicologic-Epidemiologic Evaluation of Risks (SKERFVING, S., Ed.), To be published in Nord. hyg. Tidskr., Suppl. 1(1971). [25] LINDSTEDT, G., Analyst 95 (1970) 264. [26] LIDUMS, V., ULFVARSON, U., Acta chem. scand. 22 (1968) 2150; 22 (1968) 2579. [27] FISHMAN, M.J., Analyt. Chem. 42 (1970) 1462. [28] HENRIQUES, A., Nord. hyg. Tidskr. 50 2 (1969) 164 and forthcoming paper.

110 9. THE DETERMINATION OF TRACES OF MERCURY BY SPECTROPHOTOMETRY, ATOMIC ABSORPTION, RADIOISOTOPE DILUTION AND OTHER METHODS

C.G. LAMM and J. RUZlCKA

9.1. INTRODU CTION

The methods for determining traces of mercury in food, water, bio- logical and other materials are briefly reviewed in this chapter. This treatise is not meant to be comprehensive, but critical, emphasizing modern instrumental methods which are, or may become, important for routine determination of mercury in the range of 1 to 0.001 ppm Hg as present in various materials. It is of historical interest to note that already in 1928 Stock and Zimmermann [1] investigated the possibilities of transfer of Hg, applied as a seed dressing, to the consumers. For this purpose they developed a method in which mercury was first deposited from an acid solution on a copper wire which was then sealed in a glass capillary. By heating that end of the capillary which contained the wire, mercury was distilled off and condensed in the other end collecting in one or two microdroplets, the diameters of which were then measured by means of a microscope furnished with a micrometric scale. Quantities of mercury down to 5X10"5 ppm were thus determined in various materials [2] (Table XVIII), and it is of interest that this technique was used even twenty years later for the determination of mercury in soup seasoning [3]. Since then a number of procedures based on radioactive tracer tech- niques, optical methods and gas chromatography have been suggested. Mercury-ion-selective electrodes are likely to be important in the future. Most of the methods cited in the literature aim at determining total mercury content rather than individual mercury compounds. Only a few, like gas chromatography, are suitable also for simultaneous separation and deter- mination of the individual organomercury compounds. Thus when, for example, thin-layer chromatography is being used for separation of organo- mercurials, any of the methods reviewed here can be used for determination of mercury in resulting fractions.

TABLE XVIII. ANALYTIC RESULTS CITED FROM REF. [2]

Micrograms per 100 g of material Materials Minimum Maximum

Rocks 2.5 143

Soils 0.1 170

Fossil 0.1 2.5

Rainwater 0.005 0.048

Vegetable organic matter 0.2 8.6

Animal organic matter 0.1 11.0

111 The major difficulty associated with the use of these methods for deter- mination of traces of mercury in various materials is not connected with their limits of sensitivity, which are often more than sufficient for the present purpose, but with the choice of procedures for decomposition of the sample and separation of mercury from interferences. These steps, which might at the same time preconcentrate mercury before determination, have to be performed quantitatively and also in such a way that no additional mercury is added to the sample with the reagents used. The latter source of error — the reagent blank — can easily be kept low by careful work and the use of extremely pure chemicals and can be precisely controlled by frequent check of the analytical procedure. However, the other require- ment, namely the quantitative transfer of isolated traces of mercury from the sample to a detector, is much more difficult to achieve. It is a well-known fact that serious losses of mercury can occur, e. g. due to its volatilization, and precautions are usually taken not to heat solutions which contain traces of this element to a very high temperature. If this cannot be avoided, evaporation must be done slowly and carefully. However, in a recent article by Toribara et al. [4] it was pointed out that nearly all mercury can be lost, even at , in solutions containing only traces of reducing species. This is due to evaporation of metallic mercury, which is formed by disproportionation of mercury(I). The practical way to avoid such losses is to add an oxidizing substance (e.g. permanganate) which has a higher oxidation potential than the mercury(II)/mercury(I) system. Many difficulties reported in the literature to obtain 100% recovery are likely to be a result of such losses by volatili- zation. However, losses may also be due to adsorption of traces of mercury on the walls of the vessels used, adsorption on the colloidal solid particles and adsorption on emulsions of fats present, or they may be due to an incomplete destruction of the analysed material, etc. It is, indeed, surprising that only very few investigators have used tracer techniques employing 203Hg to study the behaviour of traces of mercury [5-8] and that even fewer have attempted to check their own procedures in this way [9, 10], We therefore suggest that the intercomparison of methods, as usually done by means of recognized standard materials (IAEA standard flour [11], . kale, Bowen [12]), should in future be accompanied or combined with check- ing by means of radiomercury. This may, to a certain extent, reveal the reasons for the discrepancies observed among the different laboratories. Indeed, it should be stressed that this check with 203Hg is inevitably made on each individual sample in the cases of any radioisotope dilution method. This fact is the main advantage of dilution analyses compared with non active analytical methods. In spite of the difficulties pointed out above, we believe that the methods reviewed in this chapter will eventually become preferred to activation analysis. The reason is that they are simpler, cheaper, faster and easier to automate and therefore more suitable for routine work. The following sections treat the material according to the operations involved in each of the analytical procedures: sample decomposition, sepa- ration and pre concentration of mercury, followed by a review of individual instrumental methods. It is our hope that this presentation will enable the reader to make his own choice either of adopting a certain method in its entirety or of combining selected operations into a procedure suitable for his particular need.

112 9.2. DECOMPOSITION OF THE SAMPLES

All methods described here are destructive, which means that the samples are completely decomposed, in order to make all the mercury present available for analysis. In general, two approaches can be adopted: wet or dry decomposition. The choice of these methods depends primarily on the nature of the sample to be analysed. Thus, for example, aqueous solutions, urine, metals, soils or minerals have to be decomposed by the wet method, whereas biological samples, seed, food, paper, etc. can be decomposed by both wet and dry techniques. However, whenever a choice is possible between these alternatives, dry combustion should always be preferred.

9.2.1. Wet decomposition of solid samples

The most thoroughly investigated wet decomposition method is the one described and recommended by the British Analytical Methods Committee [10] for determination of small amounts of mercury in organic matter. In this procedure the sample is decomposed by a mixture of nitric and sul- phuric acids in a modified Bethge apparatus (Fig. 24). To avoid losses of mercury by volatilization, the distillate, which is obtained during de- composition of the sample, is collected in B. Investigations utilizing 203Hg added to various samples either as mercury(II) chloride or phenylmercury(II) acetate, led to the adoption of the following method: The sample is transferred to the oxidation flask and a cooled mixture of 20 ml of water, 5 ml of concentrated sulphuric acid and 50 ml of concen- trated nitric acid are added. After any initial reaction has subsided, the sample is carefully heated, the distillate being collected in B with tap A closed. When the temperature in the flask has reached 116°C, the col- lected distillate is emptied through tube C into a measuring cylinder. During further decomposition, the distillate is again collected in B; how- ever, when the distillation mixture darkens, the distillate is run from the reservoir back to the flask. When all the material has decomposed, the apparatus and its contents are allowed to cool, and all liquids are collected in the volumetric cylinder. One millilitre of this solution is titrated with standard sodium hydroxide to determine the normality of the sample. The sample is then diluted with water to obtain a total acidity of about 1M, heated to boiling, hydroxylammonium chloride is added and the solution is set aside to cool to room temperature. The purpose of adding hydroxyl - ammine is to prevent oxidation of dithizone, which is subsequently used for colorimetric determination of mercury. This method features the advantage of decomposing large samples like up to 100 g of potatoes. The drawbacks are that the method is slow, must frequently be attended and requires large amounts of concentrated acids resulting in high blank values. Whereas the efficiency of decomposition and distillation were checked by 203Hg, this was not the case for the sub- sequent steps of reduction and solvent extraction. This wet decomposition was used for determining mercury in potatoes, eggs and cocoa spectrophotometrically by means of dithizone [10], The present authors have also used this method of decomposition for determining mercury in flour, fish, meat, eggs, etc. by the substoichio- metric isotope-dilution method [13], However, difficulty in decomposing

113 fat present in the samples was experienced. This, together with other drawbacks, as stated above, in processing a large number of samples led us to adopt the Schoniger combustion method. A modification of the wet decomposition method utilizing has been published by the British Analytical Methods committee [ 14].

9. 2. 2. Wet decomposition of liquid samples

This is, in fact, the only alternative when large volumes of aqueous samples have to be analysed. Wet destruction is, of course, also suited for small volumes of water, blood, urine etc. The most recently published procedure, which was used in connection with atomic absorption [15] can be briefly summarized as follows: Ten millilitres of urine are pipetted

114 into a Pyrex flask, 1 ml of concentrated sulphuric acid is added and the vessel is cooled in a water bath. 15 ml of 6% wt/vol. of are added, whereafter the mixture is cautiously shaken and left loosely stoppered overnight at room temperature. The next day, excess of permanganate is reduced with 3 ml of 20% wt/vol. of hydroxyl- ammonium chloride and the solution is immediately analysed. A similar procedure, but at an elevated temperature, was used in connection with spectrophotometry by means of dithizone [16] and further in connection with substoichimetric radioisotope dilution [13]. Although the procedure is not very fast, it is simple, does not require attention, and is reliable. The only possible difficulty which might be experienced is that the per- manganate used may contain some traces of mercury resulting in a high blank value. alone has also been used [17] for de- composition of water samples. It is, however, not certain that this results in a complete decomposition of organomercurials.

9.2.3. Dry decomposition of combustible materials

Burning of organic materials in an atmosphere of oxygen has several advantages. It is fast, simple and can be performed with a very low blank value. Oxygen can be purified from mercury much more easily [18] than from mineral acids and, being commercially available in sufficiently pure form, it is also cheaper than suprapure chemicals. The volatility of mercury is an advantage here because mercury can be quantitatively re- covered even from incompletely burned samples [13]. Further, when radio- mercury is used as a tracer, isotopic equilibrium is rapidly attained in the vapour phase. Burning of organic materials can be accomplished either in a closed system (Schoniger flask) or in a stream of oxygen. Schoniger flask combustion was originally used for mercury-residue analysis by Gutenmann and Lisk [19] who also modified the apparatus for combustion of a larger sample. The procedure was later tested [13] with radiomercury and modified as follows: Briquettes (diameter approx. 14 mm, height 2-4 mm) are pressed from organic material, which may contain up to 30% humidity, and placed in a wire basket (A in Fig. 25) together with a tiny paper fuse. A Teflon®-covered magnetic stirring bar (C) is placed in the combustion flask and a rubber balloon is attached to the side arm (D). 70 ml of 0. 2M HC1 and 10 ml of 0. 02M KMn04 are added and the flask is thoroughly flushed with oxygen. The paper fuse is lit and the wire basket immediately placed in the flask. The stopper is held firmly in place until the combustion is complete, which takes about 20 sec, whereafter the content of the flask is magnetically stirred until all visible vapours have been absorbed in the splashing solution. This usually takes 10 to 15 min after which the solution, which now contains all the mercury ori- ginally present in the sample, can be further analysed. Studies on this procedure [13] have shown that mercury does not remain in the sample briquette even at incomplete combustion. Further, surprisingly enough, mercury was never found in the rubber balloon. On the other hand, sub- stantial losses of mercury occurred if the aqueous solution did not contain

enough of the oxidant (KMn04 or H2Oz) (see also Ref. [4]). Pressing of briquettes is much preferred to wrapping the sample in cellophane or paper, as these materials usually contain sufficient mercury to influence the result [13], The procedure could be further adopted to combustion of

115 FIG.25. A 2-litre oxygen flask for SchBniger combustion. A: Wire basket. B: Heating and ignition coil. C: Magnetic stirrer. D: Side arm with rubber balloon.

samples containing more than 30% humidity by reshaping the wire basket and connecting it to a transformer. Thus, the sample can first be elec- trically dried in the closed flask, already filled with oxygen, and finally burned by electric ignition (see B in Fig. 25). The Schoniger combustion has been used in connection with spectrophotometric determination of mercury by dithizone [19] and also with substoichiometric radioisotope dilution [13]. Combustion of samples in a stream of oxygen was recently suggested by Lidums and Ulfvarson [18] and used in connection with photometry of mercury vapours. Because this very interesting decomposition technique is closely connected to the subsequent separation of mercury by amalgama- tion, it is described in the section dealing with separation methods.

9.3. SEPARATION AND PRECONCENTRATION OF MERCURY

The volume of decomposed samples usually varies between 50 and 100 ml of an acid aqueous solution, which also contains different oxidants. Besides that, a number of elements in varying amounts may be present, depending on the nature of the sample. It is therefore necessary to separate and also to preconcentrate mercury before final determination. This can be done by distillation, coprecipitation, ion exchange, volatilization, amalgamation or solvent extraction. However, for the present purpose only the last three techniques have found wider application among which solvent extraction has been used most extensively.

9.3.1. Solvent extraction

Since Fisher's discovery of dithizone in 1925 [20], this reagent became, and still is, the most successful one for extraction and spectro- photometry or mercury and other heavy metals [21]. Because the extracta- bility of metal dithizonates decreases in the order palladium(II), gold(III),

116 mercury(II), silver(I), copper(II), bismuth(III), (II), (III), zinc(II), cadmium(II), cobalt(II), lead(II), (II), tin(II) and (I) [22], the separation of mercury by dithizone is very selective. Further, as the extraction constant of mercury dithizonate has a very high value

(log K = 26.8 in CC14)[22] mercury can be quantitatively extracted with an excess of dithizone (5 X 10-5 M in , chloroform or benzene) from 6M sulphuric acid up to pH4. These solutions, however, must not contain any oxidants which would decompose dithizone. There- fore urea, hydrazin or hydroxylamine hydrochloride is frequently added before extraction. Very high selectivity of separation can be achieved by extracting mercury from a solution, at pH2 to 4, containing EDTA and hydroxylamine hydrochloride [13, 215. Here, gold and palladium are re- duced to the metals, copper is masked by EDTA and silver by chloride. Quantitative separation is achieved by extracting this solution with two portions of approx. 5X10"5 M dithizone in carbon tetrachloride for about one minute. From the thus obtained organic extract, mercury can be stripped back by shaking it for about one minute with a small volume of a sodium -hydrochloric acid solution, which is then further analysed.

9.3.2. Other separation methods

Electrodeposition of mercury on a metal by formation of an amalgam was first used by Stock et al. [1] in connection with his micrometric method, and recently by Fishmann [17] in connection with atomic absorption spectrophotometry. Separation of mercury from water according to this method can briefly be summarized as follows: One hundred millilitres of a samples is measured into a 200-ml-volumetric flask and 10 ml of con- centrated hydrochloric acid are added. A coil made of thin silver wire is placed into this flask, which is then shaken overnight. The solution is decanted and the coil is rinsed with demineralized water and acetone. By electrically heating this silver coil in an absorption cell the mercury is vaporized and measured. It is reported that a minimum of 5 to 6 hours are needed for quantitative recovery (80 to 128%) from the sample at the level of 0. 5 ng of mercury per litre. Iron was reported to interfere, but the influence of other species was not studied. The method is not very fast but simple, and it would be interesting to investigate its further applicabilities. Filtration of aqueous samples containing traces of mercury through an asbestos pad impregnated with, for example, CdS is another way to concentrate mercury before its determination by atomic absorption [23], Further, volatilization of mercury has been used in its separation from samples of urine digested by the permanganate method [15]. After addition of ten drops of 20% wt/vol. tin(II) chloride solution, air is bubbled through the sample solution at a controlled flow and the mercury vapours are carried directly into the absorption cell of a photometer. Volatilization and amalgamation of mercury are combined in an inter- esting method suggested by Lidums and Ulfvarson [18]. Samples of urine, blood, plasma, egg and liver are placed in a porcelain boat which is then heated in a stream of oxygen. The gases are passed through a heated column of sodium to remove any combustion products, and — after cooling - mercury is adsorbed on a filter made of gold grains. When the whole sample has decomposed and the apparatus has been flushed with

117 oxygen a zero line is established on the recorder connected to a photometer with an absorption cell. The gold filter is now heated to glow-red and the absorbance of the volatilized mercury vapours in the cell is recorded. Approximately two samples per hour can be analysed by this technique, the weight of the samples being 50 to 200 mg.

9.4. DETERMINATION OF MERCURY

9.4.1. Non-active instrumental methods

9.4.1.1. Spectrophotometry of mercury dithizonate .

Mercury(II) dithizonate, Hg(HDz)2, is an orange, water-insoluble crystalline compound, which is soluble in most organic solvents. Extracted into carbon tetrachloride (which is the frequently used solvent), mercury(II) dithizonate strongly absorbs light at 485 nm, the molar extinc- tion coefficient here being 71. 2 X 103. Thus, considering a 5-cm long photocell, with a volume of 10 ml, a minimum of0.2-0.5/jg Hg could theoretically be determined spectrometrically in each sample [21, 22], An example of such a determination is the method recommended by the British Analytical Methods Committee [10] as it is outlined here: One millilitre of 20% wt/vol. hydroxylammonium chloride solution is added to the aqueous solution of hydrochloric acid-, which was used for stripping mercury from the organic phase (see Section 9.3.1). 1 ml of 10% wt/vol. urea and 1 ml of 2.5% wt/vol. EDTA is added, the mixture is transferred into a separatory funnel and set aside for 15 min. About IX 10"3%wt/vol. solution of dithizone in carbon tetrachloride is added in 0. 5-ml portions from a burette. The separatory funnel is shaken for about 10 sec, the layers are allowed to separate and the lower one is dis- carded into another funnel containing a small amount of 4M acetic acid. The operation is repeated until the last extract has a greyish-green colour. The volume of the thus obtained organic extract is adjusted to a nearest round value with pure carbon tetrachloride and its optical density is meas- ured at 485 nm with the blank solution as reference. The mercury content of the sample can then be estimated either by using the above given value of the molar extinction coefficient, or more precisely by comparison with a calibration curve, obtained in the identical way. Although the dithizone method might seem to be straightforward and simple to perform, it is unfortunately not so when the highest sensitivity of determination should be reached. First, not only Hg(HDz)2 [21, 22], but also a compound like Hg2(HDz)2, which is orange-yellow, violet Hg(Dz) or orange HgClHDz [24] are formed depending on the pH, the ratio of mercury to dithizone, etc. These chelates have been well studied and their formation can be avoided by following the above or any other recognized procedure [21], The next problem is connected to the reaction of organo- mercurials with dithizone, during which compounds like CH3C6H4HgHDz are formed [21], Some of them absorb the light at similar wavelengths as

Hg(HDz)2 and therefore their formation must be prevented by a complete

1 H2DZ stands for diphenylthiocarbazone, mol.wt 256.3, familiarly called dithizone.

118 decomposition of the organic material to be analysed. The last, and probably most serious problem, is connected to the formation of oxidation products of dithizone like diphenylthiocarbodiazon [21], This, and other so-called yellow oxidation products of dithizone also strongly absorb light in the vicinity of 485 nm and therefore interfere with the determination of mercury. This is why oxidants have to be so carefully reduced before solvent extraction and — as this is not easily accomplished in the first solution obtained after wet or dry decomposition — why spectrophotometric measurements of dithizone extracts obtained directly from such solutions are not recommended. With all precautions taken, amounts smaller than about 1-0.1 ppm Hg cannot be determined safely.

9.4.1.2. Atomic absorption spectrophotometry

Atomic-absorption spectrophotometers, when used for determining traces of mercury, exploit the fact that mercury vapours absorb resonance radiation at 253.7 nm. Any mercury photometers therefore consist of a light source emitting mercury resonance lines, an absorption cell (or a flame), where mercury vapours are measured, and a photoelectric detector. There are many atomic-absorption spectrophotometers on the market and any of them, fitted with a mercury lamp, can serve the present purpose. Liquid samples can thus be sprayed directly into the flame, but fumes and suspended matter in the vapour phase would cause an "apparent absorption" by physical scattering of the light. Although corrections for "nonatomic absorption" are possible, preliminary separation of mercury from the sample is always recommended [15]. Dithizone extracts can either be burned directly in the flame [25], or determined according to the so-called "cold vapour atomic absorption method" by means of a photometer, equipped with an absorption cell instead of a flame. Thus, according to Refs [26] to [29] the dithizone extract is decomposed by heat in a current of air, in order to obtain free mercury vapour, which is then transferred to a cuvette and measured. An elegant method, using reduction with tin(II) chloride and vaporization with a stream of air, which again carries the mercury vapours directly into a photocell, has already been mentioned above [15], The method of Lidums and Ulfvarson [18] employs a principle of measuring mercury vapours by utilizing a modified Beckmann DU spectro- photometer equipped with a mercury lamp. A very simple, yet very sensi- tive, mercury photometer was suggested by Ling [30], It employs a mercury resonance lamp as source of monochromatic light, thereby dis- posing of the necessity of a monochromator, a filter or a narrow-band pass detector. This makes the instrument simple and sufficiently compact to be portable. The detection limit is 0.3 ng Hg and the sensitivity can be further increased by a so-called "null technique".

