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Toxicity, bioavailability and metal speciation
C~p.~~oehe~. P!zy&l.Vol.tO6C,No.3, pp. 585-595, 1993 Printed in Great Britain
0742-8413193 $6.OUi-0.00 0 1993PergamonPress Ltd
MINI REVIEW
TOXICITY, BIOAVAILABILITY AND METAL SPECIATIO~* S.B. JONNALAGADDA~
and P.V.V.
PRASADARAO$
TDepartment of Chemistry; and SDepartment of Anatomy, University of Zimbabwe, Box MP 167, Mt. Pleasant, Harare, Zimbabwe (Fax 263-4 732-828) (Received 13 July 1993; accepted for publication 30 July 1993)
Abstract-l. Environmental toxicology emphasizes the difference from traditional toxicology in which pure compounds of interest are added to purified diets, or injected into the test animals. When the objective is to study the fate and effects of trace elements in the environment, knowledge of the speciation of the elements and their physicothemical forms is important. 2. Cadmium salts such as the sulfides, carbonates or oxides, are practically insoluble in water. However, these can be converted to water-soluble salts in nature under the influence of oxygen and acids. Chronic exposure to Cd is associated with renal toxicity in humans once a critical body burden is reached. 3. The solubility of As(II1) oxide in water is fairly low, but high in either acid or alkali. In water, arsenic is usually in the form of the arsenate or arsenite. As(II1) is systemically more poisonous than the As(V), and As(V) is reduced to the As(II1) form before exerting any toxic effects. Organic arsenicals also exert their toxic effects in V&JOin animals by first metabolizing to the trivalent arsenoxide form. Some methyl arsenic com~unds, such as di- and trimethyiarsin~, occur naturally as a consequence of biological activity. The toxic effect of arsenite can be potentiated by dithiols, while As has a protective effect against the toxicity of a variety of forms of Se in several species. 4. Selenium occurs in several oxidation states and many selenium analogues of organic sulfur compounds exist in nature. Selenium in selenate form occurs in alkaline soils, where it is soluble and easily available to plants. Selenite binds tightly to iron and aluminum oxides and thus is quite insoluble in soils. Hydrogen selenide is a very toxic gas at room temperature. The methylated forms of Se are much less toxic for the organism than selenite. However, the methylated Se derivatives have strong synergistic toxicity with other minerals such as arsenic. 5. Aquatic organisms absorb and retain Hg in the tissues, as methylmercury, although most of the environmenta Hg to which they are exposed is inorganic. The methylmercury in fish arises from the bacterial methylation of inorganic Hg. Methylmerc~ in the human diet is almost completely absorbed into the bloodstream. The nervous system is the principal target tissue affected by methylmercury in adult human beings, while kidney is the critical organ following the ingestion of Hg(II) salts.
INTRODUCTION In the recent past, considerable attention has been paid to the speciation of metals due to its importance in understanding the fate and effects of metals in the environment. The total concentration of a metal in an environment is needed for the mass balance. However, when the study relates to fate and effects, knowledge of the physico-chemical forms is required. Considering the wide scope of the subject, present discussion is restricted to the speciation of four metals, namely cadmium, arsenic, selenium, and mercury and their bioaccumulation and toxicity. The human uptake of these metals, and for that matter, most of the pollutants, occurs via the inhalation of ambient air and the ingestion of food and drinkingwater in addition to the possibility of absorption through the skin. Figure 1 shows the routes of absorption, distribution and excretion of toxicants in _ *Presented as plenary lecture at International Symposium on Trace Elements and Liver Diseases, 14-17 February 1993, Karachi, Pakistan.
the body (Casarett, 1980). Environmental toxicology emphasizes the difference from traditional toxicology in which pure compounds of interest are added to purified diets, or injected into the test animals. There is a need to consider all the aspects of a trace element and other elements/materials which can interact with the element and alter its bioavailability or toxicity. One must consider the entire exposure process, including other constituents which accompany the toxic element of interest, if one wants to evaluate en~ronmental risk of toxic elements disposed into the ecosystems (Chaney, 1988). DISCUSSION Cadmium
The mobility of cadmium in the environment and effects on the ecosystem depend to a great extent on its speciation. Cadmium has relatively high vapour pressure. The vapour is oxidised quickly to produce cadmium oxide in air. When reactive gases such as carbon dioxide, water vapour, sulfur dioxide, sulfur 585
S. B. JONNALAGADDAand P. V. V. PRA~ADARAO
586
Absorption,
Distribution
lnoestion
And
Excretion lntraperitoneal
lntrayenws
Inhalation
subcutaneous Intramuscular
Dermal
I
Kidney
Y
i-i
one
Q Bladder
?l Feces
Fig. 1. Routes of absorption, distribution and excretion of toxicants in the body (Courtesy
of L. J.
Casarett).
trioxide or hydrogen chloride are present, the Cd vapour reacts to produce cadmium carbonate, hydroxide, sulfite, sulfate or chloride, respectively. Cadmium salts such as the sulfide, carbonate or oxide, are practically insoluble in water. However, these can be converted to water-soluble salts in nature under the influence of oxygen and acids. The sulfate, nitrate and halogenates of Cd are soluble in water. The speciation of Cd in soil water (Fig. 2) and
-10.
iii .o u :: ‘0 E B
seawater (Fig. 3) is important for the evaluation of its potential hazard (WHO, 1992). Much of the cadmium entering waters from industrial sources is rapidly adsorbed by particulate matter, where it may settle or remain suspended, depending on local conditions. This can result in low concentrations of dissolved cadmium even in water bodies that receive and transport large quantities of the metal (WHO, 1992).
Cd++
-ll-
-l2-
_,j_
-14-
-15 . 4
I 4.5
5
55
6
6.5
7
1.5
0
05
9
PH Fig. 2. Inorganic
cadmium
speciation
in soil water
(Reproduced
with permission
from WHO,
1992).
Toxicity, bioavailability and metal speciation
587
CdC$
0.1
7.0
7.2
7k
7.6
7.6
0.0
6.2
1.b
6.6
6.6
9.0
PH Fig. 3. Distribution of the chemical species of ~dmium in seawater at 25°C and I atm as a function of pH (Reproduced with permission from WHO, 1992).
Most of the cadmium found in mammals, birds and fish is inorganic in nature and probably bound to protein. Marine organisms generally contain higher cadmium residues than their fresh water and terrestrial counterparts. Cadmium tends to concentrate in the viscera of vertebrates, especially the liver and kidneys, and concentrations are generally higher in older organisms (Eisler, 1985). Table I summa&es the concentrations of cadmium in biota (WHO, 1992). Microorganisms generally exhibit a high capacity to take up cadmium from water and retain the
metal in their cells. Molluscs concentrate cadmium to a high degree over a period of time, but uptake is often slow. Cadmium assimilation efficiencies in different soil invertebrates are shown in Table 2 (WHO, 1992). Metallothioneins have been known to play an important role in Cd metabolism in animals. These proteins are induced by increased levels of Zn, Cd and Cu in animal cells, especially in the intestine, liver, kidney and placenta. Formation of these cysteine-rich metal-chelating-proteins tends to limit
Table 1. Concentrations of cadmium in biota. Reproduced with permission from WU(I 119971 Organisms Concentration Marine Organisms Algae Molluscs
Crustaceans Annelids Fish Birds Mammals Freshwater organisms Piants Molluscs Annelids Fish Terre&a1 organisms Plants Annelids Birds Mammais
Part of the oraanisms
Soft parts Kidney Liver Digestive gland Whole body Whole body Whole body Kidney Kidney
Cadmium (ma/kg dry weight)
Whole plant Roots Soft parts; fresh weight Whole body; fresh weight Whole body; fresh weight
g6.1 0.2-1.4 0.5-3.2 0.01-1.04
Whole plant Grain Whole body Whole body; fresh weight Kidney; fresh weight Kidney
927.1 6251 3.ISl2.6 <0.05-0.24 67.4 68.1
588
S.