9.4.1.3. Atomic

By this method the intensity of fluorescence of mercury vapours, excited by light from a mercury lamp, is measured. In contradistinction to atomic absorption the fluorescence of mercury vapours is measured relative to a "dark" background because the beam of the exciting light is perpendicular to the light beam reaching the photocell [31]. This makes

119 it possible to reach higher sensitivity of measurement, e.g. down to 0. 002 ng Hg, when organic solvents containing mercury are directly burned in a flame. The content of mercury in urine down to 0.1 ng Hg/ml was determined by this technique [31], The disadvantage of this approach may, however, be that the calibration curve has a maximum which means that a high content of mercury could be confused with a low one [32],

9.4.1.4. Gas chromatography

Gas chromatography has so far only been used to a limited extent in determination of total mercury. Krestovnikov and Sheinfinkel [33] thus employed gas chromatography on a pretreated carbon column to determine mercury in gases. Mercury was adsorbed on the column at room tempera- ture and replaced with at increased temperature. A argon detector with a 10-mCi 147Pm source was used to detect mercury in the effluent gas stream. In analysis of various organomercurials, however, gas chromatography — often combined with thin-layer, paper or column chromatography - seems the most suitable choice. Indeed Westoo and her coworkers [34, 35] have been able to determine methylmercury compounds ranging between approx. 0.07 - 4.45 ppm Hg in various foodstuffs according to this concept. A review of these methods and a comparison between results obtained in some Swedish and Japanese laboratories is found in Ref.[36],

9.4.1.5. Ion-selective electrodes In analogy with pH measurements with a glass , the chemical activity of mercury(I) and (II) ions can be estimated in a simple way by means of a mercury-ion-selective electrode (see, for example, Ref.[37]). Thus, activities well below 10_15 M can be measured either with a "solid- state" [38] or a "liquid-state" [39] mercury-ion electrode combined with a potentiometer and a reference electrode. The chemical activity, which may be thought of as the "effective concentration" of free mercury ions in the solution, represents that fraction of mercury ions which has not been complexed by complexing agents like hydroxyl, iodide, cyanide, sulphide or other ions. It therefore nearly always constitutes only a fraction of the total mercury content in the solution; however, it may very well be that fraction which is physiologically the most important. Thus, it is a well- known fact that it is the chemical activity of calcium ions in serum, and not the total calcium content, which controls such biochemical processes as nerve excitation, conduction, muscle contraction, etc. Equally well known are differences in toxicity of various mercury compounds like HgS, an Hg2Cl2 d HgCl2, which increases with their solubility (and therefore with the activity of Mercury(I) or (II) ions in respective solutions). It is beyond the scope of this chapter either to discuss the significance of determinations of the total mercury content and the content of organo- mercurials or to suggest that also chemical activity of mercury ions in aqueous samples should be measured. However, we wish to stress that recent development in ion-selective electrodes (i.e. Selectrodes © [40]) may potentially prove useful in future work, especially in simple, automated systems. Here, not only mercury- ion activities could be determined, but also total mercury concentrations by the methods of standard addition or substraction.

120 9.4.2. Radioisotope techniques

The radioisotope 203Hg features favourable properties like a half-life of 47 days and an easily measurable radiation (/3max =0. 21 MeV, 7 = 0.28 MeV), which both make it very suitable for tracer experiments. Because this isotope can be produced by the nuclear reaction 202Hg (n, 7) 203Hg, which has a large activation cross-section, it is commercially available at a specific activity of more than 1 mCi/mg Hg at a price of less than 3$/mCi. These factors make it possible to measure precisely, by means of a simple instrumentation, less than 10~4 mCi, i.e. the activity associated with an amount of mercury smaller than 10-1° g, at a negligible expense. This favourable situation can be utilized in three different ways: for check- ing existing procedures and for determining mercury by radioisotope dilution or radioisotope exchange methods.

9.4.2.1. Control of analytic procedures

Control of any analytic procedure by means of radiomercury consists of a measurement of nonactive mercury by the radioactivity of 203Hg which has been added to the sample in trace amounts [9], It is therefore most important to have both radioactive and nonactive mercury in the same chemical form straight from the beginning of the experiment. If various compounds of mercury are found in the sample radioactive and nonactive mercury must be present in all of them in the same proportion — i. e. iso- topic equilibrium must be achieved. Generally all "nonactive" analytical procedures can be checked by labelling the mercury in the sample with a minute amount of 203Hg at the very beginning of the analytical procedure. During decomposition of the sample, isotopic equilibrium is attained and any losses of mercury during subsequent operations can easily be evaluated by comparing the originally introduced activity with that found in a final fraction. These measurements often make it possible to adjust the ana- lytical procedure in such a manner that 100% recovery is always obtained. Whenever quantitative, or at least perfectly reproducible, recoveries cannot be achieved, radioisotope dilution is the only alternative.

9.4.2.2. Isotope dilution analysis

After adding a known amount, y, of mercury, labelled with 203 Hg at a specific activity So to a sample containing the'unknown amount of nonactive mercury to be determined, x, the radioactivity-mass balance relationship at isotopic equilibrium can be expressed as

yS0 =(x + y)S X

where Sx is the specific activity of the resulting mixture. This to the well-known isotope dilution equation:

121 where S0 =Ac,/m0 and Sx = Ax/mx. Here A0 and Ax are the radioactivities of isolated amounts of mercury m0 and mx measured before and after the mixing of the radioactive and nonactive mercury. Once the specific activity of the standard radiomercury solution S0 is known, the analysis of each individual sample only involves measurement of the specific activity

Sx of mercury, isolated from it. Evidently, quantitative recovery is not necessary here, as measurement of the activity, Ax, of any fraction, mx , of mercury is sufficient. This approach is of great advantage whenever small quantities of mercury are involved, since uncontrollable amounts might be lost by volatilization, adsorption, co-precipitation, incomplete extraction, etc. These obstacles do not hamper the analytical result ob- tained by isotope dilution because radiomercury is lost in the same pro- portion as nonactive mercury. Evidently, any of the instrumental methods described above can be 203 used for measurement of mx. When Hg of high specific activity is used, the sensitivity of determination is the same as that of the instrumental method. In this context an isotope dilution analysis combined with atomic absorption seems to be a promising alternative, care being taken to avoid any release of radiomercury above the burner. As such a procedure has not yet been developed the substoichiometric isotope dilution (see, for example, Ref. [41]) seems to be a suitable alternative.

9.4.2.3. Substoichiometry and automation

In contradistinction to quantitative separation, which aims at a 100% yield of mercury from each individual sample, substoichiometric separation [41] is meant to achieve constant recovery from each sample, irrespective of its original mercury content. If the same amount of mercury is always separated from mixtures resulting after isotope dilution, then m0 =mx and the isotope dilution equation becomes:

where A0 and Ax are the activities of equal amounts of mercury, sub- stoichiometrically isolated from the standard radioisotope solution and its mixture with the sample to be analysed, respectively. By this approach the amount of mercury, x, present in the sample, only becomes a function of the amount of labelled mercury added, y, and the ratio of the sub- stoichiometrically separated activities. Ideally, the limit of detection of this method is given by the maximum specific activity of 203Hg available. The reproducibility of the method depends on the substoichiometric separation, which is accomplished by, for example, reacting mercury with always the same amount of dithizone, which is smaller than the amount stoichiometrically corresponding to y. Thus, irrespective of the total mercury content (x+y), always the same amount of mercury is extracted into the organic phase whose radioactivity is then measured. The advantage of this approach is not only its simplicity and sensitivity, but also the in- creased selectivity of the determination. Clearly, if mercury is not quantitatively extracted into the organic phase, because not enough dithizone

122 is available (substoichiometry), the fraction of mercury remaining in the aqueous solution safely prevents formation of less extractable dithizonates. With excess of dithizone, on the other hand, not only all mercury, but also silver and possibly copper, are extracted and thus interfere in the deter- mination. By this method approx. 10"5 g Hg was found in a 0. 1-g sample of zinc blende and approx. 10-6 g Hg in 200 g of mineral water [41]. By using 2X10"5 M and 4X 10"7 M dithizone in carbon tetrachloride as extractant, mercury was selectively extracted from aqueous samples, acidified with sulphuric acid, even in the presence of a 500-fold excess of cadmium, cobalt, copper, iron, , lead, thallium, zinc, silver, bismuth and nickel. Later, after being further developed and automated, the substoichio- metric isotope dilution was used for analysis of biological samples and food. Its final version [13] can be briefly outlined as follows:

(1) The sample for analysis is decomposed in the presence of ca. 50 ng of radiomercury, y. Aqueous samples are treated with permanganate, organic materials are burned in an oxygen flask. (2) The resulting aqueous solution is extracted twice with an excess of dithizone (IX 10"5 M in carbon tetrachloride) and from the thus obtained combined organic extracts, mercury is stripped back into 5 ml of hydro- chloric acid — sodium nitrite solution. (3) These aqueous samples are automatically analysed in the Technicon AutoAnalyser, equipped with a flow-through scintillation counter.

The manual operations 1 and 2 were described previously and there- fore only the automated part of the method is explained here. The aqueous sample, which contains both active and nonactive mercury (x+y) in isotopic equilibrium is transferred to a plastic cup which is then placed into the sampler (Fig. 26). This is programmed to deliver samples into the machine

FIG.26. Flow-sheet indicating automatic substoichiometric isotope dilution analysis. (Note: This figure was by mistake omitted in the original article[13], and appeared as erratum 4 months later in the same journal.)

123 at a rate of either 10 or 20 samples per hour. Once the sample has been pumped into the machine, nitrite is first reduced by hydroxylamine hydro- chloride at a slightly elevated temperature (50° C) and then buffered and masked by citric acid-EDTA solution. The next operation is the substoichiometric solvent extraction with 3 X 10"8 M zinc dithizonate in carbon tetrachloride. An aliquot of the organic phase is then separated in a simple device (Fig. 2 in Ref.[42]), "segmented" (cut into aliquots interspersed with an aqueous solution) and pumped into the flow-through cell. This segmentation ensures reproducibility of the measurement of radioactivity of the organic phase as well as a perfect wash of the flow-through cell whose background reading was very low and constant even after several months of use. The signal of the scintillation counter is integrated by a ratemeter and continuously registered. Because the sampler alternatively introduces the samples and distilled water into the machine, the record consists of a series of peaks. Radioactive standards of high specific activity give the highest peaks of a

constant height corresponding to A0, whereas the peaks of the samples are lower the more nonactive mercury, x, they contain. The value of y was chosen to be 50 ng to be close to a recommended maximum permissible value of mercury in food. The automated part of the methods takes about five minutes to complete. Organic samples burn in less than one minute, but absorption in the aqueous phase, extraction and re-extraction takes about half an hour. As most of these operations are unattended, several samples can be handled at the same time. Therefore two persons could possibly analyse about sixty samples per day. The sensitivity of the method is sufficient for most purposes. Thus, from 2 to 0. 00004 ppm Hg were determined in samples of milk, bread, flour, apples, egg, fish, beef, liver, fish-bone flour, filter paper, cellophane, sulphuric acid, water [13], etc. In the standard material of kale, 0.0159 ppm Hg was found as compared with 0.0150+ 0. 008 ppm reported by Bowen [12]. Likewise, standard cereals, distributed by the IAEA for intercomparison purposes, 0.0435 ppm Hg was found as compared with "the most probable mean value of 0.044 ±0.0014 ppm reported by Merten et al. [11]. In both cases the relative standard deviation of this automated analysis was + 5%. The method is fast, simple and very cheap to perform. Its drawback is a "memory effect" which can be well seen on Fig. 3 in Ref. [13]. This source of error, which is inherent in all AutoAnalyser systems, is due to the slight adsorption of mercury on the walls of the tubing of the machine. It can be overcome either by repetitive analysis, which means that each sample should be analysed several times by the machine (the first analysis being disregarded), by improving the wash or by further shortening of the tubes carrying the sample. The latter alternative now seems feasible as an improved technique of solvent extraction by AutoAnalyser systems was recently reported [43], For routine screening of a large number of samples a two-detector system was adopted [44], Each detector assaying the gamma radiation of 203Hg is connected to a ratemeter and a recorder (Fig. 27). The detector system I measures the total radioactivity of each sample and thus controls any mercury losses incurred during the previous manual operations. Detector II only measures the radioactivity of the substoichiometrically

124 ISAMPLE«y|

< DESTRUCTION < Z < SEPARATION z

RECORDER I yield % |SAMPLE CHANGER| -.100

IPUMPI T 50 | FLOW COUNTER|--^ u to. S 1 2 3 4 5 .DITHIZONE

[SOLVENT EXTRACTION RECORDER II < z o t— < 05

XFLOW COUNTER ! n S I 2 3 4 5 WASTE

FIG.27. diagram indicating the manual and automated operations in an substoichiometric isotope dilution analysis employing a two-detector system (from Fig.3 in Ref.[47]).

isolated amounts of mercury in exactly the same way as described above. As long as no losses of mercury have occurred all peaks on recorder I will be equally high corresponding to y, and the readings obtained by recorder II are all correct. Whenever losses of mercury occur during the manual operation, the peaks on recorder I would be smaller, However, in the case of 50% stoichiometry, i.e. the amount of zincdithizonate added to each sample being equivalent to 50% of y, the results would still be correct for losses not exceeding 50% of the total mercury present. Thus, it is seen from Fig. 27 that whereas the samples marked S (standard), 1, 2, 3 and 4 contain enough mercury, sample 5 must be re-analysed, as more than 50% of y was lost. The use of this system for a more complicated situation has also been described [44].

125 The automated substoichiometric isotope dilution analysis thus com- bines the advantage of using 203Hg both as a means of detecting and correct- ing any losses incurred and as a direct tool for determining trace amounts of mercury. This concept of individual check should also be possible to use in an automated procedure where recorder I would still measure the radioactivity and recorder II would show the signal from, for example, an atomic absorption photometer.

9.4.2.4. Isotopic exchange methods

Clarkson and Greenwood [45] have determined trace quantities of mercury in urine and biological homogenates by first adding cysteine to a fresh sample adjusted to pH7.4 in order to complex all mercury present. Subsequent addition of a minute amount of 203Hg of high specific activity results in a quick labelling of all mercury at a given specific activity. A stream of nitrogen almost saturated with nonactive mercury (200Hg, 20 mg/m3) is bubbled through the labelled solution and a rapid isotopic exchange takes place according to the reaction:

203 200 200 203 Hg (sample) + Hg (gas) : > Hg (sample) + Hg (gas)

The radioactive mercury in the gas is trapped and counted in a scintillation well-counter. Based on the first-order rate of loss of radioactivity from the sample solution and assuming that the gas achieves complete isotopic equilibrium with the solution, it can be shown that the time for the loss of half the total activity from the sample solution is directly proportional to its concentration of mercury. This method avoids all difficulties of decomposing the sample. How- ever, complete transfer of all mercury to the cysteine complex, complete labelling with 203Hg and complete isotopic equilibrium of mercury in solu- tion and gas are fundamental requirements only achieved by a proper choice of the experimental conditions. One source of error is the dissolution of mercury vapour in the sample solution. The method was tested for samples containing at least 2 /jg of mercury, but — depending on the specific activity of 203Hg available — a lower limit of detection is reported to be approx. 0.025/ng of mercury. As pointed out by Handley [46], solvent extraction techniques become particularly useful in isotope exchange and displacement methods involv- ing radioisotopes. In this case inorganic mercury present in an aqueous sample solution is determined by equilibrating this solution with an organic

phase (e.g. CC14) containing a known amount of mercury present as a complex of di-n-butyl phosphorothioic acid, {Hg [(C4H90)2 P (O) S]2} . If the mercury in one of the phases is initially labelled with 203Hg, isotopic exchange takes place according to, for example:

203Hg (organic) + 200Hg (aqueous) 200Hg (organic) + 203Hg (aqueous)

but no net transfer of mercury between the phases will occur. At radio- chemical equilibrium, characterized by the same specific activity in both

126 phases, radioassay of each phase makes it possible to calculate the distri- bution ratio D„„, where

^ _ amount of Hg in organic phase H8 amount of Hg in aqueous phase

Thus, the mercury content of the sample is a simple function of the radio- activity ratio of the two phases. This method was tested in standard solutions containing from 10"2 to 10-7 g mercury giving results within 99 to 101% of the known quantities of mercury present with a standard deviation of about 1%. Incorporation of 203Hg into the reagent rather than into the sample solutions eliminates one pipetting step, but necessitates repeated preparations of the reagent due to the limited half-life of 203Hg. When addition is made to the aqueous phase, the total amount of mercury (labelled with 203Hg) added should pre- ferably not exceed 1% of the mercury content of the sample in order to avoid a correction. Whereas sulphate, nitrate, perchlorate, phosphate or acetate do not interfere, chloride is reported to prevent complete iso- topic exchange, as may also other anions that complex mercury. Inter- fering cations like Pd2+ , Au+ or Cu+, are those having higher extraction constants than that of mercury. In the case of Ag+ an error of about - 3% is encountered when Ag+ is present in a mole ratio to Hg2+ of 1000 to 1. Cu2+, Bi3+ , Pb2+ , Cd2+ , In3+ and Zn2+ do not interfere when present in a mole ratio to Hg2+ of between 10"1 and 104 to 1. The same author [46] also reports a displacement method whereby mercury is determined indirectly. Here, a known amount of the silver

complex of di-n-butyl phosphorothioic acid, [Ag (C4H90)2 P (O) S], dissolved in for example CC14, is shaken with the sample solution containing mercury. Because of the higher extraction constant Hg2+ ions quantita- tively displace the Ag+ ions in the complex. The distribution ratio of 110 silver DAg is then easily calculated if a trace amount of Ag is either added to the aqueous phase or incorporated into the organic phase complex, followed by radioassay of aliquots of each phase after isotopic equilibration. As one Hg2+-ion displaces two Ag + -ions:

= Ag0 - 2 Hg a Ag 2 Hga where Ag0 denotes the initial number of moles of silver in the organic phase and Hga the unknown number of moles of mercury in the sample solution. This method was tested in standard solutions containing from 1.5X 10-3 to 10"6 g mercury giving results within 98 to 100% of the known quantities of mercury present, with a standard deviation between 1 - 3%. As long as these methods involve one-step procedures, as in the case of a urine analysis, they possess the advantages of being simple, sensitive, rather selective, applicable over a wide range of concentrations and could easily be automated. However, in the general case, when a decomposition of the sample and a preliminary separation of mercury is required, they do not possess the unique advantage of isotope dilution because radio- mercury is added to each sample in the final stage of the analysis. Conse- quently, any losses of mercury during, for example, decomposition of the sample will lead to erroneous results.

127 9. 5. DISCUSSION AND EVALUATION OF THESE METHODS, AND COMPARISON WITH ACTIVATION ANALYSIS

A pertinent question would be the choice of an actual procedure chosen among the methods described in this chapter. This is difficult to answer, not only because we lack personal experience with some of the methods reviewed, but also because the choice depends on the nature of the sample to be analysed, the number of analyses, the content of mercury in the sample, the equipment available and — perhaps most important — the personal taste and training of the chemist himself. We believe, however, that the following suggestions may be useful. In emergency cases, when large amounts of mercury are expected in the sample (i.e. about a hundred times more than a "maximum permissible value" of, say, 0.05 ppm), and when only a few samples should be quickly analysed, spectrophotometry of mercury dithizonate is the easiest choice. The sample should be decomposed either by the Schoniger combustion or by the permanganate method, followed by extraction with dithizone and stripping back with sodium nitrite. When amounts of mercury ranging from 1 to 0.005 ppm are to be deter- mined in a limited number of samples, atomic absorption seems to be the best choice. Also in this case we would tend to propose the Schoniger combustion of solid samples, although such a procedure has not yet been tested. In this situation the method developed by Lidums and Ulfvarson [18] seems the best choice, although it requires a special apparatus in which only about two samples can be analysed per hour. For analysis of water or urine, permanganate oxidation followed by volatilization of mercury at room temperature [15] seems suitable, especially since this method is also capable of further development. However, it must be noted here that none of these methods have as yet been tested by means of radiomercury. One does not like to recommend ones own method [13], but there seems no other choice than the automated substoichiometric radioisotope dilution when a number of samples, containing from 1 to say 0.0001 ppm Hg, have to be analysed. The Schoniger combustion or permanganate method should be used for decomposition and the mercury should be preconcentrated by dithizone extraction. For routine screening of a large number of samples the two-detector system [44] should be further developed. Finally, we believe that in future mercury-ion-selective electrodes will become important in routine screening, especially in automated systems. A comparison between the above-mentioned methods and activation analysis is now relevant. The most important criteria are naturally the precision and accuracy of the analysis which are the most valuable features of activation analysis. For the present purpose the reliability of activation analysis was demonstrated by the results obtained in the standard materials supplied by the IAEA and Bowen. Of all the methods reviewed in this chapter, only the automated substoichiometric isotope dilution analysis was tested in analyses of these same materials and, as shown above, there was good agreement. Reasonable agreement was also found in comparisons between activa- tion analysis and the atomic absorption procedure developed by Lidums and Ulfvarson [18] in samples of plasma, blood, urine, egg and liver. The precision and accuracy of results obtained by other methods described above have not yet been tested in comparison with those of activation analysis.

128 Other criteria, like speed, simplicity, cost of operation and even health hazards clearly speak in favour of the methods described in this chapter. Except for water analysis, the analytical result can be obtained within one hour, whereas irradiation of a sample in a reactor takes about one day when the same sensitivity is to be obtained. Cooling of the sample, transport and handling also require considerable time. Normally, analysis by atomic absorption or substoichiometric isotope dilution can be done for a fraction of the cost of an activation analysis. Further, the instrumenta- tion is much cheaper; even the apparatus for automated screening by means of substoichiometric radioisotope dilution is cheaper than an average multichannel analyser usually required for activation analysis. Beside the purely economic factor there is, however, still another point to consider in evaluating these methods. While activation analysis is capable of determining only the total mercury content of a sample, gas chromatography, and also atomic absorption and substoichiometry (in combination with various separation methods) are potentially suitable for determination of inorganic mercury as well as various organomercurials. This can, of course, also be achieved in activation analysis by irradiating different fractions obtained after, say, solvent extraction and thin-layer chromatography. However, this does not seem to be the best approach because such separations must be quantitative, the reagent blank must be kept low (and controlled), whereby the main advantages of activation ana- lysis are lost. Isotope dilution analysis utilizing various 203Hg-labelled mercury compounds is potentially better suited for this purpose.

REFERENCES TO CHAPTER 9

[1] STOCK, A., ZIMMERMANN, W., Geht Quecksilber aus Saatgut-Beizmitteln in das geerntete Korn und in das Mehl iiber?, Z. angew. Chem. 41 (1928) 1336. [2] STOCK, A., CUCUEL, F., Die Verbreitung des Quecksilbers, Naturwiss. 22 (1934) 390. [3] MAJER, V., WERNER, S., HOPPE, K., MARECEK, V., Traces of mercury in soup seasoning, Chem. Obz. 25 (1950) 185. [4] TORIBARA, T.Y., SHIELDS, C.P., KOVAL LARYSA, Behaviour of dilute solutions of mercury, Talanta 17 (1970) 1025. [51 TORIBARA, T.Y., SHIELDS, C.P., The analysis of submicrogram amounts of mercury in tissues, Am. ind. Hyg. Ass. J. 29 (1968) 87. [6] MAGOS, L., Radiochemical determination of metallic mercury vapour in air, Br. J. ind. Med. 23 (1966) 230. ~~ [7] CLARKSON, T.W., ROTHSTEIN, A., Excretion of volatile mercury by rats injected with salts, Hlth Phys. 10 (1964) 1115. [8] CLARKSON, T.W., GATZY, J., DALTON, C., Equilibration of mercury vapour with blood, University of Rochester Atomic Energy Project Rep. UR-582 (1960). [9] GORSUCH, T.T., Radiochemical investigations on the recovery for analysis of trace elements in organic and biological materials, Analyst, Lond. 84 (1959) 135. [10] ANALYTICAL METHODS COMMITTEE, The determination of small amounts of mercury in organic matter, Analyst, Lond. 90 (1965) 515. [11] MERTEN, D., TUGSAVUL, A., "Determination of mercury and in flour". Nuclear Techniques for Studying Pesticide Residue Problems (Panel Proc.) IAEA, Vienna (1970) 39. [12] BOWEN, H.J. M., Comparative elemental analyses of a standard plant material, Analyst, Lond. 92 (1967) 124. — [13] rOziCka , J., LAMM, C.G., Automated determination of traces of mercury in biological materials by substoichiometric radioisotope dilution, Talanta 16 (1969) 157. [14] ANALYTICAL METHODS COMMITTEE, The use of 50 per cent hydrogen peroxide for the destruction of organic matter, Analyst, Lond. 92((1967) 403. [15] LINDSTEDT, G., A rapid method for the determination of mercury in urine, Analyst, Lond. 95 (1970)264.

129 [16] NIELSEN KUDSK, F., Specific determination of mercury in urine, Scand. J. clin. Lab. Invest. 16 (1964) 670. — [17] FISHMAN, M.J., Determination of mercury in water, Analyt. Chem. 42 (1970) 1462. [18] LIDUMS, V., ULFVARSON, U., Mercury analysis in biological material by direct combustion in oxygen and photometric determination of the mercury vapour, Acta chem. scand. 22 (1968) 2150. [19] GUTENMANN, W.H., LISK, D.J., Rapid determination of mercury in apples by modified Schoniger combustion, J. agric. Fd Chem. 8 (1960) 306. [20] FISHER, H., Die Metallverbindungen des Diphenylthiocarbazons und ihre Verwendbarkeit fur die chemische Analyse, Wiss. Veroff. Siemens-Werken 4 (1925) 158. [21] IWANTSCHEFF, G., Das Dithizon und seine Anwendung in der Mikro- und Spurenanalyse, Verlag Chemie GmbH, Weinheim (1958). [22] STARY, J., The Solvent Extraction of Metal Chelates, Pergamon Press, New York (1964). [23] BALLARD, A.E., THORNTON, C.D.W., Estimation of minute amounts of mercury, Ind. Engng Chem. analyt. Edn. 13 (1941) 893. [24] BRISCOE, C.B., COOKSEY, B.G., Mercury chloride dithizonate: Its reactions, properties and stability constant, J. chem. Soc. (A) (1969) 205. [25] PYRIH, R.Z., BISQUE, R.E., Determination of trace mercury in soil and rock media, Econ. Geology 64 (1969) 825. ~~ [26] JACOBS, M.B., YAMAGUCHI, S., GOLDWATER, L.J., GILBERT, H., Determination of mercury in blood, Am. ind. Hyg. Ass. J. 21 (1960) 475. [27] JACOBS, M.B., GOLDWATER, L.J., GILBERT, H., Ultramicrodetermination of mercury in blood, Am. ind. Hyg. Ass. J. 22 (1961) 276. [28] NIELSEN KUDSK, F., Chemical determination of mercury in air, Scand. J. clin. Lab. Invest. 16 Suppl. 77 (1964) 1. — [29] ULFVARSON, U., Determination of mercury in small quantities in biologic material by a modified photometric-mercury vapour procedure, Acta chem. scand. 21 (1967) 641. [30] LING, C., Sensitive simple mercury photometer using mercury resonance lamp as a monochromatic source, Analyt. Chem. 39 (1967) 798. [31] VICKERS, T.J., MERRICK, S.P., Determination of parts per milliard concentrations of mercury by atomic fluorescens flame spectrometry, Talanta 15 (1968) 873. [32] BROWNER, R.F., DAGNALL, R.M., WEST, T.S., Studies in atomic fluorescence spectroscopy VIII, Talanta 16 (1969) 75. [33] KRESTOVNIKOV, A.N., SHEINFINKEL, I.G., Argon detector for determination of mercury in gases, Gaz. Kromatogr. Sb., Moscow 2 (1964) 53. [34] WEST66, G., Determination of methylmercury compounds in foodstuffs I, Acta chem. scand. 20 (1966) 2131. [35] WESTOO, G., Determination of methylmercury in foodstuffs II, Acta chem. scand. 21 (1967) 1790. [36] METYLKVIKSILVERI FISK, Report (National Institute of Public Health, S-10401, Stockholm) Nord. Hyg. Tidsskr. Suppl. 3 (1970) 1. (An English translation will be published as a supplement to Nord. Hyg. Tidsskr. in the beginning of 1971). [37] RECHNITZ, G.A., Ion selective electrodes, Chem. Engng News, June 12 (1967) 146. [38] RUZldKA, J., LAMM, C.G., A new type of solid-state ion selective electrodes with insoluble sulphides or halides, Analytica chim. Acta (in press). [39] rAXiCkA, J., TJELL, J.C., The liquid-state ion sensitive electrode, Analytica chim. Acta 51 (1970) 1. [40] Rl3Zl£KA, J., LAMM, C.G., S electrode © - the universal ion selective solid-state electrode. I Halides, Analytica chim. Acta (in press). [41] R02.ICKA, J..STARY, J., Substoichiometry in Radiochemical Analysis, Pergamon Press (1968). [42] RuXlCKA, J., LAMM, C .G., A new concept of automated radiochemical analysis based on substoichio- metric separation, Talanta 15 (1968) 689. [43] CARTER, J.M., NICKLESS, G., A solvent extraction technique with Technicon AutoAnalyzer, Analyst 95 (1970) 148. [44] LAMM, C.G., R£J2.I£KA, J., Advantages of a two-detector system in automated substoichiometric radioisotope dilution analysis, Talanta 16 (1969) 603. [45] CLARKSON, T.W., GREENWOOD, M.R., Simple and rapid determination of mercury in urine and tissues by isotopic exchange, Talanta 15 (1968) 547. [46] HANDLEY, T.H., Selective determination of mercury by isotopic exchange with mercury di-n-butyl phosphorothioate in an extraction system. Alternative displacement method with silver di-n-butyl phosphorothioate, Analyt. Chem. 36 (1964) 153. [47] RU?.ICKA, J., Substtfkiometriske metoder i sporstofanalyse, Forskning 78 (1969) 259.

130 10. IDENTIFICATION OF MERCURIAL COMPOUNDS

J. O'C. TATTON

10.1. INTRODUCTION

Methods for the detection and determination of mercury have been investigated by generations of analytical chemists and today a variety of methods, ranging from wet chemical procedures to neutron activation and oxygen bomb techniques, can be used to determine the mercury content of a sample. These methods, however, do not reveal the chemical nature of the mercury compound or compounds present in the sample; most of them do not even indicate whether the mercury present is in an elemental, organic or inorganic form. The nature of the mercury compound is of some importance in view of the variation in toxicity between individual organomercurials, but it is only in recent years that procedures have been developed for the identification and determination of individual organomercury compounds. Various paper-chromatographic methods [1-3] for the separation and identification of organomercury compounds have been described but, as with most paper chromatographic techniques, they have been superseded by methods based on thin-layer chromatography (TLC) that are quicker and usually more sensitive and reliable. Gas-liquid chromatography (GLC) has also been shown to provide efficient systems for identifying and determining organomercury compounds. At the moment, apart possibly from mass spectrometry, these two techniques, TLC and GLC,, provide the only rapid and satisfactory methods for these purposes and they alone will be referred to here.

10. 2. TLC AND GLC OF ORGANOMERCURIALS

One of the basic problems that affects the use of any chromatographic method for organomercurials is the variety of compounds commercially available. In general, they are of the formula R-Hg-X where R is usually methyl, ethyl, methoxyethyl, ethoxyethyl, phenyl or tolyl and X is sulphate, chloride, acetate, dicyandiamide, etc. The nature of X has a great effect on the general properties of the compound. When X is an anion, such as sulphate or acetate, the compound tends to be ionic and water-soluble but when X is a or dicyandiamide the compound tends to be non-polar and soluble in organic solvents. Workers with TLC and GLC have there- fore found it necessary to convert all these compounds so that they have a common anion for analytical purposes and to leave the identification of the original anion to be carried out by normal routine chemical tests. Johnson and Vickery [4], for example, have described a TLC method for analysing commercial organomercurials and seed-dressing preparations which depends on the prior conversion of all the mercury compounds present to their chlorides by shaking the sample with acetone containing 10% of 0. 15% aqueous . After centrifuging the mixture, the supernatant liquor is used for spotting onto a silica gel chromatoplate.

131 The recommended mobile solvent is cyclohexane-acetone (4:1) and the resultant spots are visualized by spraying the plate successively with a copper sulphate solution and a -sodium sulphite solution. The orange-brown spots reach maximum intensity after about 30 min and can be inspected by visible or light. Distances travelled on the chromatoplate by various organomercury chlorides in relation to a dimethyl yellow marker are shown in Table XIX. For identification purposes, 2. 5 fig is recommended as a suitable amount of a compound for spotting onto a plate and it can be taken as an indication of the sensitivity of the method.

TABLE XIX. R,t VALUES FOR ORGANOMERCURY CHLORIDES

Chloride Rst

Mercury(II) chloride 0.21

Methoxyethylmercury 0. 60 Ethoxyethylmercury 0. 73 Phenylmercury 0. 85 Ethylmercury 0. 93

Rst = distance moved by sample spot distance moved by a dimethyl yellow marker spot. System = Absorbent layer - silica gel 250 jjm thick Developing solvent - cyclohexane-acetone (4:1).

Westoo [5, 6] investigated the TLC of organomercury compounds because of her interest, as a residue analyst, in the identification of methylmercury residues in fish and other foodstuffs. She showed that TLC could be used successfully for some organomercurials after con- verting them to the dithizonates. A more systematic study of the TLC characteristics of these derivatives by Tatton and Wagstaffe [7] enabled them to recommend a number of TLC systems to separate the dithizonates of all those organomercury compounds in common commercial use (Table XX). All the mercury compounds they examined readily yielded their characteristic, stable, intensely yellow to red, complexes simply by shaking the organomercurial in solid form or in solution, with a chloroform solution of dithizone until a slightly green colour indicated an excess of reagent. Any inorganic mercury compound that may be present gives the usual mercury di-dithizonate under these conditions while any is at least partially converted to phenylmercury dithizonate. As little as 2 /Jg of these dithizonates are plainly self-indicating on a chromatoplate as yellow or red spots. WestQo [5, 6] also showed that methylmercury dithizonate was susceptible to GLC treatment on a 10% Carbowax column, with electron-capture detection. Teramoto and co-workers [8], also working with methylmercury compounds, used as 25% diethylene glycol succinate column. Tatton and Wagstaffe [ 7] investigated these and a number of other stationary phases, on various supports, with electron-capture detection. As with TLC, they

132 TABLE XX. THIN-LAYER CHROMATOGRAPHIC SEPARATION OF DITHIZONATES OF ORGANOMERCURY COMPOUNDS

(Rf values X100)

System Ditnizonate

Methylmercury 64 48 57 77 89 86 Ethylmercury 64 51 62 78 91 87 Methoxyethylmercury 32 16 25 44 58 49 Ethoxyethylmercury 44 23 34 55 71 67

Phenylmercury 48 34 46 62 72 69 Tolylmercury 52 40 53 69 79 76

Mercury di-dithizonate 19 9 17 28 19 15

Absorbent (250 pm thick) Mobile solvent System 1: Silica gel Hexane: acetone 9:1 2: " " " " 19:1 3: " " " " 93:7 4: " " Light petroleum: acetone 9:1 5: Alumina Hexane: acetone 19:1 6: " Light petroleum: acetone 19:1

found it far more convenient to work with the dithizonates of the compounds studied. Optimum conditions for separation of organomercury dithizonates were obtained on a 2% polyethylene glycol succinate column using Chromosorb G as support material. Table XXI(a) shows typical retention times obtained with a column of this composition. The dithizonates of the various alkyl and alkoxyalkyl mercury compounds have fairly short retention times for this system but are clearly separated. Sensitivity is good and 0. 05 ng of these compounds can easily be detected. By contrast, the arylmercury dithizonates have relatively long retention times with peaks correspondingly broader at the base. As a consequence, sensitivity to phenyl- and tolylmercury dithizonates is about 1 ng with this system. A shorter column containing only 1% of polyethylene glycol succinate was recommended for these compounds resulting in sharper peaks with shorter retention times, as shown in Table XXI(b). Sensitivity was also improved to about 0. 5 ng for these arylmercury compounds. All the GLC work described here was performed using electron- capture detector systems. The electron-capture detector in its usual form contains a tritiated copper foil and is quite sensitive to mercury compounds but care has to be taken as they tend to form a deposit on the detector foil. This deposition inhibits the beta-emission and overloading the system can result in emission falling to zero. Regular cleaning of the tritium source is obviously undesirable and can be avoided by keeping column and detector at 180°C or lower, and by restricting the mercury content of injections to 100 ng or less. It is worth noting that commercially available samples of organo- mercury compounds have often been found to be very impure and to contain

133 TABLE XXI(a). TYPICAL GLC RETENTION TIMES FOR ORGANOMERCURY DITHIZONATES (Minutes)

Column temperature (°C) Dithizonate 140 150 160 170 180

Methylmercury 3.8 2. 8 2.2 1. 6 1. 2 Ethylmercury 6.6 4. 6 3. 6 2. 7 2.0 Ethoxyethylmercury 17.0 11.6 8.7 6. 2 4.9

Methoxyethylmercury 17.4 12. 0 8.7 6. 2 4.9

Tolylmercury - - - 29. 0 19.5

Phenylmercury - - - 42. 0 27. 0

Column details - 27o polyethylene glycol succinate on Chromosorb G (acid-washed, DMCS treated, 60-80 mesh) in glass columns 1, 5 m long and 3 mm int. diam. Carrier gas — nitrogen.

TABLE XXI(b). TYPICAL GLC RETENTION TIMES FOR ORGANOMERCURY DITHIZONATES (Minutes)

Column temperature (°C) Dithizonate

170 180

Tolylmercury 6.4 3,2

Phenylmercury 10.0 5.0 Column details — l<7o polyethylene glycol succinate on Chromosorb G (acid- washed, DMCS treated, 60 - 80 mesh) on glass columns 1. 2 m long and 3 mm int. diam. Carrier gas — nitrogen.

varying amounts of inorganic mercury as well as other organomercury compounds including diphenylmercury. Indeed it is almost impossible to obtain some compounds in a sufficiently pure state for use as analytical standards for TLC and GLC purposes. In this connection TLC has been shown to be a useful method of isolating pure specimens for use as standards [7],

10.3. APPLICATION OF METHODS TO SAMPLES

The methods described by Westoo [5, 6] were designed mainly for detecting residues of methylmercury compounds in fish but were applicable to other foodstuffs. The organomercurial is converted to the chloride or bromide by treatment with hydrochloric or hydrobromic acid and then

134 extracted with toluene. Clean-up of the extract is effected by conversion of the mercurial into a water-soluble form, such as the hydroxide, sulphate or cysteine derivative, and back-extraction into benzene. These methods gave good recoveries for methylmercury compounds but are not suitable for alkoxyalkyl compounds which are usually very unstable to acids. Tatton and Wagstaffe [7] sought a more general method to cover all the commercial organomercurials in general use; one which would be applicable to fruits and vegetables for which they considered an aqueous system of extraction would not be adequate. Their method consists of macerating the sample with a mixture of propan-2-ol and alkaline cysteine hydrochloride solution. After allowing the mixture to settle, the super- natant liquor is diluted with a sodium sulphate solution and washed with ether. The washed solution is then extracted with an ethereal solution of dithizone. This extract is dried and concentrated for examination by TLC and GLC. Good recoveries are indicated for alkyl-, alkoxyalkyl- and arylmercury compounds from potatoes, tomatoes and apples and the method can be applied to other foodstuffs.

REFERENCES TO CHAPTER 10

[1] KANAZAWA, J., SATO, R., Bunseki Kagaku (Japan Analyst) 8 (1959) 322. [2] SERA, K., KANDA, M. , MURAKAMI, A., SERA, Y., KONDO, Y., YANAGI, T., Kumamoto Med.J. 15 (1962) 38. [3] CHIA-LANG KAO, MIN SUN, YU-CHINSING, Hua Hsueh Tung Pao (Chem. Bull., Peking) 11 (1963) 46. [4] JOHNSON, G.W., VICKERY, C., Analyst (Lond.) 95 (1970) 357. [5] WESTOO, G., Acta chem.scand. 20 (1966) 2131. [6] WESTOO, G., Acta chem. scand. 21 (1967) 1790. [7] TATTON, J. O'G., WAGSTAFFE, P.J., J. Chiomatog. 44 (1969) 284. [8] TERAMOTO, K., KITABATAKE, M., TANABE, M., NOGUCHI, Y., J. Chem. Soc. Japan, Ind. Chem. Sect. 70 (9) (1967) 1601.

135

11. THE RELIABILITY OF MERCURY ANALYSIS IN ENVIRONMENTAL SAMPLES

Results of an Intercomparison organized by the IAEA

J. HEINONEN, D. MERTEN and 0. SUSCHNY

11.1. INTRODUCTION

The assessment of hazards due to environmental contamination with mercury is based on the results of analyses for the metal and its compounds in a large number of different matrices. The overall error in these analyses is a large contributory factor to erroneous estimation of hazards. Determination of the degree of reliability of analytical results is, therefore, an essential step in the calculation of mercury background and contamination levels and in the pursuance of changes of these levels with time and location. Reliability is a function of the precision and the accuracy of analysis. The precision of an analytical method, namely its degree of reproducibility, is easily determined within a laboratory by repetition of analysis. When an error term is attached to mercury data quoted in a paper it nearly always refers to the degree of reproducibility obtained in two or three replicate measurements carried out on sub-samples by the same analyst in the same laboratory and with the same analytical method. Any bias in the method which may be inherent to it or due to wrong standards, contaminated reagents or just to bad practice will, as long as it is reproducible, escape detection. This way of quoting errors tells us nothing about the accuracy of the reported values, i. e. the degree of their deviation from the "true value". Determination of accuracy requires more effort. One way of going about it is to use different analytical methods and equipment upon sub- samples of the same material. This is a time-consuming technique which is not entirely fool-proof since any bias common to all methods, e. g. the use of a wrong standard, would still remain undetected. Another technique consists in the analysis of identical samples at several laboratories who report their results for evaluation. If the overall mean of these results is regarded as the most probable value, laboratories may check their results against this value and obtain useful information on their performance. If the number of laboratories taking part in such a comparison is large enough and if information on the method used by each is available, the data may be used also to evaluate different methods, determine their precision and detect their bias. Finally, standard reference materials may be used which contain a known amount of the material to be determined and permit a direct check of analytical performance. The International Atomic Energy Agency under its Analytical Quality Control scheme provides materials for intercomparison as well as standard reference materials.

137 In 1966, the Agency organized a series of intercomparisons for the determination of mercury in biological material by neutron activation and other techniques1.