B. JONNALAGADDA and P. V. V. PRASADA
RAO
‘fable 2. Cadmium assimilation efficiencies in different soil invertebrates. Reproduced with oermission from WHO (19921 Species Snail Arianta arbuslorum * Centipede Lithobius variegates Millipede Clomeris marginnta Pseudoscorpin Neobisium muscorutn Mite Platynothrus pelfifer
Insects
Orchesella cincta Notioohilus bintttutus
Food
Cadmium concentration in food (rmol/g)
i .48
Agar fsopod Hepatopan~eas
1.21-10.2
Maple leaves
Assimilation efficiency (%) 55-92 o-7.2 8.2-40.6
Collembolans
0.20
58.9
Green algae
0.15
11.2
Green algae Collembolans
0.09 0.23
3;::
‘Assimilation value for midgut gland.
further transport of Cd, and prevent Cd injury to the animal and its progeny. It has been known for several years that Fedeficiency substantially increases absorption of dietary Cd in experimental animals and humans. Iron-deficient humans absorbed as much as 24% of the Cd in an oatmeal breakfast, while strongly Fesu~cient individuals absorbed < 1.O% (Flanagan et al., 1978). An iron storage protein in intestinal mucosal cells of Fe-sufficient animals, ferritin, strongly adsorbs Cd, so much so that metallothionein is not induced in these cells. When the same diets are fed to Fe-deficient animals, i.e. with no ferritin to adsorb the Cd, the metallothionein is induced binding the Cd in the cells. Animal studies continue to show that dietary Zn and Ca are also important in Cd absorption. Increased dietary Zn apparently induces biosynthesis of metallothionein in the intestinal mucosal cells; this protein then binds Cd and Zn. Zinc may also inhibit Cd absorption by mucosal cells. Whole body and kidney Cd were reduced as dietary Zn increased (Lamphere et al., 1984). Calcium deficiency causes the intestine to increase its ability to absorb Ca, which also increases Cd absorption. Like Zn, Ca may also compete in the absorption process directly. The reason why Zn protects the organism from cadmium absorption is that zinc has a stronger tendency to react with acids than cadmium, and as a result can displace it from an acid solution: Zn+CdZ+-+ZnZ+ +Cd. Japan farm families who consumed rice with high Cd, but low Zn content suffered Fanconi syndrome, a proximal renal tubular proteinuria caused by Cd. A different story of Cd exposure is the case of New Zealanders consuming large amounts of oysters which contained about 5 mg Cd/kg fresh weight, but exhibited no Cd related health problems. A point to note is that the individuals who consumed large amounts of Cd-rich oysters in New Zealand, had higher dietary levels of Zn, Fe, and Ca levels compared to Japan farm families. Nordberg et al. (1986) found that most of the Cd in these oysters (cultured with environmental Cd dose) was bound to high
molecular weight proteins, but not as a metallothionein, as is found when acute Cd cases. Further, the oysters contained 5.3 mg Cd and 100 mg Zn/kg wet weight. The kidney analysis in deceased oyster consumers confirmed that Cd absorption has been quite low in this population, suggesting its very low bioavailability. In most Cd pollution situations, Zn accompanies Cd at 100-200 mg Zn/mg Cd, and all crops but rice tend to reduce the Cd: Zn ratio during formation of fruits and grains. In these cases, Zn, Fe and Ca in the foods help to maintain the intestine in a condition which allows little Cd absorption. The risk of soil Cd toxicity to humans depends on the crop grown, elements which accompany Cd into the edible crop tissues, and Fe, Ca and Zn levels in the normal diets of the individuals at risk. Arsenic
While arsenic has not been shown directly to be an essential element in terrestrial or fresh water systems, it certainly plays an important biological function in the sea where it is the tenth most abundant element with an average concentration of 23 x 10e9 M. Various forms of inorganic and organic arsenic are summarised in Table 3 (WHO, 1981). The most important commercial compound is arsenic(II1) oxide (As,O,) (arsenous acid anhydride). Its vapour pressure at ambient temperatures is significant (0.6 g/m3 at 25”C), a fact which is important in the transport and distribution of arsenic in the environment. The solubility of arsenic(III) oxide in water is fairly low (2% at 25°C). The resulting solution is slightly acidic and contains arsenous acid (H,AsO,). Arsenic(II1) oxide is highly sofuble in both acid and alkali. In aqueous solution, arsenic is usually in the form of the arsenite or arsenate (salts containing anions AsO:-, AsO:-, HAsO:- and H,AsO;). Reduction by organic matter of arsenic(II1) and sulfate ions in the sediments of aquatic systems is likely to be responsible for the formation of both metallic arsenic and arsenic sulfides at the same location. Lead arsenate, copper arsenate, copper(H) acetate meta-arsenate (Paris Green), and calcium arsenate, all of which have been used as insecticides,
Toxicity,
bioavailability
and metal speciation
589
Table 3. Some common inorganic and organic arsenic comoounds. Reoroduced with oermission from WHO (1981) CAS number 1327-53-3
Name Inorganic arsenic trivalent Arsenic(II1) oxide
Synonyms
Formula As,O,(or As406)
Arsenic trioxide Arsenous oxide White arsenic
H,AsO,
13464-S-9
Arsenous acid
13768-07-05
Arsenenous acid Arsenites, salts of Arsenous acid
Arsenious acid
HAsO, H,AsO, HAsO:or AsO:-
7784-34-I
Arsenic(lI1) chloride
Arsenic trichloride Arsenous trichloride
AsCI,
1303-33-9
Arsenic(II1) sulfide
Arsenic trisulfide Orpiment, auripigment
AsA
1303-28-2
Inorganic arsenic pentavalent Arsenic(V) oxide
Arsenic pentoxide
A@,
7778-39-4
Arsenic acid
Orthoarsenic acid
H,AsO,
10102-53-I
Arsenenic acid Arsenates, salts of arsenic acid (ortho)
Metaarsenic acid
HAsO, H,AsO; HAsO: or AsO:-
Methanearsonic acid
CH,AsO(OH),
Cacodylic acid
(CH,),AsO(OH)
124-58-3
Organic arsenic Methylarsonic acid
75-60-5
Dimethylarsinic acid
4964- 14- 1
Trimethylarsine oxide
593-52-2
Methylarsine
98-50-O
Arsanilic acid
p-Aminobenzene-arsenic (4-aminophenyl)-arsenic
139-93-5
Arsphenamine
4,4-Arsenobis(2-aminophenol)dihdrochloride Salvarsan
HCI H,N
0 II (OH),OAs-C,Hh--NHCNH,
(CH,),AsO CH,AsH, acid acid
121-59-5
Carbarsone
[4-(Aminocarbonyl-amino)phenyl]arsenic acid N-Carbamoylarsanilic acid
554-12-3
Tryparsamide
{4[(2-Amino-2-oxoethyl)amino]phenyl)arsonic acid
121-19-7
3-Nitro-4-hydroxyphenylarsonic acid
H,N--C,HrAsO(OH)> NH,. HCI
I HCLC,H,-As=As-
(OH) 2OAs-C 6H 4-NHiH
L ,H,-OH
2CNH 2
NO,
II
HO-C,HsAsO(OH), 98-72-6
4-Nitrophenyl arsenic acid Arsenobetaine Arsenocholine Dialkylchloroarsine Alkyldlchloroarsine
p-Nitrophenyl
are only slightly soluble in water. Organic arsenical herbicides such as DSMA (disodium methanearsonate), MSMA (monosodium methane arsonate) and cacodylic acid [(CH,),AsO(OH)] are employed as selective herbicides for controlling certain weedy grasses in the cotton fields. Sodium arsenite is extensively used as a livestock dip to control ticks and lice. Carbon-arsenic bonds are quite stable under a variety of environmental conditions of pH and oxidation potential. Figure 4 illustrates the soil-air cycle of arsenic (WHO, 1981). Some methyl arsenic compounds, such as di-and trimethylarsines, occur naturally as a consequence of biological activity. In solution, these may undergo oxidation to the corresponding methylarsonic acids. Methylarsonic acid is a difunctional acid, that forms soluble salts with alkali metals. Most environmental transformations of arsenic appear to occur in the soil, in sediments, in plants and animals, and in zones of biological activity in the
arsenic acid
O,N--C,H.--AsO( (CH,),As + CH,COOH (CH,),As + CH,CH,OH R,AsCI RASCI,
oceans. Biomethylation and bioreduction are probably the most important environmental transformations of the element, since they can produce organometallic species that are sufficiently stable to be mobile in air and water. In the natural estuarine environment, biomethylation by microbes occurs by different mechanisms depending on the pH conditions. Figure 5 shows the biomethylation of arsenic by S-adenosyl methionine functioning as the methyldonor in basic conditions, and methylcobalamin as the methyl-donor in acidic conditions (Wood, 1988). However, the biomethylated forms of arsenic produced are subject to oxidation and bacterial demethylation back to inorganic forms. It was ascertained that methylarsines were the toxic agents. Dimethylarsine [As(Ch,),H] is mainly produced by anaerobic organisms, while trimethylarsine [As(Ch,),] resulted from aerobic methylation. The probable mechanism for the methylation of arsenate to methanearsonates (McBride et al., 1978) is shown in Fig. 6. The
S. B. JONNALAGADDA and P. V. V. PRASADARAO
TRANSPORT
IN
TRANSPORT ARSENICAL
INDUSTRIAL SOURCES DUST I
I dry deposition precipitation
Ir 4
HERBICIDES
I I 1
4 uptake
+
I i
(Cl
dec y was R off
H2AsO;
Oxygen present Organic As w
+ 03,N2 0,
PLANTS
I
HAsO,
OUT
Oxygen
soil biomethylation
Fe (OH1 SO,,5
HAsO H,AsO,
I
I
-
HAS&----’
absent -)
H,S
H,AsO,,--_,HAsO,
--,As&etc...
4 1 Leaching
Fig. 4. Local air-air cycle of arsenic. Reproduced with permission from WHO (1981). proposed mechanism indicates that As(V) has to be reduced to As(M) before being methylated. Inorganic trivalent arsenic is systemically more poisonous than the pentavalent form and it is possible that pentavalent arsenic is reduced to the trivalent form before exerting any toxic effects. Organic arsenicals are also thought to exert their toxic effects in vivo in animals by first metabolizing to the trivalent arsenoxide form. It is thought that there is a blood-mammary barrier to arsenic, as evidenced
by several separate studies in which cows were fed monosodiummethane arsonate, lead arsenate and arsenic trioxide, without a significant increase of the arsenic content of their milk. The toxic effects of arsenic compounds in animals are thought to be mediated through a reaction with sulfhydryl groups of tissue proteins and enzymes (Fig. 7). Arseno compounds, (R-As=As-R) are readily oxidised, even by trace amounts of oxygen. Their action has been suggested to be due to their
5 Adenosylmethionine
S Adenosylhomocysteine
hneA&, HS -6
h
CH3
I
+3 +As CH,+
ICH,I
2e
eCH,As++ ‘\
a
KH,),As+ +e,+H+
1 KH,),A
Fig. 5. (a) Methylation of arsenic salts by S-adenosyl-methionine. synthesis
of methylarsenic
compounds.
H
(b) Mechanism for B,,-dependent
Toxicity, bioavailability and metal speciation
As~*O~~-
->
2e
591 2e
CHS' As3+033-
_ 0s-
> CIi 3Ass*0 3 *-
-
->
a-
<~ethyIarsonat~>”
cIi3+ CH3As”0
2e
->
2 *-
(CHS)SAs5*08
-> -0"
(Cacodylate) CH3* ->
(CH,),AsS+O
(CHs)sAs3*0 A> _0'-
(CH,),AsS*
Fig. 6. Methylation of arsenate to methanearsonates. conversion
to the corresponding
arsenoso
derivatives
(R-As---O). These compounds can be divided into mono-substituted and di-substituted according to their reaction with suhhydryl groups. The monosubstituted compounds, exemplified by R-As==O, react with enzymes containing sulfhydryl groups. Inhibition of different enzyme systems by these arsenic compounds was shown to be reversed by addition of an excess of a monothiol, e.g. glutathione. Some enzymes contain two thiol groups, which can react with the monosubstituted arsenic compound, thereby yielding a 5-membered ring structure. This is reversed by dithiols, e.g. 2,3-dimercaptopropanol (British antilewisite, BAL), but not by monothiols (WHO, 1981). Mahaffey and Fowler (1977) examined the effects of dietary cadmium and lead on the toxicity of arsenic administered to rats. They showed that the efficiency of food utilization was more reduced by the combination of arsenic and cadmium than by each metal alone. Selenium Selenium exists naturally in several oxidation states and some of its chemical forms are volatile. Selenium has similar chemical and physical properties as sulfur and tellurium. Many selenium analogues of organic sulfur compounds exist in nature. Selenium in the + 6 or selenate state is stable under both alkaline and oxidising conditions. Thus, in alkaline soils it is easily R-As=0
Protein
=
+ 2 R’SH
/
SH
\
SH
Protein
/
= R-As(SR’)s
+
s\ ’
available to plants. Selenium in the +4 state occurs naturally as selenite. In alkaline solution, it tends to oxidise slowly to the +6 state, if oxygen is present. It is readily reduced to elemental Se by a number of reducing agents. Selenium dioxide, an anhydride of selenious acid, sublimes at 317°C. This is important with regard to air pollution through the combustion. Selenite binds tightly to iron and aluminum oxides. Thus, it is quite insoluble in soils. In its -2 state, selenium exists as hydrogen selenide and in a number of metallic selenides. Hydrogen selenide at room temperature is a very toxic gas, a strong reducing agent and a relatively strong acid. In air, it decomposes rapidly to form Se and water. Figure 8 illustrates the biological cycle of selenium (WHO, 1987). In the estuarine environment, it is anticipated that the biomethylation of selenium will be a rapid process, and therefore most of the selenium in the sea is in the form of organoselenium compounds. This microbially mediated process has the potential to create a toxic local situation with respect to coastal marine life, if major anthropogenic inputs of selenium occur in estuaries. Of special interest to the reactivity and movement of the metalloids of arsenic and selenium, is the fact that inorganic species of these elements behave as electrophiles in acidic conditions and as nucleophiles at basic pHs. Figure 9 shows the biomethylation of selenium species by methylcobalamine (vitamin B,,) under acidic conditions (Wood, 1988).