11. 2. INTERCOMPARISON OF MERCURY DETERMINATIONS

Two different samples of flour were used in this intercomparison, one was prepared from wheat the seeds of which had been treated with mercury compounds, the other from untreated wheat. Each sample was homo- genized and sub-samples of 2 kg were sent to laboratories. Each laboratory was free to use its routine technique but was requested to inform the Agency in some detail about that technique and the results obtained with it for the provided sub-sample. Twenty laboratories participated in the intercomparison. They returned 187 results, 101 on the mercury-treated and 86 on the untreated sample. The following statistical treatment was used in the evaluation of the data:

= individual result

n = number of results

x = average within laboratories n Z/Xi X = J n

N = number of laboratories returning results

X = average between laboratories N Sx x —-— N

a = standard deviation

/£(xi -x)'

s = estimated standard deviation within laboratories

£(xt - • M (n-1)

> TUGSAVUL, A., MERTEN, D., SUSCHNY, O. , The Reliability of Low-level Radiochemical Analysis. Results of Intercomparisons Organized by the Agency During the Period 1966-1969, IAEA Report, 30 March 1970.

138 s = standard error within laboratories

s s =

S = standard deviation between laboratories

S = standard error between laboratories

t = student's factor for n-1 degrees of freedom at the 95% probability level

t(s) = 95% confidence limits of single determination

t(s) = 95% confidence limits of mean within laboratories

t(S) = 95% confidence limits of overall mean between laboratories

TABLE XXII. DETERMINATION OF MERCURY IN FLOUR

Treated with Hg Untreated

Lab. No. of Mean a Lab. No. of Mean a t(s) Ks) Method «s) t(s) Method No. determinations (ppm) No. determinations (ppb)

1 4 4. 80 0. 76 0.38 3 1 1 80.0 - - 1

2 6 3.29 0.51 0.21 3 2 6 61.3 88.4 36.0 3

3 6 1.92 4. 09 1. 67 1 3 6 105.5 49. 0 20.0 1

4 6 5. 85 0.86 0. 35 1 4 6 41.2 24. 6 10.0 1

5 6 6. 83 1.31 0.54 1 5 6 37.5 16.5 6. 8 1

6 5 2. n 0.47 0.21 1 6 5 301.4 129.0 57.6 1

7 4 4.71 0.29 0. 14 1 7 2 43.0 72. 0 51.0 1

8 6 4. 98 0.38 0.15 1 8 6 43.8 28.0 11.4 1

9 4 5. 00 1.02 0. 51 1 9 3 33.0 10.9 6.3 1

10 6 5. 59 0.34 0. 14 10 6 40.5 13.2 5.4 1

11 3 5. 10 0. 70 0.40 11 1 <40.0 - •

12 6 5. 46 0. 35 0. 14 1 12 6 51.8 19.7 8.0 1

13 5 78.0 49.2 21.9 13 1 <5000 - -

14 6 5.46 2. 50 1.02 1 14 6 30.3 12.5 5. 1 1

15 6 5.68 1.43 0. 59 1 15 6 32.3 3.9 1.6 1

16 6 5.60 0. 85 0. 35 1 16 3 32.7 15. 1 8.7 1

17 3 2.82 0.41 0. 24 1 17 3 295.3 137.5 79.5 1

18 1 2. 10 - - 2 18 1 270.0 --

19 6 4. 15 0.29 0. 12 1 19 6 247. 0 36.0 14.7 1

20 6 5. 10 0. 10 0. 04 2 20 6 25. 5 3.7 1.5 1

The following codes are used: 1 = destructive activation analysis 2 = nondestructive activation analysis 3 = chemical methods

139 Hg IN FLOUR (treated with Hg-compound) Hg IN FLOUR (not treated)

X = 4.59*1.37 X = 42.5±H.7

I IN

I-"'"* Vo I-'

! ii

20 Lab. No. 10 Lab. No.

FIG. 28. Reliability of mercury analysis. Most laboratories used neutron activation followed by chemical separation and determination of radioactive mercury isotopes. Four laboratories used non-destructive activation analysis, two used chemical methods. Separation methods reported included distillation, extraction, displacement, sulphide precipitation, reduction and isotopic exchange. The variety of techniques used was too large to allow a separate evaluation of these methods. It appears, however, that non-destructive activation analysis tends to produce high values when used on samples containing very small amounts of mercury. The results are shown in Table XXIIandFig. 28. Mercury contents of the mercury-treated sample are shown in ppm, those of the untreated one in ppb. In the figure, the overall mean of all laboratory averages is shown by a long dotted line drawn across the chart, individual laboratory averages are represented by short horizontal lines, confidence limits of single determinations and of means within laboratories are shown by thin and by thick vertical lines, respectively. The average values reported by all laboratories, except one, for the sample of mercury-treated flour were combined to calculate the overall mean and the standard deviation of the laboratories' means:

X±S = 4.59 ± 1.37 ppm Hg

(Not included is the obviously erroneous value of 78 ppm reported by one laboratory. ) The corresponding confidence limits of the mean are ± 0. 7 ppm Hg. In calculating the mean mercury content of the untreated sample, several obviously high values (>100 ppb) had to be discarded. The re- maining averages combined gave a mean and standard deviation between laboratories of:

X±S = 42.5 ±14.7 ppb Hg and a 95% confidence limit of ± 9.0 ppb Hg.

11.3. CONCLUSIONS

It is the aim of any analytical quality control to find out whether the reliability of the results meets the requirements of the investigation. Accuracy requirements differ with the type of work, with the purpose for which it is carried out and with the levels of element or compound in- vestigated. When the purpose of the measurements is to reveal a slow trend in environmental contamination levels, high precision and accuracy are normally required. When the results returned by the participating laboratories are reviewed in the light of these considerations, most laboratories documented work of sufficient accuracy and precision in the ppm-range of mercury. In the ppb-range, however, the situation is much worse and many labora- tories returned unsatisfactory results. This is most probably due to systematic errors such as contamination of the samples before activation leading to a relatively large number of too high results.

141

12. PRESENT LEVELS OF MERCURY IN MAN AND HIS ENVIRONMENT*

A. V. HOLDEN

12.1. INTRODUCTION

Although mercury has long been recognized as an element existing in low concentrations in soils generally, and in commercially-exploitable quantities in certain minerals such as cinnabar, its presence in biological systems, and the toxic effects resulting from its discharge in industrial wastes, has only been demonstrated during the past two decades. Following incidents in Minamata, Japan [9, 18],and the discovery of high mercury levels in wild birds and fish in Sweden [ 35], investigators in several countries have examined the position regarding mercury levels in both the terrestrial and aquatic environments, and the possible sources of contamination. It has now become important to assess the natural levels of mercury in the environment, and to be able to detect any increase due to the effects of mercury discharges, as well as the effects, if any, of such increases both on eco-systems and on man himself. This chapter attempts to present the evidence so far available, in published reports, on the concentrations at which mercury has been found to exist in various components of the terrestrial and aquatic eco-systems. Data are given for air, water, plant and animal species, and for man in circumstances other than those involving contamination by employment in industry, or from medical treatment. The levels of mercury in soils and foodstuffs are dealt with more fully in other chapters. One major problem which arises in the comparison of published data on mercury levels is that methods of analysis are still being developed and improved to attain the required sensitivity and accuracy. Data obtained by one method are not necessarily comparable to those given by another. Where possible, the methods of analysis used by investigators are identified in this chapter in conjunction with the data given. Unless otherwise stated, the data refer to total mercury.

12. 2. SOURCES OF MERCURY IN THE ENVIRONMENT

Apart from the natural occurrence of mercury in elemental form or as minerals, there are several man-made sources which give rise to environ- mental contamination. These have been reviewed by Lofroth [29], Ackefors et al. [13] and Larsson [4] for Sweden, and Fimreite [36] for Canada, and include:

1. Fungicides used in agriculture 2. Fungicides used in the paper and pulp industries 3. Mercury electrodes in the chlorine-alkali industry

5 The author of this review is greatly indebted to the many individuals who kindly supplied reprints and references.

143 4. Electrical apparatus such as rectifiers, switches and batteries 5. Uses in medicine 6. Combustion of fossil fuels (oil and coal) containing traces of mercury 7. Refining of mercury from ores 8. Use in recovering other metals from minerals 9. Mercury compounds used in anti-fouling and mildew-proofing paints 10. Mercury or its salts used as catalysts, e.g. in the production of acetaldehyde and vinyl chloride 11. Use in thermometers

The relative importance of these sources will clearly vary from one country to another. In Sweden the pulp and paper industry, in Canada and the USA the chlorine-alkali industry, and in Japan the vinyl chloride process, have been cited as major causes of pollution.

12.3. MERCURY IN AIR AND WATER

12.3.1. Air

According to Eriksson [ 1 ], concentrations of mercury in air are around 20 ng/m3, equivalent to about 20 ng/cm2 over the surface of the earth. This mercury probably arises from the evaporation of metallic mercury in soils, and is believed to be in metallic form in air, not easily washed out by rain. In the neighbourhood of industries using mercury, the concentra- tions in air could be expected to be much higher. Byrne and Kosta [ 2], using neutron activation, found mercury deposits as high as 50 (jg/cm2 on exposed surfaces near the distillation plant of a mercury mine in Yugoslavia. Mercury in air is now being analysed routinely in Sweden by the International Meteorological Institute [4]. The maximum acceptable concentration of mercury in air is fixed at 100 fxg/m3 most countries, although the limit in the Soviet Union is 10 /ng/m3 , according to Axelsson and Friberg [ 19] .

12.3.2. Rain and snow

Eriksson [ 1] gives the concentration in rain as about 200 ng/litre, although data are scarce. Stock and Cucuel [ 3], using a microscopic method, reported values for rain in the range 50- 500 ng/litre in 1934, but in view of recent evidence of contamination by industrial pollution of the atmosphere these analyses may include mercury other than of natural origin. Johnels et al. [ 5] found 0. 07 ng/g of mercury in the surface layer of snow on Lake Usken, Sweden, and 0. 21 ng/g in a layer below, deposited a month earlier (neutron activation). Byrne and Kosta [2], using a similar technique, found that mercury concentrations in melted snow, which had been centrifuged to remove solids, were 1-3 ng/g both near a mercury mine and about 1 km away, but the solids fraction contained 100 to 1000 times as much mercury near the mine (and distillation plant) as was found at the second site.

144 TABLE XXIII. MERCURY IN FRESH-WATERS

Concentration Source Method Reference (ng/litre)

Well water (Germany) 10 - 50 M [3]

River water (USSR) 400 - 2 800 [10]

River water (Sweden) 130 NA [5]

River water (Sweden) 250® NA [5]

River water (Sweden) 40 NA [20] a River water (Sweden) 510 NA [20]

River water (Sweden) 270a NA [20]

River water (Yugoslavia) 63 NA [6]

Tap water (Yugoslavia) 100 - 200 NA [2]

Spring water (Yugoslavia) 300 NA [2]

Reservoir (Yugoslavia) 107 NA [2]

River (Yugoslavia) 100 - 1300 NA [2]

a a River (Yugoslavia) 4 000 - 13 000 NA [2]

Agano River (Japan) 100 GC [9]

Ohmi River (Japan) <10 C [15]

Shibue River (Japan) 80 C [15]

Seki River (Japan) <10 C [15]

Detroit River (USA) 30 0003 NA [7] a Contamination known to exist. Methods: M = microscopic measurement NA = neutron activation GC = gas chromatography C = colorimetry

12.3.3. Fresh-waters

Stock and Cucuel [3] reported 10-50 ng/litre in well-waters in Germany in 1934, but 100 ng/litre in the river Rhine. In recent years, several more analyses have been reported in the literature, and these are given in Table XXIII. Most of the uncontaminated waters contained concen- trations below 200 ng/litre, but values up to 30 000 ng/litre of total mercury have been found in waters polluted by effluents containing mercury. At least a part of this mercury may have been in suspended matter, as in the samples of Byrne and Kosta [ 2] taken below the mercury plant.

145 12.3.4. Sea-water

Stock and Cucuel [3] found 30 ng/litre of mercury in sea-water. Sillen [8] reported the same value, and considered that the mercury probably exists as negatively charged mercuric chloride complexes. Hosohara [16] examined samples of water at different depths in the Pacific Ocean; at the surface he found concentrations from 80 to 150 ng/litre, at 500 metres from 60 to 240 ng/litre and at 3000 metres from 150 to 270 ng/litre (photometric method). Sea-water in Minamata Bay, Japan, sampled in the same year (1960), contained 80 to 660 ng/litre ([17], photometric method). Kiyoura, quoted by Ui [9], found less than 1000 ng/litre and mostly about 100 ng/litre in the same area, where methylmercury was being discharged in the effluent from an acetaldehyde plant.

12.4. MERCURY IN SOILS AND SEDIMENTS

The level of mercury in soils will be dealt with more fully in another chapter, but as its occurrence in soils may be relevant to the levels found in underwater sediments, an indication of the natural soil concentrations is appropriate here. Most of the available data have been obtained by Swedish and Canadian workers. In Sweden the mercury content of samples of soils of various types was found [ 11 ] to range from 20 to 920 ng/g, with a mean of 70 ng/g, the highest values being found in organic soils. Stock and Cucuel [3] regarded 100 ng/g as normal but gave ranges of 100 - 290 ng/g for forest soils, 140- 1000 ng/g for cultivated soils, 30- 34 ng/g for clay soils and 1-29 ng/g for sand. Warren and Delavault [12] in Canada considered normal levels to be 10- 60 ng/g, and values above 250 ng/g to be anomalous. Where mercuric sulphide (cinnabar) deposits occurred in British Columbia, they found as much as 1000- 10 000 ng/g in soils. In the vicinity of gold, and base-metal deposits, soils were found to contain 50- 250 ng/g, but some- times up to 2000 ng/g. Certain British soils examined by the same workers contained 250- 15 000 ng/g, the mercury apparently being of geological origin and not due to agricultural fungicidal practices. Few analyses have been made of mercury in the sediments of rivers, lakes or the sea, but the levels could be expected to be much higher than those in the overlying waters. Much of that detected has probably been derived from the deposition of eroded soils from the adjacent catchment area. Hasselrot [ 20] found 34 - 168 ng/g (dry weight) by neutron activation in river sediments upstream of factories using mercury, levels which are of the order of one thousand times greater than those in unpolluted waters. Below a paperboard mill using a mercurial fungicide the sediment was found to contain 18 400 ng/g at 1. 6 km, 4 300 to 10 100 ng/g at 5 km and 3 500 - 8 000 ng/g at 7 km, mostly in the upper 2 cm of sediment. Similarly, below a chlorine-alkali plant, he found 11 600 - 26 500 ng/g in the upper 4 cm of sediment 550 m downstream and 1200 ng/g in the top 1 cm of sediment 750 m downstream. Even 7 km downstream 440 ng/g were found. Similar analyses have been reported from the United States [ 7] where contaminated sediments below a chlorine-alkali plant contained 5 400- 86 000 ng/g. Other areas, less contaminated, contained 600 - 4000 ng/g. Saito [15] reported 350- 3730 ng/g in river muds near Japanese industrial plants (processes

146 unspecified), the overlying water generally having less than 10 ng/g (dithizone method). All the above analyses are in terms of dry weight. Marine sediments near the Swedish and Japanese coasts have been examined. Sediments sampled in the Sound between Denmark and Sweden, adjacent to the Swedish coast, were found to contain high mercury levels, up to 2000 ng/g (dry weight), apparently due to pollution [13]. Other marine sediments examined near the Swedish coast contained 1 000 - 1 500 ng/ g. Sediments in the region of the discharge of mercury-containing effluents in Minamata Bay, Japan, were found [15] to contain from 7 160 - 801 000 ng/g (dry weight, dithizone method). The form in which the mercury occurs in freshwater or marine sedi- ments has not been identified in the analyses quoted, but Jensen and Jernelov [ 14] reported that inorganic mercury can be converted to methyl- mercury by micro-organisms in freshwater sediments in aquaria. Thus, mercury in fish or invertebrates may enter directly in the methyl form.

Wood et al. [42] showed that a non-enzymatic process involving vitamin B12 can produce methane and methylmercury.

12.5. MERCURY IN VEGETATION

12.5.1. Terrestrial vegetation

This section is concerned mainly with the levels of mercury in vegetation other than that to which mercurial compounds have been applied deliberately, as in agriculture and horticulture. Very little published information appears to be available on the natural levels of mercury in plants. Byrne and Kosta [ 2] examined grass, various wild plants, trees and vegetables near a mercury-processing factory and at a distance of one kilometre from the factory, where the soil had a high mercury content. Other areas, where contamination or high soil levels were absent, were also sampled, all analyses being made by neutron activation. In the factory area, deposition of mercury on tree bark was as high as 50 jug/cm2, with lower concentrations on vegetation exposed for a shorter time. The mercury contents of sub-surface tissues were 1000 ng/g wet weight for Crocus albilorus, 1100 ng/g for Sambucus nigra (elderberry) stems, and 44 ng/g for Tussilago farfara (coltsfoot) shoots. The bark of Prunus avium (cherry) in the factory area contained 59 000 ng/g, in the high-mercury soil area 6 000 ng/g and in the uncontaminated area 63 ng/g. Values in peeled thick wood were 300, 84 and 2.3 ng/g respectively. Other data are given for root vegetables and cabbages, and the concentrations in the factory area were approximately one order higher than in the high- mercury soil area (soil concentrations being 0. 02- 0. 06%). The vegetables in the factory area contained from 7 to over 800 ng/g (wet weight) and in the other area from 1 to 102 ng/g, carrots showing a large capacity for accumulating mercury from soil. The seeds of tomatoes, apples and wal- nuts were also found to accumulate more mercury than the flesh of the fruit. None of the plants examined showed any evidence of damage from the presence of mercury. Kosta and Byrne [6], using neutron activation, found 137 ng/g in grass and only 2. 5 ng/g in wheat flour, but the origin of the samples is unspecified.

147 Saito [15] recorded concentrations of mercury (determined by neutron activation) in paddy rice, ranging from 72 - 99 ng/g on untreated crops in Japan to 134 - 530 ng/g where mercurials had been used. Rice from the Philippines and Thailand contained 34 - 50 ng/g. Grass in Japan contained 65 ng/g. Tobaccos, presumably treated with mercurials during cultivation in Japan, were found to contain 980 - 1 600 ng/g. Residues in sprayed fruit and vegetables were reviewed by Smart [ 10] .

12. 5. 2. Aquatic vegetation

The accumulation of mercury in aquatic vegetation seems to have received little attention, although Johnels et al. [5] reported 140 ng/g in Nite 11a spp. in apparently uncontaminated fresh-water. A sample of Fontinalis spp. above a paper mill contained 76 ng/g, and below the mill 3 700 ng/g, indicating the considerable accumulation resulting from the discharge of an organomercury fungicide. There is no indication as to whether the samples referred to were dried, or analysed in terms of wet weight. Hannerz[21], using radioactively labelled organomercury compounds, found that submerged parts of plants in fresh-water ponds had concentrations of mercury 10-20 times higher than in the emergent parts, presumably due to surface adsorption. The uptake in the plant tissues seldom gave a concentration factor (from water) exceeding 100, and was the same order of magnitude in different species. Adsorption to the submerged parts varied considerably with species, being related to the surface area exposed per unit weight of the plant. For algae, the blue-green Nostoc pruniformes contained low concentrations and the filamentous Oedogonium spp. high concentrations. In the marine environment, Stock and Cucuel I 3] found 30- 37 ng/g in the sea-weed Laminoria hyperborea, and 18 - 23 ng/g in Fucus vesiculosus.

12.6. MERCURY IN INVERTEBRATES

12.6.1. Terrestrial invertebrates

The main attention in the problem of mercury contamination of the terrestrial environment has been given to the accumulation of residues by seed-eating or fish-eating birds, and the extent to which terrestrial invertebrates may absorb mercury seems to have been virtually ignored so far. The fruit fly Drosophila melanogaster has been used experimentally [22] in the investigation of genetic effects produced by organomercury compounds, and adult females were found to concentrate methylmercury chloride from 5 jug/ml in the feed to 90 Mg/g as mercury in the females, the concentration decreasing to 30 ng/g in 7 - 8 days. The eggs from these females contained 110- 120 Mg/g.