Cl,
+ H,O
AsCH=CHCI
AsCH=CHCl
+
2HCl
s’
Fig. 7. Reaction of arseno compounds with sulfbydryl groups of proteins.
592
S. B. JONNALAGADDA
and P.V.V. PRASADA RAO
Fig. 8. Biological selenium cycle: well-established pathways are indicated by solid lines, those needing further substantiation by dotted lines. Reproduced with permission from WHO (1987).
The intestinal absorption of soluble selenium compounds by rats is highly efficient. It has been shown that these animals absorbed the doses of selenate (92%), selenomethionine (91%) and selenocystine (81%) (Thomson et al., 1975). Only limited information is available regarding the absorption of Se occurring naturally in foods by animals. Poor bioavailability of selenium to animals is reported with certain fish products containing selenium. Studies carried out on human volunteers showed that intestinal absorption of oral doses of selenium was 94% for selenate selenium, 97% for selenomethionine, and 60% for sodium selenite. Only 55% of selenium as it occurs naturally in fish was absorbed, when fish was consumed in diet (Thomson et al., 1986). Hydrogen selenide, which is a volatile compound, is found to be deposited in liver and kidney when inhaled. Sodium selenite and selenium oxychloride
Fig. 9. Synthesis of alkylselenium compounds from selenium salts.
are absorbed through the skin. The distribution of Se appears to be relatively independent of the form and route of administration. The accumulation in human organs was found to be in the following descending order in the tissues: kidney > liver > spleen > pancreas > testes > heart muscle > intestine > lung > brain (Schroeder et al., 1970). Under normal conditions, selenide is methylated to form trimethyl selenonium ion, the main urinary metabolite of selenium. In case of Se toxicity, this pathway is overloaded and dimethyl selenide is produced. These methylation reactions are considered detoxication steps, and the methylated end products are much less toxic for the organism than selenite (Obermeyer et al., 1971). However, both of these methylated selenium derivatives have strong synergistic toxicity with other minerals. Vitamin E deficiency increased the susceptibility of the rats to chronic poisoning by Se as selenite. Selenium poisoning caused by feeding rats seleniferous wheat was decreased by giving sodium arsenite. However, arsenic potentiates the toxicity of the trimethyl selenonium ion. Dietary sulfate partially counteracts the toxicity of selenate, but it has no effect against selenite or organic forms of selenium. The discovery that Se is a component of the enzyme glutathione peroxidase (Rotruck et al., 1973) provided a logical explanation for the nutritional interaction of vitamin E and selenium, a puzzle that perplexed scientists for many years. The estimated safe and adequate range of selenium intake in humans is shown in Table 4 (WHO, 1987).
Toxicity, bioavailability and metal speciation Table 4. Estimated safe and adequate range of selenium intake. Reproduced with permission from WHCl (1987) Group Infants Children
Adults
O-Q.5 0.5-I
Daily selenium intake ocg) IO-40 20-60
1-3 46 7+
20-80 30-120 S&200
Age (a)
5&200
a = years.
The ecological evidence strongly favours a relationship between low selenium status and the incidence of Keshan disease (an endemic cardiomyopathy) in children in China (Yang et al., 1984), and Kashin-Beck disease (an endemic osteoarthropathy) in children in China and Eastern Siberia (Sokoloff, 1985). When selenium and arsenic interact, arsenic has a protective effect against the toxicity of a variety of forms of selenium in several species. The most logical hypothesis is that arsenic combines with selenium by reacting with selonol (-SeH) groups to form a detoxification conjugate that passes readily into bile, enhancing the biliary excretion of selenium (Levander, 1977). A study carried out on mice demonstrated that arsenic(II1) was more efficient in protecting against selenium toxicity than arsenic(V). Mercury Various toxic effects of mercury compounds can be best understood by considering the speciation and the ways in which they are handled by the body. For this purpose the mercurial compounds may be divided into the following main groups: elemental mercury vapour, divalent inorganic mercury salts, short-chain alkyl and other organic compounds; and aryl and alkyl mercury compounds. Global transport, bioaccumulation, and transformation of inorganic
593
mercury arise from the conversion of mercury compounds into methylmercury as well as the exposure to methylmercury through sea-food and other foods. There is a well recognized global cycle for mercury, whereby emitted mercury vapour is converted to soluble forms and deposited by rain onto soil and water. Figure 10 illustrates the global cycle of mercury. Metallic mercury is oxidised in water to the divalent mercuric ion Hg’+. Much of this precipitated as insoluble mercuric sulphide, especially in anaerobic environments, becomes oxidised to sulfate, releasing mercuric ion again. Despite the uncertainties concerning speciation, the global cycle of mercury is believed to involve the inorganic forms almost exclusively. Mercuric salts and organic mercury are readily taken up by organisms in water. The change in speciation of mercury from inorganic to methylated forms is the first step in the aquatic bioaccumulation process. Methylation can occur non-enzymatically or through microbial action. Aquatic organisms and fish take up the metal and retain it in the tissues, as methylmercury, although most of the environmental mercury to which they are exposed is inorganic. Table 5 summarises the methylmercury levels in the muscle tissue of various species of fish in different regions (WHO, 1990). Environmental levels of methylmercury depend upon the balance between bacterial methylation and demethylation. There is little information that fish themselves either methylate or demethylate mercury. Methylmercury is efficiently absorbed by all living organisms due to its great affinity for sulfhydryl groups. Most of it becomes protein bound and very little is excreted. As a result of food-chain biomagnification, highest levels are found in the tissues of such predatory species as fresh water trout, pike, bass and ocean tuna, swordfish, and shark. The bioconcentration factor, i.e. the ratio of the concentration of
‘-4s
BOTTOM SEDIMENT (20x lo-6g/kgI
Fig. IO. The global cycle of mercury. Reproduced with permission from WHO (1990).
S. B. JONNALAGADDA
594
and P. V. V. PRASADA
Uo
Table5.The range of published average values of methylmercury (mg mercury/kg wet weight) in the muscle tissue of various species of fish. Reproduced with permission from WHO (1990) Species Non-predators Mackerel Sardine Unspecified Number of edible species Predators Tuna Swordfish Shark, ray, do&h
Atlantic ocean
Pacific Ocean
Indian Ocean
Mediterranean Ocean
0.07-0.2 0.03-0.06
0.16-0.25 0.03
0.005 0.006
0.24 0.15
0.08-0.27
0.07-0.09
0.02-0.16
0.1-0.3
0.03-0.8 0.8-1.3
0.3 1.6
0.064-6.4 -
1.2 1.8
1.0
0.7-I. 1
0.094-1.5
1.8
methylmercury in fish tissue to that in water, is usually between 10,000 and 100,000 (U.S. EPA, 1990). There is no evidence in the literature for the synthesis of organomercury compounds in human and mammalian tissues (WHO, 1976). The conversion of methylmercury to inorganic mercury is considered a key step in the process of excretion of mercury after exposure to methylmercury (WHO, 1990). Mercury vapour is more soluble in plasma, whole blood and haemoglobin than in distilled water (Hursh, 1985) and it readily crosses the blood-brain barrier. The oxidation of metallic mercury vapour to divalent ionic mercury is not fast enough to prevent the passage of elemental mercury through the bloodbrain barrier, the placenta, and other tissues. The oxidation serves as a trap to hold the mercury and leads to accumulation in brain and foetal tissues (WHO, 1976). The neurotoxic effect seen after exposure to metallic mercury vapour is attributable to the divalent mercury ion formed through oxidation in the brain tissue. Results of both human and animal studies indicate that about 80% of inhaled metallic mercury vapour is retained by the body, whereas liquid mercury is poorly absorbed via the gastrointestinal tract. Inhaled inorganic mercury aerosols are deposited in the respiratory tract and absorbed, the rate depending on the particle size (Hursh et al., 1976). The reduction of divalent mercury to metallic mercury has been demonstrated both in animals (mice and rats) and humans (Sugata et al., 1979). Inorganic mercury compounds are probably absorbed from the human gastrointestinai tract to a
level of less than 10%. The kidney is the main depository of mercury after the administration of elemental mercury vapour or inorganic mercury compounds (SO-90% of the body burden of animals). In humans, methylmercury in the diet is almost completely absorbed into the blood stream and is distributed to all tissues in about 4 days (Kershaw et al., 1980). ~ethylmercury is converted to inorganic mercury, assumed to be Hg2+ in mammals. The nervous system is the principal target tissue for the effects of methylmercury on adult human beings. Renal damage is one of the most frequently described non-neural effects of methylmercury. The kidney is the critical organ following the ingestion of inorganic bivalent mercury salts. It has been estimated that humans have a daily intake of about 2.4pg methylmercury from all sources. The total daily intake of all forms of mercury from ail sources has been estimated to be about 6.7 pg. The average daily intake and retention of total mercury and mercury compounds in the general population not occupationally exposed to mercury is shown in Table 6. Metals have been used by mankind for thousands of years, but many of the most useful metals are toxic, and the price of their utility has often been high. The increased demand for metals of all kinds that followed the Industrial Revolution was accompanied by the appearance of metal-induced occupational diseases on a large scale. Even the so called essential elements are also characterized by a surprisingly narrow range of optimal activity. A scant 4-5 fold change in concentration is su~cient to convert overt
Table 6. Estimated average daily intake and retention (pg/day) of total mercury and mercury compounds in the general population not occupationally exposed to mercury. Reproduced with permission from WHO (1990) Exposure Air Food Fish Non-fish Drinking water Dental amalgams Total
Elemental mercury vapour
Inorganic mercury com~unds
Methylme~ury
0.030 (0.024)
0.002 (0.001)
0.008 (0.0064)
0 0
0.600 (0.042) 3.6 (0.25) 0.050 (0.0035) 0 4.3 (0.3)
2.4 (2.3) 0 0 0 2.41 (2.31)
3.8%ZP(317) 3.9-21 (3.1-17)
Figures in parentheses represent the estimated amount retained in the body of an adult.
Toxicity, , ,I 1Swvival $!eficiency
,IToxicity
Optimal
bioavailability ;Letholity
i
(lJ---~ ESSENTIAL
)1g/d -10 “9
/d -.5
ELEMENT.
DOSE
-50
-Se-200-103-10~ -
-2
-
- F -
P
10 -20
-100
Fig. 11.Dose-response relationship for an essential element. Estimates for specific daily requirements are in pg/day for selenium and in mg/day for fluorine.
signs of deficiency to overt signs of toxicity. A further change of ltss than an order of magnitude in either direction is sufficient to cause death. Figure 11 illustrates the dose-response relationship of essential elements, selenium and fluorine. In general, toxicity is usually caused by a few specific chemical species. The effect is further complicated by other metals and ligands in the environment, requiring a multi-variable approach. One must consider the entire exposure process, including other constituents that accompany the element of interest, which can interact with the element and alter its bioavailability or toxicity. Further, the oriental approach to the treatment of illness employing the natural products, i.e. the concoctions and decoction containing various species, inclusive of certain toxic trace elements, provokes thoughts towards the importance of the speciation and interactions of various elements; and their bioavailability and toxicity. REFERENCES
Casarett L. J. (1980) The Basic Science of Poisons In Casarett and Doull’s Toxicology. (Edited by Doull J., Klassen C. D. and Amdur M. 0.). 2nd Edn., Chap. 3, p. 39. Macmillan Pub. Co., Inc., New York. Chaney R. L. (1988) Metal Speciation: Theory, Analysis and Applicafion (Edited by Kramer J. R. and Allen H. E.), pp. 219-260. Lewis Publishers, Inc., Chelsea, U.S.A. Eisler R. (1985) Cadmium hazards to fish, wildlife and invertebrates: A synoptic review, Washington, DC, U.S. Dept of the Interior, Fish and Wildlife Service, Biological Report No. 85. Flanagan P. R., McLellan J. S., Haist J., Cherian G., Chamberlain M. J., and Valberg L. S. (1978). Increased Dietary Cadmium Absorption in Mice and Human Subjects with Iron Deficiency. Gastroenterology 74, 841-846. Hursh J. B., Clarkson T. W., Cherian M. G., Vostal J. V., and Mallie R. V. (1976) Clearance of mercury (Hg-197, Hg-203) vapor inhaled by human subjects. Archs. envir. Health. 31, 302-309. Hursh J. B. (1985) Particle coefficients of mercury (203 Hg) vapour between air and biological fluids. J. appl. Toxic. 5, 327-332. Kershew K. G., Clarkson T. W. and Dhahir P. H. (1980) The relationship between blood levels and dose
and metal speciation
595
of methylmercury in man. Archs. envir. Healih 35, 28-36. Lamphere D. N., Dorn C. R., Reddy C. S. and Meyer A. W. (1984) Reduced Cadmium body burden in cadmiumexposed calves fed supplemental zinc. Envir. Res 33, 119-124. Levander 0. A. (1977) Metabolic interrelationships between arsenic and selenium. Envir. Health Perspect. 19, 159-164. McBride B. C., Merilees H., Cullen W. R. and Pickett W. (1978) Anaerobic and aerobic alkylation of arsenic. In Organometals and Organomeralloids (Edited by Brinckman F. E. and Bellama J. M.), pp. 94-115, ACS symp. Ser. 82. Am. Chem. Sot., Washington, DC. Mahaffey K. R. and Fowler B. A. (1977) Effects of concurrent administration of lead, cadmium and arsenic in the rat. Envir. Health Perspect. 19, 165-171. Nordberg M., Nuottaniemi I., Cherian M. G., Nordberg G. F., Kjellstrom T. and Garvey J. S. (1986) Characterization studies on the cadmium binding proteins form two species of New Zealand Oysters. Envir. Health Perspect. 65, 57-62. Obermeyer B. D., Palmer I. S., Olson 0. E. and Halverson A. W. (1971) Toxicity of trimethylselenonium chloride in the rat with and without arsenite. Toxic. appl. Pharmac 20, 135-146. Rotruck J. T., Pope A. L., Ganther H. E., Swanson A. B., Hafeman D. G. and Hoekstra W. G. (1973) Selenium: biological role as a component of glutathione peroxidase. Science 179, 588-590. Schroeder H. A., Frost D. V. and Balassa J. J. (1970) Essential trace metals in man: Selenium. J. chron. Dis. 23, 227-243. Sokoloff I. (1985) Endemic form of osteoarthritis. Clin. rheum. Dis. 11, 187-202. Sugata Y. and Clarkson T. W. (1979) Exhalation of mercury-Further evidence for an oxidation-reduction cycle in mammalian tissues. Biochem. Pharmac 28, 3474-3476. Thomson C. D., Robinson B. A., Stewart R. D. H. and Robinson M. F. (1975) Metabolic studies of 75Se selenocystine and ‘?3e selenomethionine in the rat. Br. J. Nutr. 34, 501-509. Thomson C. D. and Robinson M. F. (1986) Urinary and faecal excretions and absorption of a large supplement of selenium as selenite or as selenate. Am. J. C/in. Nutr. 42, 1015-1022. U.S. EPA (1990) Ambient Water Quality Criteria for Mercury. Criteria and Standards Division (EPA-600/479049), Washington, DC, U.S.A. WHO (World Health Organization) (1976) Mercury (Environmental Health Criteria l), p. 131. WHO (World Health Organization) (1981) Arsenic (Environmental Health Criteria 18) Geneva. WHO (World Health Organization) (1987) Selenium (Environmental Health Criteria 58). WHO (World Health Organization) (1990) Methylmercury (Environmental Health Criteria 101). WHO (World Health Organization) (1992) CadmiumEnvironmenfal aspects (Environmental Health Criteria 135). Wood J. M. (1988) Transport, bioaccumulation and toxicity of metals and metalloids in microorganisms under environmental stress. In Metal Speciation, Theory, Analysis and Application (Edited by Kramer J. R. and Allen H. E.), pp. 295-314. Lewis Publishers, Chelsea, Michigan. Yang G., Chen J., Wen Z., Ge K., Zhu L., and Chen X. (1984) The role of selenium in Keshan disease. In Advances in Nufritional Research (Edited by Draper H. H.), pp. 203-231. Plenum Press, New York.