12.6.2. Aquatic invertebrates

Some measurements of the concentrations of mercury in fresh-water invertebrates have been made by Swedish workers as part of their investi- gations into the accumulation of mercury residues by fish in rivers receiving effluents from paper mills. The mills used phenylmercury compounds for

148 TABLE XXIV. MERCURY CONTENT OF AQUATIC INVERTEBRATES IN UNPOLLUTED AND POLLUTED WATER, NEAR A SWEDISH PAPER MILL

Mercury (ng/g) Aquatic invertebrate Above mill Below mill

Trichoptera 52 - 54 5 600 - 17 000

Plecoptera (Isoperla spp.) 72 2400

Hirudinea (Helobdella spp.) 25 2350 - 4400

Neuroptera (Sialis spp.) 49 - 52 4 800 - 5 500

Asellus aquaticus 59 - 65 1 900

(From Johnels et al. [5]).

controlling slime. Pulp mills have used similar compounds as for stored pulp. Data on invertebrate residues from Johnels et al. [ 5] are summarized in Table XXIV, the analyses being made by neutron activation. The concentrations in the uncontaminated area are in the range 25 - 72 ng/g, but in the contaminated area are about two orders higher (1 900 - 17 000 ng/g). Some of the values were obtained three months after use of the mercury compound was discontinued, but there was no evidence of any decrease in the level of contamination. These levels of mercury, moreover, appear to have been without effect on the organisms concerned, but could have been one source of intake for fish such as pike (Esox lucius) which were living in the river. Paper fibres discharged from the mill in question were found to contain 2 400 ng/g in a wet sample, and may have formed part of the food of the invertebrate fauna. The same workers also examined various species of zoo-plankton, and found a mean of 140 ng/g in an area not directly polluted. In similar areas the muscle tissue of crayfish (Astacus fluviatilis) contained 140 ng/g and Chironomus plumosus larvae 230 ng/g. Hannerz [21], in experiments with labelled organomercury compounds, used a number of different invertebrate species, including molluscs, insect larvae, leeches, beetles and Crustacea. The concentration factors from water were very variable, depending on the food intake and nature of food, and on the activity of the species concerned, but values from a few hundred to several thousand times were typical. Marine invertebrates have received little attention, but during investi- gations into the cause of the Minamata poisoning in Japan [ 18] contaminated shellfish were analysed. Samples from an area of low toxicity contained 2 7 000 ng/g of mercury, and in an area of high toxicity about 100 000 ng/g. The species of shellfish were not disclosed.. Oysters from the area con- tained 39 000 ng/g of mercury. The values given above are in terms of dry weight of tissue, and are approximately 2.5 times the corresponding wet weight value. Concentrations in terms of wet weight of tissue found in oysters from Galveston and Chesapeake Bays, on the eastern coast of the

149 United States, were of the order of 300 - 1 000 ng/g. In both of these areas, as in the Minamata area, vinyl chloride production is carried out using a mercuric-chloride catalyst, but the processes differ significantly, and much less mercury evidently enters the marine environment from the US plants [18]. Data from other areas are few, but a Canadian report [44] refers to 150 ng/g of mercury in a and 400 ng/g in a queen crab taken as isolated samples in the Gulf of St. Lawrence (neutron activation analysis). In experimental work [83] the mussel Mytilus edulis and the short-necked clam Venerupis philippinarum absorbed radioactively-labelled mercuric chloride rapidly in a few days from a concentration of 0. 05 mg/litre in sea- water. Increases of 660 and 190 times, respectively, were measured in soft tissue. Other studies using labelled mercury [28] demonstrated large differences between concentrations in different organs of Venerupis philippinarum. Concentrations of mercury measured in polluted shellfish were 15 000 - 63 000 ng/g.

12.7. MERCURY IN AQUATIC

12.7.1. Fresh-water vertebrates

Most of the investigations into mercury contamination of the fresh- water environment have been made in Sweden [5, 13, 24, 25, 56], where the use of organomercury fungicides, particularly phenylmercuric acetate, in the paper and pulp industry has led to serious contamination of lakes and rivers. Other sources of discharge to fresh-waters include the chlorine-alkali industry, in which mercury electrodes are used, and the processing of mercury ores to recover the metal. Most of the analyses have been made by neutron activation [ 5, 25], but gas chromatography [ 24, 26, 56] has also been used to determine methylmercury. The first references, in the literature, to analyses of fresh-water fish for mercury give concentrations (in terms of wet weight) of 30 - 180 ng/g [ 3] in 1934, and 76 - 167 ng/g [23] in 1948. These values are similar to those currently regarded as indicative of residues of natural origin. The distribution of mercury residues found (by neutron activation) in various organs and tissues of pike (Esox lucius) is shown in Table XXV, from Johnels et al. [5]. Apart from the scales, the range of concentrations is less than four to one, and the muscle tissue, constituting the major proportion of the total weight and the part consumed by man or other pre- dators, contains almost the highest concentration. Johnels and his co- workers found a relation between total weight and mercury content of muscle tissue for pike in each of several Swedish lakes and, for comparison, calculated the mercury content of a 1-kg fish from each of many localities sampled. The relationship applies to age and size, but age estimation is more difficult in routine investigations. At levels below 200 ng/g the relation is not significant. The concentrations in muscle tissue, calculated for a 1-kg pike from samples (numbering 3 to 11 fish) taken in 49 different localities in Sweden, Finland, Norway and Switzerland, ranged from 60 to 2 500 ng/g. In the few areas believed to be uninfluenced by human activities involving mercury the values were 60 - 140 ng/g. A larger number of areas, in which mercurial

150 TABLE XXV. MERCURY IN THE ORGANS OF A PIKE (Esox lucius) (Tido-Lindo, Lake Malaren, Sweden, 1.7.64)

Organ Mercury (ng/g)

Heart muscle 1000

Axial muscle 850

Liver 780

Kidney 640

Intestine 610

Ovary 560

Epidermal finrays 390

Gill 300

Brain 290

Spleen 280

Scales 104

(From Johnels et al. [5]).

seed dressings were used, or into which such chemicals could have been airborne, gave values of 100- 300 ng/g. Most of the localities were believed to be influenced by industrial discharges, often some distance away, or by seed dressings, but in a few cases the high values obtained, 250- 1400 ng/g, were unexplained. The final group, where direct discharge from industry was recognized, gave values of 450 - 2 500 ng/g. Pike from a contaminated locality, where the concentration in water was 0.25 ± 0.07 ng/g, contained the equivalent of 2 300 ng/g in a 1-kg fish, representing a concentration factor of 9 200 from water to fish. The Swedish workers found that concen- tration factors varied among different localities, but were generally about 3000. Lower mercury levels have been found in Sweden in fish from eutrophic waters than in those from oligotrophic waters, and the latter thus appear to be more susceptible to the effects of mercury contamination [ 25] . The highest mercury concentration so far recorded in Swedish pike is 9 800 ng/g, in a fish caught in southern Sweden [ 29]. None of the concentrations found appear to have had any significant biological effect on the fish, but as many samples contained more than the maximum acceptable limit for human food in Sweden (1 mg/kg = 1 000 ng/g), the consumption of fish from many areas was severely restricted [29], Analyses of pike in Sweden have also been reported by Westoo [ 24], Johnels et al. [ 25] and Tejning [49] . Pike from Finland [ 30] were found to contain abnormally high concentrations in many areas. The data from

151 TABLE XXVI. MERCURY LEVELS IN PIKE (Esox lucius) FROM VARIOUS SOURCES (Concentrations in ng/g in muscle tissue, wet-weight basis)

Country Range Mean Method Reference Remarks

a Sweden 60 - 140 90 NA U) Uncontaminated

Sweden 100 - 3003 190 NA t5] Slight contamination

Sweden 250 - 1400" 630 NA [51 Moderate contamination

Sweden 450 - 2 5003 1200 NA [5] High contamination

Sweden 154 - 1340 360 NA [20] Above pulp mill

Sweden 4400 - 7 500 6 600 NA [20] Below pulp mill

Sweden 83 - 2300 - NA [25] --

Sweden 150 - 5 200 1090 NA [24] --

Norway 120 - 1270 560 NA [34] --

Finland 60 - 5 800 - NA [30] 1967 data

Finland 19 - 5 320 - NA [30] 1968 data

Canada (Cedar Lake) - 750 AA [32] 25 fish

Canada (Lake Winnipeg) - 490 AA [32] 19 fish

b Canada (Manitoba Lakes) 130 - 680 - AA [32] 56 fish

Canada () - 1430 AA [32] 3 fish

Canada (N. Saskatchewan River) 700 - 1 600 1150 AA [31] 4 fish

Canada (S. Saskatchewan River) 1000 - 10 600 1100 AA (31) 4 fish

United States (Lake Erie) - 640 AA [7] 3 fish

Scotland (lowland lake) 78 - 118 102 NA [33] 5 fish

Scotland (hill lake) 330 - 1260 760 NA [33] 5 fish

Sweden (Dalsland, VSstergStland) 252 - 2366 b 962 NA [49] 64 fish

Sweden (Lake VSnern) 716 - 1182b 870 NA [49] 65 fish

Method: NA = neutron activation AA = atomic absorption

a Calculated for 1 kg pike (see text)

^ Range of mean values.

these reports, together with information from pike analysed in Canada, the United States, Norway and Scotland, are summarized in Table XXVI. Many more pike have been analysed by Westoo and RydSl [56]. The values from Canada and the United States include samples from river and lake systems polluted by mercury discharges from chlorine-alkali plants, but the Scottish hill lake which gave high values is in an area where no industry exists and no use of mercury is known. It is possible that the geological strata have an above-normal mercury content. Table XXVIindicates that mercury levels in pike from uncontaminated areas, or those receiving only slightly airborne pollution, are generally not greater than about 300 ng/g, but in waters receiving significant pol- lution from mercury discharges the concentrations in pike muscle may be as high as 10 000 ng/g. The upper limit of natural mercury concentrations in fish has been variously estimated at 200 ng/g [29], 150 ng/g [44] and even 100 ng/g in Japan [ 50].

152 TABLE XXVII. MERCURY LEVELS IN VARIOUS SPECIES OF FRESH- WATER FISH IN SWEDEN (Concentrations in ng/g in muscle tissue, wet-weight basis)

Species Range Reference

Perch (Perca fluviatilis) 130 - 3 950 [24] 130 - 1030 [25]

Walleye (Stizostedion spp.) 170 - 2 550 [24]

Brown trout (Salmo trutta) 22 - 130 [24]

Salmon (Salmo salar) 112 - 130 [25]

Pike-perch (Stizostedion lucioperca) 51 - 710 [25]

Whitefish (Coregonus spp.) 70 - 1400 [24] 34 - 650 [25]

Eel (Anguilla anguilla) 34 - 880 [25]

All analyses by neutron activation.

(From WestBO [24] and Johnels et al.[25]).

Several other species of fresh-water fish have been examined in .Sweden [24, 25, 26], Finland [30], Norway [34], Japan [50], Canada [ 31, 32, 46, 47] and the United States [ 7] . In most instances only a few fish of each species have been analysed, but the data from Westoo [24] and Johnels et al. [25], which relate to at least six fish of each species, are given in Table XXVII and indicate the ranges of values which may be found among uncontam- inated and contaminated environments. (Many more analyses were reported by Westoo and Rydal [ 56] ). The salmonids, brown trout and salmon, contained relatively low levels, although it is not certain whether this is due to the fish having been obtained only from unpolluted rivers. For the same species, Eades [37] reported less than 15 ng/g in organic form in specimens found dead in Ireland (Gage colorimetric method), although death was not thought to be due to mercury poisoning. The Japanese data given by Ui [50] are for species mostly different from those examined in Europe and North America. The samples were taken in the Agano River, Niigata, where an outbreak of mercury poisoning similar to Minamata disease was detected in 1965. Methylmercury chloride from an acetaldehyde plant was the cause of contamination. Although much less than 100 ng/litre of the compound was found in the river water, the concentrations in the various species of fish were mostly between 0 and 5 000 ng/g, with a few values up to 23 000 ng/g. Invertebrates in the river contained mercury in the range 0 - 10 000 ng/g. Johnels et al. [5] found evidence that the concentration of mercury in pike muscle increased with the weight (and age) of the fish, at least above

153 200 ng/g. Above 1 000 ng/g there was apparently no relation between the concentration and age of the fish, and from the data presented the concen- tration/weight relationship could only be regarded as approximate. How- ever, Fimreite [47] also tested the concentration/weight relationship for a number of species of fish in Canada, and in most instances found a significant positive correlation. Experimental work by Hannerz [21], using radioactively-labelled mercury compounds, showed that differences exist in the pattern of distri- bution of residues between the various organs and tissues, according to the organic form in which the mercury is complexed, and also whether fresh- water or brackish water is used. The mercury is accumulated rapidly but eliminated slowly, giving concentration factors of several thousands. The highest concentrations were found in the liver and kidneys, rather less in the heart, spleen, gills and brain, and low levels in muscle and bone. Uptake rates varied among the organs. Methylmercury was most rapidly assimilated, a concentration factor of 9 000 being found in the kidneys of pike, and 2 000 in muscle. The route of assimilation appeared to be mainly through the outer epithelia. Similar studies by Backstrom [45], who injected labelled mercury compounds into several species of fresh- water fish, showed that uptake was high in the gills, kidneys, liver, spleen and thyroid, but lower in muscle. Yet analyses of pike in Swedish lakes and rivers [5] have shown that levels in muscle are among the highest of any organ or tissue (Table XXV). Mercurial compounds have been used for many years as therapeutic agents in fish hatcheries. Rucker [38] examined pyridylmercuric acetate (PMA) and 6.25% ethylmercury phosphate (Lignasan) in treatment of bac- terial gill disease and parasitic protozoans. PMA was unacceptably expen- sive and toxic to some species of fish, but Lignasan was more satisfactory. Sockeye salmon (Oncorhynchus nerka) exposed for one hour per week in 1-1.33 mg/litre of Lignasan over four months gave mercury residues in whole fish of 8. 1 mg/kg and in eviscerated fish of 5. 9 mg/kg. These fish were examined one month after treatment ceased, and further samples indicated that the level did not return to normal for three months. The normally expected mercury concentration was up to 0. 05 mg/kg (50 ng/g). Thus, the whole fish examined had 160 times the normal content of mercury. Rucker and Amend [ 39] examined rainbow trout (Salmo gairdneri), sockeye salmon (Oncorhynchus nerka) and chinook salmon (O. tshawytscha) after exposure to ethylmercury phosphate (EMP) and pyridylmercuric acetate. The concentration of mercury in the gills and blood increased over a period of 1 - 2 days and then declined, while levels in the kidney and liver were maintained for several weeks after a single exposure to EMP. Following eleven daily one-hour exposures of rainbow trout to 2 mg/litre of Timsan (6.25% EMP), blood concentrations reached 22.8 mg/kg with 16. 7 mg/kg in liver, 19. 3 mg/kg in kidney and 4.0 mg/kg in muscle. Twelve once-weekly exposures produced higher levels in the kidney but similar values in the other tissues. Exposure to 2 mg/kg of PMA also produced similar values. The therapeutic use of these mercurials is thus likely to result in high mercury concentrations in fish which may be destined for human consumption. The maximum acceptable level of mercury in human food is still a subject of discussion in the various countries which have identified high mercury levels in fish destined for human consumption. Following the

154 occurrence of Minamata disease in Japan, estimates were made of the consumption of fish and shellfish by the victims. Lofroth [ 29] discussed the reasoning, based on this information, which resulted in a Swedish decision to regard fish containing more than 1 mg/kg (= 1000 ng/g) of mer- cury as unfit for human consumption. This limit was considered to be too high, and in 1968 the Swedish authorities recommended that fish from fresh and coastal waters where no general ban on use existed should only be eaten once a week. Subsequently, further calculations suggested that, as the earlier limits had been proposed on the assumption that the original Japanese data were for wet fish, when in fact they were for dried fish, the Swedish limit should have been reduced to 0.4 mg/kg or even 0.2 mg/kg. Following a study of mercury in the human body, Tejning [ 40] also favoured 0. 2 mg/kg wet tissue as the maximum acceptable limit. A Canadian report [44] mentions that a "working level" of 0.5 mg/kg is used by the Food and Drug Directorate, in the absence of any legal limit in Canada. The data given above for the levels found in various species of fish in several countries have indicated that this suggested limit of 0. 2 mg/kg (= 200 ng/g) would be exceeded by fish in many areas, often perhaps as the result of naturally occurring high mercury levels in the environment. Methylmercury has been found to be the most toxic form of organic bonding, and in Sweden commonly more than 90% of the mercury in fish has been found to be in the methyl form [ 26, 41, 56] . Samples exchanged between Swedish and Japanese analysts showed that in both countries methylmercury constituted 82 - 86% of the total in the samples examined. Mercury intro- duced into the environment in the phenyl form can be converted to the more toxic methyl form in different ways. Phenylmercury can be converted to the inorganic form as can methoxyethyl mercury. Methylation of the in- organic mercury has been shown to occur in sediments and in dead fish [ 14]. It has also been shown to be a non-enzymatic process [42]. If the form of mercury, rather than the total mercury content, is more significant in human food, the data provided by methods such as neutron activation will require to be supplemented by techniques such as gas chromatography to identify the organic combination present. The persistence of mercury contamination in the aquatic environment, after use and discharge of mercury or mercury compounds has ceased, is a subject which will be studied intensively in the next few years, as polluting discharges are controlled. The evidence of Fimreite [47], however, suggests that rapid improvement of the situation is unlikely. He found high levels in fish below a mercury mine 25 years after it had been closed (although presumably the high natural level in the soil might have been partly responsible). Fish and fish-eating birds sampled in the vicinity of pulp mills which had discontinued the use of mercury fungicides 5-10 years previously also contained elevated mercury levels. Unless the mercury can be bound in an unavailable form, its continued presence in the biota of polluted water seems to be assured. The physiological effect of mercury residues on fish has received relatively little attention, at least with organomercury compounds. Toxicity tests using fish have generally given information only on the aqueous concen- trations of mercury compounds which are lethal in short-term tests, as in the work of Boetius [73]. He found, however, that mercuric chloride and phenylmercuric acetate may be toxic at any concentration, with no evidence of a lower limit. The aqueous concentrations of mercury compounds which

155 are acutely toxic to fish are high relative to those which have been measured in natural waters, even most of those which are polluted, but the accumu- lation of residues by long-term exposure to low aqueous concentrations, or from food, may be sufficient to result in tissue concentrations potentially damaging to fish. Goldfish (Carassius auratus) killed in 0.5 mg/litre of mercury as mercuric chloride [82] contained 2 300 - 4 600 ng/g of mercury in wet tissue (whole body). By comparison, several species of fish in Japanese rivers contained 30- 3 720 ng/g. Chum salmon (Qncorhynchus keta) contained up to 3410 ng/g in liver and up to 450 ng/g in muscle tissue. Little information is available on chronic toxicity, on sub-lethal effects, or on residue levels at which such effects occur. Experiments with daily one-hour exposure of rainbow trout to ethylmercury phosphate solutions [ 39] produced slight hypertrophy of gill epithelial cells when the mercury content of blood had reached 16 200 ng/g (about 1 500 ng/g in muscle), and when the blood content reached 22 000 ng/g, more extensive hypertrophy and some hyperplasia were present. No abnormal histological changes were found in red blood cells, liver or kidney (where the mercury contents were 15 000 - 17 000 ng/g). Other experiments in which exposures were given for only one hour per week resulted in slightly lower gill concentrations (up to 15 700 ng/g), but higher kidney concentration (up to 39 600 ng/g), although no abnormalities were detected in kidney or spleen. Hyperplasia of the gill epithelium was the only pathological condition seen. Toxicity is also influenced by the dissolved oxygen and hardness of the water [ 84] . These observations relate only to ethylmercury phosphate, but methyl- mercury is generally considered to be the most toxic organic form of mercury. Marine fish and shellfish analysed in the Minamata area in 1959 were found to contain 9 000 - 24 000 ng/g of mercury, and most specimens were dead [ 48], although Ui [50] reported that only 20 - 40% of the mercury was in the methyl form. No reports of analyses of fresh-water fish killed by mercury poisoning have been found in the literature. Current research on the effects of methylmercury on fish in Sweden [4] has indicated a reduc- tion in the percentage of eggs hatching successfully. Other Swedish research is concerned with possible behavioural effects. Apart from the analyses of fish, the only other fresh-water vertebrates which have been mentioned in the literature are a species of seal in Finland. These are referred to in a later section dealing with mammals.

12.7.2. Marine vertebrates

There are a number of references to the levels of mercury found in various species of marine fish, notably in Japanese and Scandinavian litera- ture. Stock and Cucuel [3] were the first to report on mercury residues in sea-fish, in 1934, and Raeder and Snekvik [51] found similar levels in 1941, namely 44 to 155 ng/g. Such values can be regarded as typical of unpolluted sea areas but, following the discovery that mercury was respon- sible for the so-called Minamata disease in Japan [9], high concentrations were found in fish in Minamata Bay. Up to 50 000 ng/g were found in fish in the Shinrui Sea. Later, several Scandinavian workers examined various species of marine fish, and these data, together with those of Canadian and other workers, are summarized in Table XXVIII. Most of the fish have been found to contain less than 200 ng/g, and such low levels are similar to those presumed to be within the natural range for

156 TABLE XXVIII. MERCURY LEVELS IN MARINE FISH FROM VARIOUS SOURCES (Concentrations in ng/g in muscle tissue, wet-weight basis)

Source Species Range Method Reference

North Sea Cod 105 - 110 M [3] North Sea Whiting 38 - 55 M [3]

Norway Various 44 - 155 M [51] Norway Halibut 75 3 GC [26] Norway Haddock 14 - 423 GC [26] Norway Cod 22 - 793 GC [26] Norway Cod 50 - 1460 NA [34] Norway Saithe 50 - 570 NA [34] Norway Haddock 60 - 220 NA [34] Norway Flounder 130 - 680 NA [34]

Denmark Plaice w t o GC [26] Denmark Whiting 36a GC [26] Denmark Cod 40a GC [26] Denmark Cod 141 - 1290 NA [54] Denmark Plaice 38 - 496 NA [54] Denmark Flounder 32 - 893 NA [54]

Greenland Cod 13 - 26 NA [54]

North Sea Cod 158 - 195 NA [54]

Baltic Sea Herring 26 - 41 NA [53]

Sweden Cod 20 - 3603 GC [24] Sweden (Baltic) Cod 42 - 90 NA [25] Sweden (GCteborg) Cod 32 - 72 NA [25] Sweden (Oresund) Cod 245 - 2 700 NA [25] Sweden (Baltic) Plaice 31 - 76 NA [25] Sweden (Skagerrak) Plaice 203 - 290 NA [25] Sweden (Oresund) Plaice 71 - 3100 NA [25]

Indian Ocean Tunny 360a GC [52] a New Zealand Sea bream sp. no GC [52]

Africa Sea bream sp. 223 GC [521 a S. Pacific Ocean Sea bream sp. s GC [52]

E. China Sea Sea bass 96 3 GC [52] E. China Sea Grey mullet 32" GC [52) a Bering Sea Herring 8 GC [52] 3 Bering Sea Flatfish 6 GC [52]

Japan Various 8 - 35a GC [52]

Canada, W. coast Sebastodes spp. 176 - 876 NA [47] Canada, E. coast Eel 283 - 382 NA [47] Canada, E. coast Herring 25 - 57 NA [47] Canada, E. coast Cod 129 - 428 NA [44] Canada, E. coast Plaice 140 NA [44] Canada, E. coast Flounder 123 - 344 . NA [44] Canada, E. coast Herring 363 NA [44] Canada, E. coast Mackerel 104 NA [44] Canada, E. coast Various 117 - 371 NA [44]

United States Haddock 17 - 23 AA [77]

Methyl mercury

Methods: M = microscopic measurement GC = gas chromatography NA = neutron activation AA = atomic absorption.