0742-8413193 $6.OUi-0.00 0 1993PergamonPress Ltd
MINI REVIEW
TOXICITY, BIOAVAILABILITY AND METAL SPECIATIO~* S.B. JONNALAGADDA~
and P.V.V.
PRASADARAO$
TDepartment of Chemistry; and SDepartment of Anatomy, University of Zimbabwe, Box MP 167, Mt. Pleasant, Harare, Zimbabwe (Fax 263-4 732-828) (Received 13 July 1993; accepted for publication 30 July 1993)
Abstract-l. Environmental toxicology emphasizes the difference from traditional toxicology in which pure compounds of interest are added to purified diets, or injected into the test animals. When the objective is to study the fate and effects of trace elements in the environment, knowledge of the speciation of the elements and their physicothemical forms is important. 2. Cadmium salts such as the sulfides, carbonates or oxides, are practically insoluble in water. However, these can be converted to water-soluble salts in nature under the influence of oxygen and acids. Chronic exposure to Cd is associated with renal toxicity in humans once a critical body burden is reached. 3. The solubility of As(II1) oxide in water is fairly low, but high in either acid or alkali. In water, arsenic is usually in the form of the arsenate or arsenite. As(II1) is systemically more poisonous than the As(V), and As(V) is reduced to the As(II1) form before exerting any toxic effects. Organic arsenicals also exert their toxic effects in V&JOin animals by first metabolizing to the trivalent arsenoxide form. Some methyl arsenic com~unds, such as di- and trimethyiarsin~, occur naturally as a consequence of biological activity. The toxic effect of arsenite can be potentiated by dithiols, while As has a protective effect against the toxicity of a variety of forms of Se in several species. 4. Selenium occurs in several oxidation states and many selenium analogues of organic sulfur compounds exist in nature. Selenium in selenate form occurs in alkaline soils, where it is soluble and easily available to plants. Selenite binds tightly to iron and aluminum oxides and thus is quite insoluble in soils. Hydrogen selenide is a very toxic gas at room temperature. The methylated forms of Se are much less toxic for the organism than selenite. However, the methylated Se derivatives have strong synergistic toxicity with other minerals such as arsenic. 5. Aquatic organisms absorb and retain Hg in the tissues, as methylmercury, although most of the environmenta Hg to which they are exposed is inorganic. The methylmercury in fish arises from the bacterial methylation of inorganic Hg. Methylmerc~ in the human diet is almost completely absorbed into the bloodstream. The nervous system is the principal target tissue affected by methylmercury in adult human beings, while kidney is the critical organ following the ingestion of Hg(II) salts.
INTRODUCTION In the recent past, considerable attention has been paid to the speciation of metals due to its importance in understanding the fate and effects of metals in the environment. The total concentration of a metal in an environment is needed for the mass balance. However, when the study relates to fate and effects, knowledge of the physico-chemical forms is required. Considering the wide scope of the subject, present discussion is restricted to the speciation of four metals, namely cadmium, arsenic, selenium, and mercury and their bioaccumulation and toxicity. The human uptake of these metals, and for that matter, most of the pollutants, occurs via the inhalation of ambient air and the ingestion of food and drinkingwater in addition to the possibility of absorption through the skin. Figure 1 shows the routes of absorption, distribution and excretion of toxicants in _ *Presented as plenary lecture at International Symposium on Trace Elements and Liver Diseases, 14-17 February 1993, Karachi, Pakistan.
the body (Casarett, 1980). Environmental toxicology emphasizes the difference from traditional toxicology in which pure compounds of interest are added to purified diets, or injected into the test animals. There is a need to consider all the aspects of a trace element and other elements/materials which can interact with the element and alter its bioavailability or toxicity. One must consider the entire exposure process, including other constituents which accompany the toxic element of interest, if one wants to evaluate en~ronmental risk of toxic elements disposed into the ecosystems (Chaney, 1988). DISCUSSION Cadmium
The mobility of cadmium in the environment and effects on the ecosystem depend to a great extent on its speciation. Cadmium has relatively high vapour pressure. The vapour is oxidised quickly to produce cadmium oxide in air. When reactive gases such as carbon dioxide, water vapour, sulfur dioxide, sulfur 585
S. B. JONNALAGADDAand P. V. V. PRA~ADARAO
586
Absorption,
Distribution
lnoestion
And
Excretion lntraperitoneal
lntrayenws
Inhalation
subcutaneous Intramuscular
Dermal
I
Kidney
Y
i-i
one
Q Bladder
?l Feces
Fig. 1. Routes of absorption, distribution and excretion of toxicants in the body (Courtesy
of L. J.
Casarett).
trioxide or hydrogen chloride are present, the Cd vapour reacts to produce cadmium carbonate, hydroxide, sulfite, sulfate or chloride, respectively. Cadmium salts such as the sulfide, carbonate or oxide, are practically insoluble in water. However, these can be converted to water-soluble salts in nature under the influence of oxygen and acids. The sulfate, nitrate and halogenates of Cd are soluble in water. The speciation of Cd in soil water (Fig. 2) and
-10.
iii .o u :: ‘0 E B
seawater (Fig. 3) is important for the evaluation of its potential hazard (WHO, 1992). Much of the cadmium entering waters from industrial sources is rapidly adsorbed by particulate matter, where it may settle or remain suspended, depending on local conditions. This can result in low concentrations of dissolved cadmium even in water bodies that receive and transport large quantities of the metal (WHO, 1992).
Cd++
-ll-
-l2-
_,j_
-14-
-15 . 4
I 4.5
5
55
6
6.5
7
1.5
0
05
9
PH Fig. 2. Inorganic
cadmium
speciation
in soil water
(Reproduced
with permission
from WHO,
1992).
Toxicity, bioavailability and metal speciation
587
CdC$
0.1
7.0
7.2
7k
7.6
7.6
0.0
6.2
1.b
6.6
6.6
9.0
PH Fig. 3. Distribution of the chemical species of ~dmium in seawater at 25°C and I atm as a function of pH (Reproduced with permission from WHO, 1992).
Most of the cadmium found in mammals, birds and fish is inorganic in nature and probably bound to protein. Marine organisms generally contain higher cadmium residues than their fresh water and terrestrial counterparts. Cadmium tends to concentrate in the viscera of vertebrates, especially the liver and kidneys, and concentrations are generally higher in older organisms (Eisler, 1985). Table I summa&es the concentrations of cadmium in biota (WHO, 1992). Microorganisms generally exhibit a high capacity to take up cadmium from water and retain the
metal in their cells. Molluscs concentrate cadmium to a high degree over a period of time, but uptake is often slow. Cadmium assimilation efficiencies in different soil invertebrates are shown in Table 2 (WHO, 1992). Metallothioneins have been known to play an important role in Cd metabolism in animals. These proteins are induced by increased levels of Zn, Cd and Cu in animal cells, especially in the intestine, liver, kidney and placenta. Formation of these cysteine-rich metal-chelating-proteins tends to limit
Table 1. Concentrations of cadmium in biota. Reproduced with permission from WU(I 119971 Organisms Concentration Marine Organisms Algae Molluscs
Crustaceans Annelids Fish Birds Mammals Freshwater organisms Piants Molluscs Annelids Fish Terre&a1 organisms Plants Annelids Birds Mammais
Part of the oraanisms
Soft parts Kidney Liver Digestive gland Whole body Whole body Whole body Kidney Kidney
Cadmium (ma/kg dry weight)
Whole plant Roots Soft parts; fresh weight Whole body; fresh weight Whole body; fresh weight
g6.1 0.2-1.4 0.5-3.2 0.01-1.04
Whole plant Grain Whole body Whole body; fresh weight Kidney; fresh weight Kidney
927.1 6251 3.ISl2.6 <0.05-0.24 67.4 68.1
588
S.
B. JONNALAGADDA and P. V. V. PRASADA
RAO
‘fable 2. Cadmium assimilation efficiencies in different soil invertebrates. Reproduced with oermission from WHO (19921 Species Snail Arianta arbuslorum * Centipede Lithobius variegates Millipede Clomeris marginnta Pseudoscorpin Neobisium muscorutn Mite Platynothrus pelfifer
Insects
Orchesella cincta Notioohilus bintttutus
Food
Cadmium concentration in food (rmol/g)
i .48
Agar fsopod Hepatopan~eas
1.21-10.2
Maple leaves
Assimilation efficiency (%) 55-92 o-7.2 8.2-40.6
Collembolans
0.20
58.9
Green algae
0.15
11.2
Green algae Collembolans
0.09 0.23
3;::
‘Assimilation value for midgut gland.
further transport of Cd, and prevent Cd injury to the animal and its progeny. It has been known for several years that Fedeficiency substantially increases absorption of dietary Cd in experimental animals and humans. Iron-deficient humans absorbed as much as 24% of the Cd in an oatmeal breakfast, while strongly Fesu~cient individuals absorbed < 1.O% (Flanagan et al., 1978). An iron storage protein in intestinal mucosal cells of Fe-sufficient animals, ferritin, strongly adsorbs Cd, so much so that metallothionein is not induced in these cells. When the same diets are fed to Fe-deficient animals, i.e. with no ferritin to adsorb the Cd, the metallothionein is induced binding the Cd in the cells. Animal studies continue to show that dietary Zn and Ca are also important in Cd absorption. Increased dietary Zn apparently induces biosynthesis of metallothionein in the intestinal mucosal cells; this protein then binds Cd and Zn. Zinc may also inhibit Cd absorption by mucosal cells. Whole body and kidney Cd were reduced as dietary Zn increased (Lamphere et al., 1984). Calcium deficiency causes the intestine to increase its ability to absorb Ca, which also increases Cd absorption. Like Zn, Ca may also compete in the absorption process directly. The reason why Zn protects the organism from cadmium absorption is that zinc has a stronger tendency to react with acids than cadmium, and as a result can displace it from an acid solution: Zn+CdZ+-+ZnZ+ +Cd. Japan farm families who consumed rice with high Cd, but low Zn content suffered Fanconi syndrome, a proximal renal tubular proteinuria caused by Cd. A different story of Cd exposure is the case of New Zealanders consuming large amounts of oysters which contained about 5 mg Cd/kg fresh weight, but exhibited no Cd related health problems. A point to note is that the individuals who consumed large amounts of Cd-rich oysters in New Zealand, had higher dietary levels of Zn, Fe, and Ca levels compared to Japan farm families. Nordberg et al. (1986) found that most of the Cd in these oysters (cultured with environmental Cd dose) was bound to high
molecular weight proteins, but not as a metallothionein, as is found when acute Cd cases. Further, the oysters contained 5.3 mg Cd and 100 mg Zn/kg wet weight. The kidney analysis in deceased oyster consumers confirmed that Cd absorption has been quite low in this population, suggesting its very low bioavailability. In most Cd pollution situations, Zn accompanies Cd at 100-200 mg Zn/mg Cd, and all crops but rice tend to reduce the Cd: Zn ratio during formation of fruits and grains. In these cases, Zn, Fe and Ca in the foods help to maintain the intestine in a condition which allows little Cd absorption. The risk of soil Cd toxicity to humans depends on the crop grown, elements which accompany Cd into the edible crop tissues, and Fe, Ca and Zn levels in the normal diets of the individuals at risk. Arsenic
While arsenic has not been shown directly to be an essential element in terrestrial or fresh water systems, it certainly plays an important biological function in the sea where it is the tenth most abundant element with an average concentration of 23 x 10e9 M. Various forms of inorganic and organic arsenic are summarised in Table 3 (WHO, 1981). The most important commercial compound is arsenic(II1) oxide (As,O,) (arsenous acid anhydride). Its vapour pressure at ambient temperatures is significant (0.6 g/m3 at 25”C), a fact which is important in the transport and distribution of arsenic in the environment. The solubility of arsenic(III) oxide in water is fairly low (2% at 25°C). The resulting solution is slightly acidic and contains arsenous acid (H,AsO,). Arsenic(II1) oxide is highly sofuble in both acid and alkali. In aqueous solution, arsenic is usually in the form of the arsenite or arsenate (salts containing anions AsO:-, AsO:-, HAsO:- and H,AsO;). Reduction by organic matter of arsenic(II1) and sulfate ions in the sediments of aquatic systems is likely to be responsible for the formation of both metallic arsenic and arsenic sulfides at the same location. Lead arsenate, copper arsenate, copper(H) acetate meta-arsenate (Paris Green), and calcium arsenate, all of which have been used as insecticides,