157 fresh-water fish, but the Scandinavian workers found that in the area between Denmark and Sweden levels of up to 1 290 ng/g occurred in cod muscle. Other species, such as flounders, plaice and saithe (all inshore feeders) also contained high concentrations. By comparison, cod from Greenland contained very low levels, 13 - 26 ng/g, and from the North Sea 158 - 195ng/g [54], Fish from the Italian coast, sampled by Ui [ 9] in an area polluted by a discharge from an acetaldehyde factory (as at Minamata), contained 530 - 4 920 ng/g of total mercury in dried samples. The corresponding wet- tissue concentrations would be about 100- 1 000 ng/g. Ui reported the percentage of methylmercury to be very variable, in the range 13 - 84%. A second group of fish gave values for dried tissue of 170- 7 390 ng/g, with 20- 88% in the methyl form. Recent Canadian investigations [44, 47] have indicated that there may be localized contamination of marine fish off both Pacific and Atlantic coasts. Some species from British Columbia and the Gulf of St. Lawrence have contained concentrations over 400 ng/g, well over the expected natural range in those areas of up to 200 ng/g. Further analyses of marine fish by Jervis et al. [ 46 ], examining samples of various sea-foods, gave values from 27 to 310 ng/g. Recent evidence of high levels of mercury in Alaskan fur seals [ 74] suggests that these may have been accumulated from above- normal levels in the fish populations of the Pacific coast of North America. Hannerz [ 21], in studies on the accumulation of various radioactively- labelled organomercury compounds, examined cod in both brackish and salt-water, and found differences in the rates of uptake for methyl- and methoxyethylmercury, the rates for both being slower in brackish water. Concentration factors from water to most organs and tissues were 500- 1 000 in brackish water, but mostly 1 000 - 2 500 in salt-water. The difference between inorganic and organic mercury compounds was marked, mercuric nitrate giving very low concentration factors, usually between one and 50, except for the gills, where, perhaps by reaction'with mucus, factors up to 5 500 were found. The discharge of organomercury compounds into coastal waters from industrial effluents can clearly result in a marked concentration into fish tissues, perhaps even more rapidly than from similar discharges to fresh water.

12.8. MERCURY IN BIRDS

The first indication of mercury-induced ecological changes arose from the evidence of Swedish ornithologists, in the late 1950s, that many species of birds, and particularly raptors, were declining in numbers, and breeding success was declining [4], The mercury was believed to have been derived , from seed dressings, for which purpose alkylmercury compounds were introduced into Sweden about 1940 [ 35] . The marked increase in the mercury content of the feathers of several species of seed-eating and predatory birds was demonstrated by Berg et al. [ 57] who found, for example, that the level of mercury in the feathers of the peregrine (Falco peregrinus) rose from a mean of 2. 5 mg/kg, in the period 1834-1940, to values over 40 mg/kg in the period 1941-1965. Similar increases were found in eagle owls (Bubo bubo), and white-tailed eagles (Haliaetus albicilla). The goshawk (Accipiter gentilis) also showed [ 58] a marked increase in the mercury content of feathers, from a pre-1947 mean of 2. 2 mg/kg to a mean in the

158 period 1948-1965 of 29 mg/kg, including some values over 80 mg/kg. (All analyses were by neutron activation.) A comprehensive examination of the effects of alkylmercury poisoning of terrestrial wildlife, and particularly of birds, was made by Borg et al. [59]. Initially a photometric method was used, but later neutron activation was employed for total mercury, with some determinations of methyl- mercury by gas-liquid chromatography. Mixed liver and kidney tissue was analysed and, of seed-eating birds found dead, 48% had more than 2 mg/kg of mercury, and 13% more than 20 mg/kg. Of seed-eating birds shot, 41% had more than 2 mg/kg and 4% more than 20 mg/kg. Of predatory birds, 62% had more than 2 mg/kg and 11% more than 20 mg/kg. The greater proportion of the mercury was found to be in the methyl form, most samples containing more than 75% as methylmercury. Unhatched eggs contained 3-11 mg/kg of mercury. Experimental studies on several species of seed- eating birds confirmed that high concentrations of mercury accumulated in the muscle, liver and kidneys, at levels similar to or slightly higher than those found in wild specimens, and clinical and histological evidence was similar. Concentrations of mercury in the liver, or mixed livers and kidneys, of birds which died were 30- 200 mg/kg. The eggs of pheasants containing 1.3-2.0 mg/kg of mercury showed a much reduced hatchability. Elimination of mercury on changing to a mercury-free diet was slow, but there was evidence of decreasing mercury levels in wildlife after alkyl- mercury use in seed dressings was discontinued in 1966 [60]. Investigations of residues in birds in other countries have been more limited, but Holt [ 61 ] , using at first a photometric method and later neutron activation on liver, kidney, muscle and brain, found much lower levels of mercury in most Norwegian specimens than had been found in Sweden. The use of mercury-dressed seed was much lower in Norway. In wood-pigeon (Columbus palumbus) only 7.1% contained more than 2 mg/kg in kidneys, in hooded crows (Corvus corone cernix) 12. 9% and in birds of prey 27. 6%. Eggs rarely had more than 1 mg/kg, even in predatory species. Mercury was believed responsible for the low fertility and rapidly declining popula- tion of white-tailed eagles in Finland [8.0], although high concentrations of the organochlorine DDE were also found in the birds. In six birds found dead, concentrations of mercury in the liver were 4.6- 27. 1 mg/kg, in kidney 48.6 - 123. 1 mg/kg and in muscle 1.9-8.5 mg/kg. Two ovaries had 8.0 and 24. 7 mg/kg. In Finnish game birds [81] pheasants contained up to 13.4 mg/kg in the flesh in spring and summer, and slightly less in winter. Among aquatic birds mergansers contained up to 5.2 mg/kg, the eider (Somateria mollissima) up to 2.2 mg/kg and the mallard (Anas platyrhynches) up to 2.7 mg/kg. All the Finnish analyses were made by the dithizone method. Experimental work [62] on domestic hens fed with grain dressed with methylmercury showed that the mercury content of eggs rose to at least 8 mg/kg, while up to about 4 mg/kg were found in muscle tissue, 8-24 mg/kg in liver and about 8 mg/kg in kidneys. No influence on the health or laying ability of the birds was found, although the hatchability of the eggs was not examined. Other experimental work, using radioactively-labelled mercury compounds on quail (Coturnix coturnix) was reported by Backstrom [ 45]. Methylmercury was rapidly absorbed, and was stable, in contrast to phenyl- mercury which was rapidly decomposed to inorganic mercury. Methyl- mercury was also evenly distributed among most organs and tissues, and

159 only slowly excreted, whereas other forms of mercury were differently distributed. The methyl form, in contrast to other forms, was also highly concentrated in the brain. Marked sex differences in distribution among tissues was also noted. A few analyses of mercury levels in birds in Great Britain have been published [63] . With the exception of the livers of two sparrow-hawks (Accipiter nisus) in which 3. 9 and 3. 0 mg/kg of mercury were found, all values were less than 1 mg/kg. Eades [37] examined various tissues of birds found dead in Ireland, but only pheasants (Phasanius colchicus), in which 2. 53 - 5. 15 mg/kg were found, and pigeons (Columba spp.), in which 0. 015 - 5. 33 mg/kg were found, contained high levels. Death may have been related to mercury content in most specimens of these two species. 0.6 mg/kg were found in the eggs of a guillemot (Uria aalge) and Kittiwake (Rissa tridactyla). An investigation into the causes of death in birds of prey in the Netherlands [64] revealed high levels of mercury in the kidneys and livers of several specimens (determined by neutron activation). The kidneys of four kestrels (Falco tinnunculus) contained 9 - 125 mg/kg, while in buzzards (Buteo buteo) 0. 65 - 93 mg/kg were found in kidneys and 0. 57 - 99 mg/kg in livers. Other species of predators contained 6. 7 - 68 mg/kg in kidneys and 8.3-66 mg/kg in livers. One crane (Grus grus) had 18 mg/kg in the kidneys. Concentrations over 60 mg/kg were regarded as consistent with death of the birds by mercury poisoning, after considering the concentrations of organochlorine compounds also present. Recently the presence of significant contamination of the North American environment by mercury has been recognized. Most of the evidence so far available has been published in Canada, although the United States Bureau of Sport Fisheries and Wildlife reported [ 65] that 26 ducks sampled in the Lake St. Clair — area contained 0. 038 - 2. 26 mg/kg in breast muscle and 0.217- 7.5 mg/kg in livers. In Michigan, 39 scaup contained 0.01 - 1. 76 mg/kg in muscle. In Canada alkylmercury compounds are the predominant fungicides used for seed dressing [36]. The analysis for mercury (using neutron activation) of the livers of seed-eating birds and the eggs of birds of prey in Canada was reported by Fimreite et al. [66], the birds having been shot, rather than found dead (as in the case of many Swedish birds examined by Borg et al. [59]). Birds from Alberta, where mercury seed dressings are common, were compared with birds from Saskatchewan, where seed dressing is less common. In the livers of seed-eating birds in Alberta 0. 02 - 10. 2 mg/kg of mercury were found, and in Saskatchewan 0. 02 - 4. 6 mg/kg. The livers of predatory birds contained 0. 04 - 11.3 mg/kg and 0. 03 - 0. 95 mg/kg respect- ively, while the eggs of the predators contained 0. 01 - 1. 71 mg/kg and 0.01 - 0.43 mg/kg respectively. The highest levels among seed-eaters occurred in pheasants, patridges, pigeons and horned larks (Eremophila alpestris), but the percentage of seed-eaters containing more than 2 mg/kg (23% in Alberta, 4% in Saskatchewan) was far smaller than that found in Sweden (41%) by Borgetal. [59], The Swedish workers found that concen- trations exceeding 30 mg/kg in the livers of pheasants were correlated with death of the birds, but the highest amount found in Canadian pheasants was 5.9 mg/kg. The mean value for Alberta pheasants was 2.83 mg/kg, but pheasants poisoned experimentally, and found to lay eggs with reduced hatchability, had levels in the liver of 3 - 13 mg/kg. Such an effect on

160 reproduction may thus be present in wild Alberta pheasants. An independent investigation of mercury residues in pheasants and partridges from Alberta, Ontario and British Columbia [46] did not confirm the high values in the Alberta birds; only one of those sampled contained more than 0. 1 mg/kg in breast muscle and another had 0.22 mg/kg in the liver. The method used was neutron activation. Fimreite [47] has also examined mercury concentrations in Canadian fish-eating birds from rivers where mercury pollution exists. The livers of several species of inland waterfowl contained 0. 02 - 17.4 mg/kg, the highest values being in the red-necked grebe (Podiceps grisigena) from Pinchi Lake, in the vicinity of a mercury mine. Species sampled in coastal waters contained 0. 12 - 11.3 mg/kg in liver, the highest values being found in cormorants (Phalacrocorax spp.) (1.22-11.3 mg/kg) and 4. 53 mg/kg in a great blue heron (Ardea herodias). Concentrations in the livers of common terns (Sterna hirundo) were also high (2.28- 2.70 mg/kg). Tern and mer- ganser eggs contained an average of 0. 66 mg/kg, a level which Fimreite ' considers may affect hatchability. It is difficult to establish, from the published data referred to, the natural levels of mercury to be expected in birds, but there are numerous references to values below 0. 1 mg/kg in liver, kidney and muscle tissue. Thus, a range of 0. 01 - 0.10 mg/kg, i.e. 10-100 ng/g, may be normal for birds in uncontaminated areas, a range similar to that for fish. How- ever, the ability of avian species to acquire mercury through food chains which may be contaminated at various stages by use of the element in agri- culture or industry could result in the enhanced mercury levels reported in many areas up to 1 mg/kg. Direct ingestion of dressed seed can give rise to concentrations one, or even two, orders greater than this, as demonstrated by values such as 125 mg/kg in the kidneys of a Dutch kestrel, and 270 mg/kg in the liver of a Swedish owl. The lethal level of mercury in the livers of Swedish pheasants was considered to be about 30 mg/kg and in the Netherlands, values above 60 mg/kg in the livers or kidneys of pre- datory birds were regarded as lethal. Hatchability of pheasant eggs in Canada was affected by more than 0. 5 mg/kg of mercury [ 66] and the birds laying such eggs had liver levels of 3 mg/kg or more. The distribution of residues among the organs and tissues of wild birds appears to be broadly similar to that in fish, with the highest concentration in liver and kidney, somewhat less in muscle and still less in eggs, the overall range being within one order of magnitude. The concentrations in birds which may result in death appear to be similar to those reported for fish, namely in the region of 20 mg/kg in muscle tissue.

12. 9. MERCURY IN MAMMALS INCLUDING MAN

Apart from residues in man himself, the published information on mercury residues in terrestrial mammals relates mainly to species in Sweden and Canada. Borg et al. [59] examined dead or dying specimens of seven mammalian species from various parts, of Sweden collected during 1956 - 63, and found that herbivorous animals such as roe deer (Capreolus capreolus) and hares (Lepus timidus, L^ europaeus) contained amounts of mercury consistently below 1 mg/kg in mixed liver and kidneys, and frequently below 0.4 mg/kg. Carnivorous animals, however, contained

161 much higher levels. In one marten (Martes martes), one polecat (Mustela putorius) and one fox (Vulpes vulpes) the concentrations all exceeded 30 mg/kg, but one weasel (Mustela nivalis) contained less than 0.2 mg/kg, all in liver or mixed liver and kidney tissue. Other martens and polecats had 0. 5 - 11.0 mg/kg. Some evidence of symptoms suggestive of mercury poisoning was found. Samples taken in 1964 mostly contained up to 5 mg/kg in liver, but two foxes and one small rodent contained over 40 mg/kg. More recently Borg, in his annual report for 1968- 69 to the National Swedish Veterinary Institute, stated that in 63 of 64 wild mink examined, mercury concentrations in liver exceeded 2 mg/kg and in 21 of these the amounts exceeded 10 mg/kg. Five others contained over 5 mg/kg in liver. The residues were largely obtained from fish, but some of the mink were feeding on waterfowl. In Canada, Jervis et al. [46] examined the liver, hair and nails of muskrats and found 0. 040- 0. 251 mg/kg in the liver, 0. 363 - 0. 874 mg/kg in hair and 1.970 mg/kg in nails. Fimreite et al. [66], in an investigation of residues in seed-eating birds and mammals, examined two species of rodents. Six specimens from Alberta contained an average of 1.248 mg/kg in liver, while five specimens from Saskatchewan averaged only 0.180 mg/kg, reflecting the difference in usage of organomercury seed dressings. Small rodents have been examined in Sweden [ 76] and 130 out of 184 specimens contained 0.5 mg/kg or less, while in 12 the values exceeded 2 mg/kg. Suzuki [78] found that radioactive methylmercury injected into mice resulted in the highest concentrations being found in the kidney, followed by liver, brain and blood. The threshold level for neurological effects was 20 mg/kg (wet tissue) in the brain, and death occurred when a level of 30 mg/kg was exceeded. In man, death occurred at brain concentrations over 50 mg/kg (presumably also on a wet-weight basis). Apart from the data on terrestrial mammals, some information has been published on marine mammals. In ringed seals in Finland [55], the muscle tissue of adult specimens from an inland lake contained mercury concentrations (by a photometric method) averaging 62.4 mg/kg, and in liver 137.8 mg/kg, although juveniles had mean values of only 1.6 mg/kg in muscle and 6.8 mg/kg in liver. For comparison, the means in muscle and liver of adults sampled in the were 0. 9 and 11.8 mg/kg respectively. The high values found in the inland specimens apparently resulted from pollution by pulp mills, the highest concentration recorded in muscle being 197 mg/kg. These figures suggest that over a period of several years the seals may have concentrated residues from fish by two orders of magnitude. Berglund and Berlin [ 79] summarized the data on mercury distribution in mammals and noted that, in the rat, brain levels were unusually low by comparison with other mammals. Neurological symptoms, however, were noted, for all species examined, to occur when the mercury concentration was within the range 10-60 mg/kg in brain. The presence of mercury in man has generally been investigated in circumstances where the mercury has been assimilated as the result of contamination while working in an industrial process, from direct poisoning or from the use of mercurial preparations for medical purposes. Such instances are the subject of a separate chapter, but this section is concerned with levels of mercury in man acquired from his environment, principally from residues in food.

162 The manner in which mercury compounds are distributed "among various tissues, metabolized and excreted depends greatly on the particular compound involved. The concentration in urine has commonly been used as an indication of mercury poisoning, but is now considered as of limited value, except perhaps for ingestion of inorganic mercury compounds. Goldwater [ 67] reported that it is not unusual to find 20- 30 ng/ml of mercury in urine, and up to 50 ng/ml in blood. He also found [68] that, of 810 people examined in 15 countries, 80% had less than 0.5 ng/ml in urine, and 4% had more than 20 ng/ml, the maximum acceptable level in many countries being 300 ng/ml. In the blood of 575 people examined from 16 countries, 76% had less than 3 ng/ml and 4% had more than 50 ng/ml. According to Lofroth [29], the maximum allowable concentration of mercury proposed for human blood is 100 ng/ml of whole blood, equivalent to about 185 ng/g in corpuscles. Mercury in blood and hair of humans in Sweden has been studied by Birke et al. [69] and Tejning [40], and some of the data are quoted by Lofroth [ 29] . Birke et al. found a linear relationship between the intake of methylmercury and the concentration in blood, and concluded that a daily consumption of fish containing 5-6 mg/kg of mercury (5 000 - 6 000 ng/g) could be lethal. Tejning found that the mean levels of mercury in the blood of individuals not subjected to excessive intake of mercury from food were 10. 06 ± 2.63 ng/g in corpuscles and 2. 27 ± 1. 08 ng/g in plasma, females having slightly more than males. Two groups of individuals consuming an average of three fish meals per week had mean values of 44 and 77 ng/g in blood corpuscles, 5.7 and 9.8 ng/g in plasma, and 6 227 and 10131 ng/g in hair. High levels in hair were also found by Sumari et al. [70], who reported 3 000 - 56 000 ng/g in the hair of individuals consuming fish, 300 - 4 300 ng/g in three people who did not. The corresponding levels in blood corpuscles were 47 - 522 ng/g and 6-117 ng/g, in plasma 7-69 ng/g and 6-15 ng/g, and in urine 3 - 228 ng/g and 14 - 65 ng/g. None of these individuals had any symptoms of poisoning. Suzuki [ 78] found that, in two positive cases of ethylmercury poisoning, the mercury concentrations in the blood were 1 500 and 1 720 ng/g. Patients dying of methylmercury poisoning had calculated concentrations in the brain of over 50 000 ng/g. The concentration in blood is, on the basis of experiments with mice, slightly less than in the brain, but these figures suggest that the symptoms of mercury poisoning may occur when the concen- tration in the blood is of the order of one twentieth of the lethal level, say 1 000- 2 000 ng/g. Some of the fish-eaters examined by Sumari, as quoted above, were found to have mercury levels in whole blood not far below this range. Saito [ 71] reported that mercury levels in the hair of twelve persons from twelve countries excluding Japan ranged from 890 to 4 190 ng/g. Twelve Japanese residents had concentrations in the range 4 100-146 000 ng/g. Values obtained from the hair of 22 Japanese associated with the Minamata disease incident were in the range 15 600 - 763 000 ng/g, and one patient, with 430 000 ng/g in hair, had 1 320 ng/g in blood and 46 ng/g in urine. (This last value would not itself be regarded as unacceptable. ) Concentrations of mercury occurring normally in other human tissues have rarely been determined, and it is therefore difficult to compare data from other vertebrates with those from man, although some data are given by Berglund and Berlin [ 79]. Kurland et al. [18] reported that in patients

163 dying of organomercury poisoning the concentrations of mercury found in hair were 14 000 - 39 000 ng/g, in kidney 3 000 - 48 000 ng/g and in brain 5 000- 12 000 ng/g. Radioisotope studies [ 75], using methylmercury on human volunteers, indicated that the mercury was excreted mainly in faeces, but with a steady increase in urinary excretion over a 30-day period. About 50% of the total body activity was located in the liver, and 10% in the head, possibly in the brain, with part in the hair. The biological half-life from whole-body measurements was found to be 70- 74 days. The pattern of distribution found was considered to be similar to that in post-mortem examinations after methylmercury poisoning. The accumulation of excessive mercury by man has also been attributed to the consumption of food other than fish. Two individuals in the United States who consumed pork from an animal fed on grain dressed with a methylmercury fungicide had 210 and 160 ng/ml of mercury in the urine [ 72]. In Finland, a woman believed to be suffering from chronic mercury poisoning, and who had slightly elevated mercury levels in urine and hair, had been eating eggs of the merganser, Mergus merganser. Forty eggs examined contained 0.5-4.0 mg/kg of mercury (Ref. [ 27] and Wahlberg, personal communication). In 1970 a report from the United States Public Health Service (quoted in Ref. [ 74]), indicated that the inhabitants of the Pribilof Islands, in the Bering Sea, had mercury levels in hair which were unusually high. One group of thirteen persons had concentrations of 5 000 - 6 000 ng, apparently derived from the consumption of muscle and liver from fur seals containing very high mercury levels. These examples indicate that it is possible for man to acquire unusually high concentrations of mercury by consuming various forms of wildlife, which themselves give no indication, by any abnormality, of an enhanced mercury content. Fresh-water and marine fish, birds, the eggs of birds and seals have so far been implicated.

12.10. CONCLUSIONS

Unlike many toxic substances produced synthetically by man, mercury occurs naturally in organic combination in living organisms. With the addition of mercury in wastes discharged to the environment from mining operations, by industrial uses, through the disposal of medical preparations, or by its incorporation in agricultural chemicals, populations of many species of wildlife now have elevated levels of mercury. In some areas, the original natural levels of mercury in the flora and fauna may now be difficult to determine. In living tissues mercury appears to exist almost entirely as methyl- mercury, probably bound to proteins. Bacterial or enzymatic processes can convert other inorganic or organic forms to methylmercury. The natural mercury levels in both fresh-water and marine fish are generally less than 0.2 mg/kg in muscle tissue, and the natural level in birds is probably of the same order. Most analyses of birds have, however, been made on liver and kidney, rather than on muscle tissue as used for field investigations on fish. Relatively fewer uncontaminated samples'of birds have been examined. The distribution of mercury between tissues, in fish, birds and mammals, is such that kidneys and liver contain the highest concentrations, those in

164 muscle, brain and blood being significantly lower, although this is only a generalization. Many populations of fish and birds examined in Scandinavia and North America, in areas where mercury is used by industry or in agriculture, have been found to contain concentrations at least one order higher than the upper limit of the estimated natural range. Individual birds found dead, or dying in experimental work on mercury poisoning, have contained 30 mg/kg or more in liver. Concentrations of a similar order have been found in humans dying of organomercury poisoning, and there is some evidence that the same level has been present in small mammals dying in the field. Marine fish found dead off Japan contained about 20 mg/kg in muscle tissue. The concentrations of mercury present in some populations of birds and fish have been found to be abnormally high as the result of pollution but, with the exception of some species of birds inSweden, there is little evidence that these elevated concentrations have as yet had any direct toxic effect whereby the species have been placed at risk. However, the levels some- times found in fish and birds may render them unacceptable as human food. The maximum permissible levels of mercury established in some countries are 0.5 or 1.0 mg/kg, values only 2.5 or 5 times the approximate upper limit estimated for many natural populations. Thus, such populations can tolerate only small increases in mercury concentrations if they are to remain acceptable for human consumption.

REFERENCES TO CHAPTER 12

[1] ERIKSSON, E., Mercury in nature, Oikos, Suppl. 9 (1967) 13. [2] BYRNE, A.R., KOSTA, L., Studies on the distribution and uptake of mercury in the area of the mercury mine at Idrija, Slovenia. I. Plants, vegetables and other environmental samples, Vest. slov. kem. DruSt. (in press). [3] STOCK, A., CUCUEL, F., Die Verbreitung des Quecksilbers, Naturwiss. 22 (1934) 390. [4] LARSSON, J. E., Environmental mercury research in Sweden, Report of Swedish Environment Protection Board (June 1970) 44 pp.

[5] JOHNELS, A.G., WESTERMARK, T., BERG, W., PERSSON, P. I., SJOSTRAND, B.t Pike (Esox lucius L.) and some other aquatic organisms in Sweden as indicators of mercury contamination in the environment, Oikos 18 (1967) 323. [6] KOSTA, L., BYRNE, A.R., Activation analysis for mercury in biological samples at nanogram level, Talanta 16 (1969) 1297. [7] ANON., Investigations of mercury in the St. Clair River — Lake Erie systems, Report of Federal Water Quality Administration, US Dept. of Interior (May 1970) 108 pp. [8] SILLgiN, L.G., Svenskkem. Tidskr. 75 (1963) 161. [9] UI, J., Mercury pollution of sea and fresh water. Its accumulation into water mass, Paper given to 4th Collegium for Medical Oceanography, Naples, Italy, Oct. 2 -5, 1969. [10] SMART, N. A., Residues of mercury compounds, Res. Rev. 23 (1968) 1. [11] ANDERSSON, A., Mercury in Swedish soils, Oikos, Suppl. 9 (1967) 13. [12] WARREN, H. V., DELAVAULT, R. E., Mercury content of some British soils, Oikos 20 (1969) 537. [13] ACKEFORS, H., LOFROTH, G., ROSEN, G.G., A survey of the mercury pollution problem in Sweden with special reference to fish, Oceanog. Mar. Biol. Ann. Rev. 8 (1970) 203. [14] JENSEN, S., JERNELOV, A., Biological methylation of mercury in aquatic organisms, Nature, Lond. 223 (1969) 753. [15] SAITO, N., Levels of mercury in environmental materials, Paper presented at IAEA Expert Meeting on Mercury Contamination in Man and His Environment, Amsterdam, 1967. [16] HOSOHARA, K., Mercury content of deep sea water, Nippon Kagaku Zasshi 82 (1961) 1107. [17] HOSOHARA, K., KURODA, R., HAMAGUCHI, H., Photometric determination of mercury in sea water, Nippon Kagaku Zasshi 82 (1961) 347.

165 [18] KURLAND, L.T., FOAR, S.N., SEIDLER, H., Minamata disease, Wld Neurol. 1 (1960) 370. [19] AXELSSON, B., FRIBERG, L., On the tolerable limits of mercury in the atmosphere and biological milieus, Occup. Hlth Rev. 15 (1963) 18. [20] HASSELROT, T.B., Report on current field investigations concerning the mercury content in fish, bottom sediment and water, Rep. Inst. Freshwat. Res. Drottningholm 4£(1968) 102. [21] HANNERZ, L., Experimental investigations on the accumulation of mercury in water organisms, Rep. Inst. Freshwat. Res. Drottningholm 48_(1968) 120. [22] RAMEL, C., MAGNUSSON, J., Hereditas (in press, 1969). [23] RAEDER, M. G., SNEKVIK, E., Mercury contents of fish and other aquatic organisms, K. Vidensk. Selsk. Forh. 21 (1948) 105. [24] WESTOO, G., Kvicksilver i fisk, Var ffida (1) (1967) 1 (Swedish). (Translation No; 1350 in English by Fish. Res. Bd Canada.) [25] JOHNELS, A. G., OLSSON, M., WESTERMARK, T., Kvicksilver i fisk, Var fSda (7) (1967) 67 (Swedish). [26] NOREN, K., WESTOO, G,, Metylkvicksilver i fisk, Var fSda (2) (1967) 13 (Swedish). (Translation No. 1351 in English by Fish. Res. Bd Canada.) [27] WAHLBERG, P., HENRIKSSON, K., KARPPANEN, E., Flyttfaglar som giftvektorer, Nord. Med. 84 (1970) 889. [28] YOSHIDA, T., KAWABATA, T., MATSUE, Y., Transference mechanism of mercury in marine environment, J. Tokyo Univ. Fish. 53 (1967) 73. [29] LOFROTH, G., Methylmercury. A review of health hazards and side effects associated with the emission of mercury compounds into natural systems, Ecological Research Committee Bulletin No. 4 (1969) as published by the Swedish Natural Science Research Council. [30] SJOBLOM, V., HASANEN, E., Kvicksilver i fisk i Finland, Paper presented at Nordic Symposium on Mercury, Nordforsk, 10-11 Oct. 1968; Nord. hyg. Tidskr. 50 (2) (1969) 37. [31] WOBESER, G., NIELSEN, N. O., DUNLOP, R.H., Mercury concentrations in tissues of fish from the Saskatchewan River, J. Fish. Res. Bd Can. 27 (1970) 830. [32] BLIGH, E.G., Mercury and the contamination of freshwater fish, Manuscript Rep. No. 1088, Fish. Res. Bd Canada (1970). [33] BLIGH, E.G., Rep. Directorate Fish. Res. for 1968, Dept. Agric. and Fish, for Scotland, p. 147. [34] UNDERDAL, B., Mercury determination in some foods, Paper presented at Nordic Symposium on Mercury, Nordforsk, 10-11 Oct. 1968; Nord. hyg. Tidskr. 50 (2) (1969) 60. [35] BORG, K., WANNTORP, H., ERNE, K., HANKO, E., Mercury poisoning in Swedish wildlife, J. appl. Ecol. 3 (Suppl.) (1966) 171. [36] FIMREITE, N., Mercury uses in Canada and their possible hazards as sources of mercury contamination, Envir. Pollut. 1. (2) (1970). [37] EADES, J.F., Pesticide residues in the Irish environment, Nature, Lond. 210 (1966) 650. [38] RUCKER, R. R., Effects of mercurial compounds on fish and humans, Bull. Off. int. Epiz. 69 (1968) 1431. [39] RUCKER, R. R., AMEND, D.F., Absorption and retention of organic mercurials by rainbow trout and Chinook and sockeye salmon, Prog. Fish Cul. (1969) 197. [40] TEJNING, S., On the food hygiene limit for mercury in fish and fish products, Paper presented at the National Institute of Public Health, Stockholm, 2 Feb., 1968. [41] WESTOO, G., Determination of methylmercury compounds in foodstuffs, Acta chem. scand. 21 (1967) 1790. [42] WOOD, J.M., KENNEDY, F. S., ROSEN, C. G., Synthesis of methylmercury compounds by extracts of a methanogenic bacterium, Nature, Lond. 220(1968) 174. [43] WESTOO, G., Determination of methylmercury salts in various kinds of biological material, Acta, chem. scand. 22 (1968)2277. [44] SPRAGUE, J. B., CARSON, W.G., Spot-checks of mercury residues in some fishes from the Canadian Atlantic coast, Manuscript Rep. No. 1085, Fish. Res. Bd Canada (1970) 16 pp. [45] BACKSTROM, J., Distribution studies of mercuric pesticides in quail and some fresh-water fishes, Acta pharmac. tox. 27 Suppl. 3 (1969) 1. [46] JERVIS, R.E., DEBRUN, D., LEPAGE, W., TIEFENBACH, B., Mercury residues in Canadian foods, fish, wildlife. Summary of Progress, National Health Grant Project (1970) No. 605-7-510, University of Toronto, Canada. [47] FIMREITE, N., Mercury contamination of Canadian fish and fish-eating birds (1970). MS Rep., University of Western Ontario, Canada. [48] KITAMURA, S. et al., Kumamoto Igakkai Zasshi 34 (1960) 593.

166 [49] TONING, S., Mercury content of blood corpuscles, blood plasma and hair in heavy fish eaters from different areas of Lake VSnern, Translation Series No. 1362, Fish. Res. Bd Canada (1970) from Rep. 670831 of Occupational Medicine Clinic, University Hospital, Lund, 1967. [50] UI, J., A short history of Minamata disease research and the present situation of mercury pollution in Japan, Paper presented at Nordic Symposium on Mercury, Nordforsk, 10-11 Oct. 1968; Nord. hyg. Tidskr. 50 (2) (1969) 139. [51] RAEDER, M. G., SNEKVIK, E., Quecksilbergehalt mariner Organismen, K. Vidensk. Selsk. Forh. _13 (1940) 169. [52] SUMINO, K., Analysis of organic mercury compounds by gas chromatography. II. Determination of organo-mercury compounds in various samples, Kobe J. med. Sci. 14 (1968) 131. [53] CHRISTELL, R., ERWALL, L. G., LJUNGGREN, K., SJOSTRAND, B., WESTERMARK, T., Methods of activation analysis for mercury in the biosphere and foods (1965). Proc. 1965 International Conference on Modem Trends in Activation Analyses. [54] DALGAARD-MIKKELSEN, S., Mercury occurrence in the Danish environment, Paper presented at Nordic Symposium on Mercury, Nordforsk, 10-11 Oct. 1968; Nord. hyg. Tidskr. 50 (2) (1969) 34. [55] HENRIKSSON, K., KARPPANEN, K.E., HELMINEN, M.. Kvicksilverhalter hos insjo- och havssalar, Nord. hyg. Tidskr. 50 (2) (1969) 54. [56] WESTOO, G., RYDAL, M., Kvicksilver och metylkvicksilver i fisk och krSfter, Var fBda. 3 (3)(1969) 19. [57] BERG, W., JOHNELS, A., SJOSTRAND, B., WESTERMARK, T., Mercury content in feathers of Swedish biKis from the past 100 years, Oikos r7 (1966) 71. [58] EDELSTAM, C., JOHNELS, A., OLSSON, M., WESTERMARK, T., Ecological aspects of the mercury problem, Nord. hyg. Tidskr. 50 (2) (1969) 14. [59] BORG, K., WANNTORP, H., ERNE, K., HANKO, E., Alkyl mercury poisoning in terrestrial Swedish wildlife, Viltrevy 6 (4) (1969) 301. [60] WANNTORP, H., BORG, K., HANKO, E., ERNE, K., Mercury residues in wood-pigeons (Columba p. palumbus L.) in 1964 and 1966, Nord. Vet. Med. 19 (1967)474. [61] HOLT, G., Mercury residues in wild birds in Norway 1965-1967, Nord. Vet Med. 21 (1969) 105. [62] SMART, N.A., LLOYD, M. K., Mercury residues in eggs, flesh and livers of hens fed on wheat treated with methylmercury dicyandiamide, J. Sci. Fd Agric. 14 (1963) 734. [63] ANON., Report of the Government Chemist, 1967, 108, H.M. S. O., London (1968). [64] KOEMAN, J. H., VINK, J.A.J., DE GOELJ, J.J.M., Causes of mortality in birds of prey and owls in The Netherlands in the winter of 1968-1969, Ardea 57 (1969) 67. [65] ANON., Mercury residues, American Fisheries Society, July - August Newsletter 14 No. 67 (1970) 6. [66] FIMREITE, N., FYFE, R., KEITH, J. A., Mercury contamination of Canadian prairie seed-eaters and their avian predators, (Manuscript) Canadian Wildlife Service. [67] GOLDWATER, L.J., The toxicology of inorganic mercury, Occup. Hlth Rev. lj> (1963) 14. [68] GOLDWATER, L.J., Mercury in our environment, J. Fd. Cosmet. Toxicol. 3 (1965) 120. [69] BIRKE, G., JOHNELS, A. G., PLANTIN, L.O., SJOSTRAND, B., WESTERMARK, T., Metylkvicksilver- ffflrgiftning genom fSrtSring av fisk! Svenska LSkartidn. 64 (1967) 3628. [70] SUMAR1, P., BACKMAN, A.-L., KARLI, P., LAHTI, A., HSlsoundersflkningar bland fiskkonsumenter i Finland, Nord. hyg. Tidskr. 50 (2) (1969) 97. [71] SAITO, N., Levels of mercury in man, Paper presented at IAEA Meeting on Mercury Contamination in Man and His Environment, Amsterdam, 13 May 1967. [72] ANON., Organomercury poisoning. Morbidity and Mortality 19 (3) (1970) 25. Report of the National Communicable Disease Center, US Dept. of Health, Education and Welfare. [73] BOETIUS, J., Lethal actions of mercuric chloride and phenylmercuric acetate on fishes, J. Medd. Danmarks Fiskeri- og Havunders8gelser 3 (4) (1960) 93. [74] PRESS REPORT, Scotsman, Edinburgh, 7 Nov. 1970. [75] AiERG, B., EKMAN, L., FALK, R., GREITZ, U., PERSSON, G., SNIHS, J.-O., Metabolism of methylmercury(203Hg) compounds in man, Archs envir. Hlth 19 (1969) 478. [76] LIHNELL, D., STENMARK, A., Bidtag till kSnnedomen om fSrekomsten av kvicksilver i smagnagare, Statens VSxstskyddsanstalt Meddelanden 13 (1967) 110. [77] PAPPAS, E.G., ROSENBERG, L. A., Determination of sub-microgram quantities of mercury in fish and eggs by cold vapour atomic absorption photometry, J. Ass. off. analyt. Chem. 49 (1966) 792. [78] SUZUKI, T., "Neurological symptoms from concentrations of mercury in the brain", Chemical Fallout (MILLER, M. W., BERG, G.G., Eds), C.C. Thomas, USA (1969) 245. [79] BERGLUND, F., BERLIN, M., "Risk of methylmercury cumulation in man and mammals, and the relation between body burden of methylmercury and toxic effects", Chemical Fallout (MILLER, M. W., BERG, G.G., Eds), C.C. Thomas, USA (1969) 258.

167 [80] HENRIKSSON, K., KARPPANEN, E., HELMINEN, M., High residue of mercury in Finnish white-tailed eagles, Ornis fenn. 43 (1966)38. [81] KARPPANEN, E., HENRIKSSON, K., HELMINEN, M., Mercury content of game birds in Finland, Nord. Med. 84 (1970) 1097. [82] HAGA, Y., HAGA, H., HAGINO, T., KARIYA, T., Studies on the post-mortem identification of the pollutant in fish killed by water pollution - XII, Bull. Jap. Soc. scient. Fish. 36 (1970) 225. [83] TSURUGA, H., Tissue distribution of mercury orally given to fish, Bull. Jap. Soc. scient. Fish. 29 (1963) 403. [84] AMEND, D.F., YASUTAKE, W.T., MORGAN, R., Some factors influencing susceptibility of rainbow trout to the acute toxicity of an ethylmercury phosphate formulation (Timsan), Trans. Am. Fish. Soc. 98 (1969) 419.

168 13. PRESENT LEVELS OF MERCURY IN MAN UNDER CONDITIONS OF OCCUPATIONAL EXPOSURE

M. BERLIN

13.1. INTRODUCTION

When evaluating the literature on concentrations of mercury in persons occupationally exposed to mercury compounds, it is necessary to keep in mind two facts: (1) The analysis of mercury in biological material requires advanced skills in chemical analysis. Many methods previously used have considerable weaknesses which often cause large errors. (2) Depending on the chemical form in which mercury appears in the body or is absorbed, the distribution and retention in different tissues and organs of the body varies. For example, there are considerable differences in the toxicity distribution and retention of mercury with exposure to elementary mercury vapour as compared with exposure to inorganic mercury salts (Berlin et al., 1969). Phenylmercury acetate (PMA), which is often used in industrial processes and as a fungicide, has a different toxicology and metabolism than the inorganic mercuric salts. A special problem involves the use of alkylmercury compounds, like methylmercury salts and ethylmercury salts (see Chapter 6 on toxicology). In this chapter an attempt is made to summarize the available data in the literature concerning the concentrations of mercury found in human organs following different types of exposure. There are few data available, even concerning those types of exposure which are rather common. This means that the conclusions that can be drawn from the available data will be rather generalized, although to acertain extent some quantitatively precise recommendations can be made.

13. 2. ANALYSIS OF MERCURY

In the author's experience, it is essential that the analysis adopted should give a reasonably accurate determination of a known amount of mercury in vivo accumulated in the tissue. Mercury is toxic in the organism at concentrations from 1 to 10 ppm. The natural concentration of mercury found in animal tissues is less than 0. 01 ppm. Therefore, the method for mercury analysis must be sensitive enough to permit the analysis of a few nanograms per gram. According to the author's experience, the only method that is generally acceptable for mercury analysis in all tissues is activation analysis. When correctly adapted, this method permits analysis in all biological tissues. WestOS (1968) has described a method for the analysis of methylmercury which, if modified for different kinds of tissues, has been shown to give reproducible results with good accuracy at the concentration levels required. Other published methods have so far not been shown to be generally usable with every type of organ or tissue, although good results can be obtained for certain kinds of tissues. It must be emphasized that data from mercury

169 analyses by means of the activation analysis method are very rare in the literature, especially in connection with a defined type of exposure. It is, therefore, necessary to try to interpret data in the literature that are often based on the results of other methods. The most extensive data which also permit a certain number of quantitative conclusions have been collected in connection with exposure to methylmercury, although a lesser part of this data refers to occupational exposure to methylmercury.

13. 3. EXPOSURE TO MERCURY VAPOUR

Although exposure to mercury vapour has occurred since ancient times and its toxic effects have been known since then, it has not been understood until recently that the clinical signs of exposure to mercury vapour differ from those of exposure to inorganic mercury salts. After exposure to mercury vapour, mercury penetrates into the brain more easily than after exposure to mercury salts and is retained there for a long time (Berlin et al., 1969). Occupational exposure to mercury is often a mixed exposure to mercury vapour and to aerosols or dust containing mercuric salts. It can be anticipated that the concentration of mercury in organs like the kidney and brain, as well as the concentration in the blood and urine, will vary with the type of exposure, especially with respect to the form in which the mercury occurs. All facts indicate that the most sensitive organ to mercury-vapour exposure is the brain. Data on mercury concentration in the brain after exposure to mercury vapour are scarce. Brigatti (1949) reported concentrations of 6 - 9 ppm in the brain of two cases exposed for several years to mercury vapour and who died of other etiology. Both cases had shown pronounced signs of mercuri- alism some years before. He also refers to reports in the literature con- cerning the occurrence of mercury in the brains of men with no known exposure to mercury. Concentrations exceeding 0. 5 ppm in the brain have never been reported in man without known mercury exposure. Grant etal. (to be published) reported a case of spongy degeneration of brain cortex. A reinvestigation of the case history revealed considerable mercury exposure 13 years before death. The mercury concentration in this brain was about 19 ppm as analysed in xylol-extracted, paraffin-embedded tissue, which means that the concentration in wet tissue was probably lower. Watanabe (1970) reported on two workers in mercury mines who showed a high content of mercury in the brain 10 years after exposure ceased. In one case, concentrations in different parts of the brain ranged from 3 to 34 ppm of Hg. The highest values were found in occipital cortex (34 ppm) and substantia nigra (23 ppm). This patient showed signs of mercurialism until his death which was due to encephalomalacia and silico-tuberculosis. In the other case, mercury concentrations from 5 to 18 ppm were found in the brain, the highest (18 ppm) in substantia nigra. According to the author, however, this worker never showed any signs of classical mercurialism. Smith and co-workers (1969) reported on the mercury concentration in the blood of workers exposed to mercury vapour in the chlor-alkali industry. They found 0. 01 - 0.1 ppm of mercury in the blood of 567 workers and less than 0. 05 ppm in the blood of 382 controls. In this population, a dose- response relationship could be demonstrated with an increasing occurrence of symptoms such as loss of weight, nervousness, tremor, , etc.

170 Concentrations of mercury in the urine varied between 0.1-1 ppm, while 96% of the controls had less than 0. 1 ppm of mercury in the urine. There were variations in exposure conditions and in the concentration of mercury in the air, which ranged from 0. 01 to 0.2 7 mg/m3. Smith and his co-workers found a good correlation between blood and urine values and the exposure data in their material, although there were large variations in the urine excretion between and within individuals over days. Interpreting their regression lines, they found that an air concentration of 0. 1 mg/m3 corres- ponds to a blood level of 0. 06 ppm and a urine level of 0. 2 ppm of mercury, corrected to 1. 018 specific gravity. The mercury exposure in the chlor-alkali industry.is probably a mixed exposure to vapour and to sublimated dust due to the presence of chlorine in the air. However, the authors estimated that the concentration of sub- limate did not exceed 10% of the total mercury concentration. Goldwater (1964) reported on 60 workers exposed to concentrations of mercury vapour in air between 0. 02 - 0. 2 mg/m who showed values in blood between 0-0.08 ppm Hg and in urine 0-0.1 ppm Hg. Similar values from exposure to mercury vapour have also been reported from Japan by Suzuki et al. (1970).

13. 4. EXPOSURE TO INORGANIC MERCURY COMPOUNDS

Pure exposure to inorganic mercuric salts is probably not very common in industry today. Considerable evidence has been collected from cases of sublimate poisoning which accumulated at the end of the last century and at the beginning of this century. This evidence and the results of experimental

TABLE XXIX. CONCENTRATION OF MERCURY IN THE KIDNEY OF CASES WITHOUT KNOWN OCCUPATIONAL EXPOSURE TO MERCURY

No. of cases ppm Hg in kidney Method of analysis Reference

3 0.17-0.58 M Bodn&r et al. (1939) 11 0.03-5.1 M Stock (1940) 69 0.75 D Butt and Simonsen (1950) 15 0-2 D Takeuchi et al. (1962) and (1968)

10 0.6 1 Fujimura (1964)

4 0.1-0.3 D Dal Cortivo et al. (1964)

11 0.05-0.5 D Matsumoto et al. (1965) 17 0.7-3 AAS Matsumoto et al. (1965) 6 0.8 i 0.7 D Mori et al. (1965) 39 2.8 (0 -26) D Joselow et al. (1967) 5 0.79, 0.5-2.1 NAA Schicha et al. (1969)

M = Micrometric method D = Dithizone method NAA = Neutron activation analysis AAS = Atomic absorption spectrophotometry

171 TABLE XXX. CONCENTRATION OF MERCURY IN THE KIDNEY OF CASES POISONED WITH MERCURIC SALTS

Interval between „ , , , , Type of exposure, ppm Hg in Compound absorbed , last exposure ... Reference r dose j it kidney and death

HgCl2 Acute poisoning 3 days 70 Lomholt (1928)

HgClz Acute poisoning 6 days 16

HgCl2 Acute poisoning, 2.5 g 6 days 35 Sollman and Schreiber (1936) HgC-lj Acute poisoning, 5.5 g 9 days 33

HgCl2 Acute poisoning, 1.0 g 8 days 24 Mercuric and salts Occupational exposure 25 Kazantzis et al. (1962)

Mercuric oxides and salts Occupational exposure Biopsy 10 Kazantzis et al. (1962) investigations indicate that the kidney is the critical organ in exposure to inorganic mercuric salts. The cause of death with acute poisoning is kidney damage; with chronic intoxication, a nephrotic syndrome appears due to tubular injury. Mercury in the kidney can be shown to occur in people who have not been specially exposed to mercury compounds in large amounts. Table XXIX summarizes the published data on mercury concentration in kidney in persons with no known exposure. The mean values in the material range from 0. 1 to more than 3 ppm. This large variation may reflect low accuracy or errors in mercury analysis of kidney tissue. In cases of known poisoning due to mercuric salts, mercury concentrations in kidney between 10 and 70 ppm have been reported (see Table XXX). Because of problems in the analysis of mercury in kidney tissue it is difficult to compare data from different authors since the possibility that mercury is lost during analysis with some methods cannot be ignored. It may be pointed out that those cases published by Kazantzis et al. (1962) refer to biopsy samples from the kidney. It is known from experimental investigations that mercury concentration in the kidney varies very much and that the total concentration in the kidney reflects, to a limited degree, the concentration in the proximal tubules where the damage first appears (Berlin, 1963 and Berlin et al. , 1963). Mercury shows a very complex distri- bution in the kidney with varying distribution patterns at different times after a single injection in animal experiments. We have found no reports that permit conclusions regarding the concentration of mercury in blood or in urine after chronic exposure to mercury salts in the form of dust. ' There is, however, little reason to assume that the concentration would be less than that after exposure to mercury vapour, with respect to the total dose absorbed. Goldwater reports on 90 cases of occupational exposure to a mixture of mercury vapour and dust of mercuric salts. The concentration in the air ranged from 0. 1 - 2 mg Hg/m3; the mercury in blood, from 0 - 0.93 ppm, and in urine, from 0-1.3 ppm.

13. 5. EXPOSURE TO ORGANIC MERCURY COMPOUNDS

Among those organic mercury compounds which give rise to occupational exposure are the arylmercury compounds like phenylmercuric acetate, and the alkylmercury compounds like methyl, ethyl and methoxiethyl mercury. This latter compound, methoxiethyl mercury, has replaced methylmercury compounds in seed treatment since it was discovered that ethyl- and methyl- mercury compounds accumulate in the fauna. Both phenylmercuric acetate and methoxyethyl mercury compounds seem to be metabolized rapidly to inorganic mercury in the mammalian body and excreted in that form. Few reports are to be found in the literature on poisoning from exposure to methoxyethyl mercury compounds and on the mercury concentrations in blood and urine that occur with this kind of exposure. Those reports available, Wilkening and Litzner (1952), D§robert and Marcus (1956), confirm that the kidney is the critical organ. A nephrotic syndrome and intestinal signs characterize the clinical appearance of chronic poisoning. Conclusive quantitative data are so far lacking. Goldwater reports on seven workers exposed for 2 months to methoxyethylmercuric salts in the form of dust. They had blood concentra- tions of mercury between 0. 34 - 1. 1 ppm and urine concentrations between

173 0. 16-1.2 ppm but no signs of poisoning. However, these workers had been exposed to mercuric compounds for a long time before this exposure. Very few cases of poisoning with phenylmercuric compounds have been reported. Irritant and burning effects on the skin were mentioned by Lecompte (1949) and an acute case of poisoning reported by Goldwater (1964) showed signs of mild kidney damage. Goldwater (1964) reported on 66 workers exposed to phenylmercuric salts occurring as dust in concentrations of 0 - 0. 5 mg/m3 air. These workers had mercury concentrations in blood up to 0. 55 ppm and in urine up to 0. 65 ppm. Another group of 65 workers exposed to a mixture of phenyl- mercuric salts, mercury vapour, and mercuric salts in concentrations up to 5. 1 mg/m3 air had urine values of mercury up to 3. 0 ppm and blood values up to 0. 55 ppm. None of these workers showed any signs of poisoning. In a study of 26 workers exposed over 6 years to phenylmercuric acetate in concentrations of 0,24- 3.2 mg/m3, Massman (1957) found concentrations of mercury in the urine up to 6 mg/litre, but found no case in which signs indicated chronic intoxication. These reports may indicate that exposure to phenylmercuric acetate is less dangerous than exposure to mercury vapour or to inorganic mercury in the form of dust. However, this still remains to be proved. Owing to the fact that methylmercury has grown to be an environmental toxicological problem of great seriousness, a considerable amount of work has been done to summarize and to collect data that relate the mercury concentration in biological material after exposure to methylmercury compounds to the toxic effects observed. Recently, a report was published by a Swedish Expert Group (1971)1 of which the author is a member, that relates mercury exposure and mercury concentrations in the blood, urine, hair and other organs to the effects of methylmercury poisoning. A total of 20 cases of alkylmercury poisoning due to occupational exposure to alkylmercury compounds has been described. The most extensive material from human beings exposed to methylmercury is to be found in Japan from the intoxication epidemics in Minamata and Niigata. Among adults, children, and foetuses (22 cases) exposed during pregnancy, 152 cases of methylmercury poisoning due to the consumption of fish containing methylmercury occurred. With methylmercury poisoning, the central nervous system is the critical organ. The lowest observed concentration in the brain with lethal methyl- mercury poisoning is around 5 mg/g of brain tissue. The foetal brain is probably more sensitive to methylmercury. The observed concentrations of mercury in the blood of populations exposed to methylmercury varies from normal values of about 0. 01 ppm of mercury to the lowest observed concen- tration,^ cases with clinical signs, about 0. 2 ppm, and to the values from lethal cases in the Japanese epidemics, which in the majority of cases probably exceeded 1 ppm of mercury in whole blood. For the most part, methylmercury in blood is to be found in the red cells. The partition coefficient between cells and plasma is 10/1. It is important, therefore, to analyse the cells and plasma separately. For practical purposes, the blood cells are the most suitable samples for mercury analysis. In the

1 Methyl mercury in fish. A toxicologic epidemiologic evaluation of risks. Report from an expert group, Nord. hyg. Tidskr., Suppl. 4(1971).

174 exposed population during the Japanese epidemics, a number of individuals who had a high concentration of mercury in the blood did not show clinical signs. Values up to 0. 7 ppm of mercury in whole blood have been reported in clinically-healthy individuals who consumed fish containing methylmercury. The available clinical, epidemiological, and experimental data indicate that there is a linear correlation between the concentration of mercury in blood and hair and the concentration in brain tissue, provided that the hair values relate to the time of formation of the part of the pile analysed. A blood concentration of 0. 2 ppm, the lowest observed level in connection with clinical signs, corresponds to about 50 ppm in hair and about 2 ppm in brain tissue. It requires a daily intake of 4 mg Hg/kg body weight per day, which corresponds to 8 hours exposure to about 0. 02 mg methylmercury/m3 of air. Less than 10% of the total excretion of methylmercury, which is about 1% of the body burden in man, is excreted in the urine. The mercury concentration in urine is, therefore, not a satisfactory index of either the degree of exposure or the burden of methylmercury. The 1 : 10 ratio between the plasma and red blood cells after exposure to methylmercury makes possible a certain degree of differential diagnosis, as compared with other types of mercury exposure. After exposure to organic-mercury compounds this ratio is around 1:1.

13. 6. SUMMARY AND CONCLUSION

The problems in connection with mercury analysis in biological material are not fully realized. According to available reports, it seems that a mercury concentration in blood exceeding 0. 02 ppm after exposure to mercury vapour is connected with a certain risk of unspecific subjective symptoms in human beings. This value corresponds to about 0. 02 mg/ m3 of air. At blood levels exceeding 0. 1 ppm (0. 1 mg/m3 of air), signs of classical mercurialism can be expected. Blood concentrations exceeding 0. 03 ppm of mercury can be regarded as signifying a definite exposure to mercury. The same conclusion should be drawn concerning concentrations of mercury in the urine exceeding 0. 1 ppm. There is too little quantitative data on which to base conclusions regarding the lowest concentrations in blood and urine from poisoning due to phenylmercury compounds or inorganic mercuric salts, nor is the lowest concentration in the critical organ, the kidney, known at which the clinical signs of poisoning appear. However, there are data indicating that the mercury concentration in urine and blood can be higher without causing clinical signs than with exposure to mercury vapour. More precise data are available in connection with exposure to methyl- mercury compounds. Concentrations exceeding 0. 1 ppm in whole blood are to be regarded as evidence of serious and dangerous exposure. This concentration in blood corresponds to a daily intake of 2 mg Hg/kg body weight per day or exposure to about 0. 01 mg Hg/m3 of air for 8 hours a day. Women should not be occupationally exposed to alkylmercury compounds during their most fertile years.

175 REFERENCES TO CHAPTER 13

BERLIN, M., On estimating threshold limits for mercury in biological material, Acta med. scand., Suppl. No. 396 (1963). BERLIN, M., ULLBERG, S., Accumulation and retention of mercury in the mouse. I. An autoradiographic study after a single intravenous injection of mercuric chloride, Archs envir. Hlth (> (1963) 489. BERLIN, M., FAZAKERLY, J., NORDBERG, G., The uptake of mercury in the brains of mammals exposed to mercury vapour and to mercuric salts, Archs envir. Hlth 18^ (1969) 719. BODNAR, J., SZEP, O., VESZPREMY, B., Uber den natllrlichen Quecksilbergehalt des menschlichen Organismus, Biochem. Z. 302 (1939) 384. BRIGATTI, L., II contenuto in mercurio degli organi di soggetti senza e con precedento absorbimento mercuriale, Medna Lav. 40 (1949) 233. BUTT, E.M., SIMONSEN, D. G., Mercury and lead storage in human tissues, Am. J. clin. Path. 20 (1950) 716. DAL CORTIVO, L.A., WEINBERG, S., Bl., GLAQUINTA, P., JACOBS, M.B., Mercury levels in normal human tissue, J. forens. Sci. 9 (1964) 501. DEROBERT, L., MARCUS, O., Intoxication professionelle par inhalation de compose organique mercurielle antiparasitaire, Annls Med. lfeg. 36 (1956) 294. FUJIMURA, Y., Studies on the toxicity of mercury. Urinary excretion of mercury and its deposition in organs in non-exposed man, Jap. J. Hyg. 1£(1964) 33 (in Japanese with English summary). GOLDWATER, L.J., Occupational exposure to mercury, The Harben Lectures, 1964, Jl. R. Inst, of publ. Hlth (Dec. 1964). GRANT, C., MOBERG, A., WESTOO, G., (to be published). JOSELOW, M.M., GOLDWATER, L.J., WEINBERG, S.B., Absorption and excretion of mercury in man. XI. Mercury content of "normal" human tissues, Archs envir. Hlth 15 (1967) 64. KAZANTZIS, G., SCHILLER, K., ASSCHER, A., DREW, R. G., Albuminuria and the nephrotic syndrome following exposure to mercury and its compounds, Q. Jl. Med. 29 (1962) 1. LECOMPTE, J., Action vesicante des derives organiques mercuriels, C.r. Seanc. Soc. Biol. 143(1949) 1296. LOMHOLT, S., Quecksilber, Theoretisches, Chemisches and Experimentelles, in Handbuch der Haut- und Geschlechtskrankheiten, Vol. 18 (JADASSOHN, J. Ed.) Springer, Berlin (1928) (Quoted by Sollman and Schreiber). MASSMAN, W., Beobachtungen beim Umgang mit Phenyl-Quecksilberbrenzkatechin, Zentbl. ArbMed. 7 (1957) 9. MATSUMOTO, H., KOYA, G., TAKEUCHI, T., Foetal Minamata disease, A neuropathological study of two cases of intrauterine intoxication by methylmercury compound, J. Neuropath, exp. Neurol. 24 (1965) 563. MORI, W., TSUNAKAWA, S., OOTA, K., TAGUCHI, H., Chemical and morphological pathology of mercury deposit following administration of a mercurial oncostatic, Acta path. jap. 15 (1965) 317. SCHICHA, H., KLEIN, H.J., KASPEREK, K., RITZL, F., Aktivierungsanalytische quantitative Bestimmung mehrerer Elemente in verschiedenen Organen und im Karzinomgewebe, Beitr. path. Anat. 138 (1969) 245. SMITH, R.G., VORWALD, A.J., PATIL, L. S., MOONEY, T.F.Jr., A study of the effects of exposure to mercury in the manufacture of chlorine, Communicated at AIHA Meeting, May 15, 1969, Denver, Colorado. STOCK, A., Der Quecksilbergehalt des menschlichen Organismus, Mitteilung liber Wirkung und Verbreitung des Quecksilbers, Biochem. Z. 304 (1940) 73. SUZUKI, T., MIYAMA, T., KATSUNUMA, H., Mercury contents in the red cells, plasma urine and hair from workers exposed to mercury vapour, Ind. Hlth £(1970) 39. SOLLMAN, T., SCHREIBER, N.E,, Chemical studies of acute poisoning from mercuric bichloride, Archs intern. Med. 57 (1936) 46. TAKEUCHI, T., MORIKAWA, N., MATSUMOTO, H., SHIRAISHI, Y., A pathological study of Minamata disease in Japan, Acta Neuropath. 2^(1962) 40.

176 TAKEUCHI, T., Pathology of Minamata disease, in Minamata Disease (KUTSUNA, M., Ed.) Study group of Minamata disease, Kumamota University, Japan (1968) 141. WATANABE, S., Mercury in the body treated for 10 years after long term exposure to mercury, Proc. 16th Int. Congr. Occup. Health, Tokyo, Department of Public Health, Medical School of Hokkaido University, Sapporo, Japan (in press, 1970). WESTOO, G., Determination of methylmercury salts in various kinds of biological material, Acta chem. scand. 22_ (1968) 2227. WILKENING, H., LITZNER, S., Uber Erkrankungen insbesondere der Niere durch Alkyl-Quecksilberverbindungen, D. med. Wschr. 77 (1952) 432.

177

LIST OF AUTHORS' ADDRESSES

M. Berlin Institute of Hygiene, University of Lund, S-220 05 Lund, Sweden

P.E. Berteau Food Additives Unit, World Health Organization, OH-1211 Geneva 27, Switzerland

D.J. Clegg Division of Toxicology, Food and Drug Directorate, Dept. of National Health and Welfare, Ottawa. Canada

R.E. DeSimone Biochenlistry Department, School of Chemical Sciences, University of Illinois, Urbana, 111.61801, United States of America

V.P. Guinn Department of Chemistry, University of California, Irvine, Calif., United States of America

J. Heinonen Division of Research and Laboratories, International Atomic Energy Agency, P.O. Box 590, A-1011 Vienna, Austria

A.V. Holden Dept. of Agriculture and Fisheries for Scotland, Freshwater Fisheries Laboratory, Faskally, Pitlochry, Perthshire, United Kingdom

S. Jensen The Environmental Protection Board, Wallenberglaboratoriet, Lilla Frescati, S-104 05 Stockholm 50, Sweden

A. Jernelöv Institutet för Vatten- och Luftvârdsforskning, Box 5607, S-114 86 Stockholm, Sweden

179 C.G. Lamm Chemistry Laboratory A, The Technical University of Denmark, Lyngby, Denmark

K. Ljunggren Isotope Techniques Laboratory, Drottning Kristinas Vag 45-47, S-114 28 Stockholm, Sweden

F.C. Lu Food Additives Unit, World Health Organization, CH-1211 Geneva 27, Switzerland

D. Merten Division of Research and Laboratories, International Atomic Energy Agency, P.O. Box 590, A-1011 Vienna, Austria

J.K. Miettinen Department of Radiochemistry, University of Helsinki, Finland

M.W. Penley Biochemistry Department, School of Chemical Sciences, University of Illinois, Urbana, 111.61801, United States of America

Kristina Rissanen Department of Radiochemistry, University of Helsinki, Finland

J. Rúzicka Chemistry Laboratory A, The Technical University of Denmark, Lyngby, Denmark

N. S aito Department of Chemistry, Faculty of Science, The University of Tokyo, Tokyo, Japan

O. Suschny Division of Research and Laboratories, International Atomic Energy Agency, P.O. Box 590, A-1011 Vienna, Austria

180 J. O'G. Tatton Laboratory of the Government Chemist, Cornwall House, Stamford Street, London S.E.I, United Kingdom

T. Westermark Division of Nuclear Chemistry, Royal Institute of Technology, Stockholm 70, Sweden

F.P.W. Winteringham Joint F AO/IAEA Division of Atomic Energy in Food and Agriculture, International Atomic Energy Agency, P.O. Box 590, A-1011 Vienna, Austria

J.M. Wood Biochemistry Department, School of Chemical Sciences, University of Illinois, Urbana, 111.61801, United States of America

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