Toxicity,
bioavailability
and metal speciation
589
Table 3. Some common inorganic and organic arsenic comoounds. Reoroduced with oermission from WHO (1981) CAS number 1327-53-3
Name Inorganic arsenic trivalent Arsenic(II1) oxide
Synonyms
Formula As,O,(or As406)
Arsenic trioxide Arsenous oxide White arsenic
H,AsO,
13464-S-9
Arsenous acid
13768-07-05
Arsenenous acid Arsenites, salts of Arsenous acid
Arsenious acid
HAsO, H,AsO, HAsO:or AsO:-
7784-34-I
Arsenic(lI1) chloride
Arsenic trichloride Arsenous trichloride
AsCI,
1303-33-9
Arsenic(II1) sulfide
Arsenic trisulfide Orpiment, auripigment
AsA
1303-28-2
Inorganic arsenic pentavalent Arsenic(V) oxide
Arsenic pentoxide
A@,
7778-39-4
Arsenic acid
Orthoarsenic acid
H,AsO,
10102-53-I
Arsenenic acid Arsenates, salts of arsenic acid (ortho)
Metaarsenic acid
HAsO, H,AsO; HAsO: or AsO:-
Methanearsonic acid
CH,AsO(OH),
Cacodylic acid
(CH,),AsO(OH)
124-58-3
Organic arsenic Methylarsonic acid
75-60-5
Dimethylarsinic acid
4964- 14- 1
Trimethylarsine oxide
593-52-2
Methylarsine
98-50-O
Arsanilic acid
p-Aminobenzene-arsenic (4-aminophenyl)-arsenic
139-93-5
Arsphenamine
4,4-Arsenobis(2-aminophenol)dihdrochloride Salvarsan
HCI H,N
0 II (OH),OAs-C,Hh--NHCNH,
(CH,),AsO CH,AsH, acid acid
121-59-5
Carbarsone
[4-(Aminocarbonyl-amino)phenyl]arsenic acid N-Carbamoylarsanilic acid
554-12-3
Tryparsamide
{4[(2-Amino-2-oxoethyl)amino]phenyl)arsonic acid
121-19-7
3-Nitro-4-hydroxyphenylarsonic acid
H,N--C,HrAsO(OH)> NH,. HCI
I HCLC,H,-As=As-
(OH) 2OAs-C 6H 4-NHiH
L ,H,-OH
2CNH 2
NO,
II
HO-C,HsAsO(OH), 98-72-6
4-Nitrophenyl arsenic acid Arsenobetaine Arsenocholine Dialkylchloroarsine Alkyldlchloroarsine
p-Nitrophenyl
are only slightly soluble in water. Organic arsenical herbicides such as DSMA (disodium methanearsonate), MSMA (monosodium methane arsonate) and cacodylic acid [(CH,),AsO(OH)] are employed as selective herbicides for controlling certain weedy grasses in the cotton fields. Sodium arsenite is extensively used as a livestock dip to control ticks and lice. Carbon-arsenic bonds are quite stable under a variety of environmental conditions of pH and oxidation potential. Figure 4 illustrates the soil-air cycle of arsenic (WHO, 1981). Some methyl arsenic compounds, such as di-and trimethylarsines, occur naturally as a consequence of biological activity. In solution, these may undergo oxidation to the corresponding methylarsonic acids. Methylarsonic acid is a difunctional acid, that forms soluble salts with alkali metals. Most environmental transformations of arsenic appear to occur in the soil, in sediments, in plants and animals, and in zones of biological activity in the
arsenic acid
O,N--C,H.--AsO( (CH,),As + CH,COOH (CH,),As + CH,CH,OH R,AsCI RASCI,
oceans. Biomethylation and bioreduction are probably the most important environmental transformations of the element, since they can produce organometallic species that are sufficiently stable to be mobile in air and water. In the natural estuarine environment, biomethylation by microbes occurs by different mechanisms depending on the pH conditions. Figure 5 shows the biomethylation of arsenic by S-adenosyl methionine functioning as the methyldonor in basic conditions, and methylcobalamin as the methyl-donor in acidic conditions (Wood, 1988). However, the biomethylated forms of arsenic produced are subject to oxidation and bacterial demethylation back to inorganic forms. It was ascertained that methylarsines were the toxic agents. Dimethylarsine [As(Ch,),H] is mainly produced by anaerobic organisms, while trimethylarsine [As(Ch,),] resulted from aerobic methylation. The probable mechanism for the methylation of arsenate to methanearsonates (McBride et al., 1978) is shown in Fig. 6. The
S. B. JONNALAGADDA and P. V. V. PRASADARAO
TRANSPORT
IN
TRANSPORT ARSENICAL
INDUSTRIAL SOURCES DUST I
I dry deposition precipitation
Ir 4
HERBICIDES
I I 1
4 uptake
+
I i
(Cl
dec y was R off
H2AsO;
Oxygen present Organic As w
+ 03,N2 0,
PLANTS
I
HAsO,
OUT
Oxygen
soil biomethylation
Fe (OH1 SO,,5
HAsO H,AsO,
I
I
-
HAS&----’
absent -)
H,S
H,AsO,,--_,HAsO,
--,As&etc...
4 1 Leaching
Fig. 4. Local air-air cycle of arsenic. Reproduced with permission from WHO (1981). proposed mechanism indicates that As(V) has to be reduced to As(M) before being methylated. Inorganic trivalent arsenic is systemically more poisonous than the pentavalent form and it is possible that pentavalent arsenic is reduced to the trivalent form before exerting any toxic effects. Organic arsenicals are also thought to exert their toxic effects in vivo in animals by first metabolizing to the trivalent arsenoxide form. It is thought that there is a blood-mammary barrier to arsenic, as evidenced
by several separate studies in which cows were fed monosodiummethane arsonate, lead arsenate and arsenic trioxide, without a significant increase of the arsenic content of their milk. The toxic effects of arsenic compounds in animals are thought to be mediated through a reaction with sulfhydryl groups of tissue proteins and enzymes (Fig. 7). Arseno compounds, (R-As=As-R) are readily oxidised, even by trace amounts of oxygen. Their action has been suggested to be due to their
5 Adenosylmethionine
S Adenosylhomocysteine
hneA&, HS -6
h
CH3
I
+3 +As CH,+
ICH,I
2e
eCH,As++ ‘\
a
KH,),As+ +e,+H+
1 KH,),A
Fig. 5. (a) Methylation of arsenic salts by S-adenosyl-methionine. synthesis
of methylarsenic
compounds.
H
(b) Mechanism for B,,-dependent
Toxicity, bioavailability and metal speciation
As~*O~~-
->
2e
591 2e
CHS' As3+033-
_ 0s-
> CIi 3Ass*0 3 *-
-
->
a-
<~ethyIarsonat~>”
cIi3+ CH3As”0
2e
->
2 *-
(CHS)SAs5*08
-> -0"
(Cacodylate) CH3* ->
(CH,),AsS+O
(CHs)sAs3*0 A> _0'-
(CH,),AsS*
Fig. 6. Methylation of arsenate to methanearsonates. conversion
to the corresponding
arsenoso
derivatives
(R-As---O). These compounds can be divided into mono-substituted and di-substituted according to their reaction with suhhydryl groups. The monosubstituted compounds, exemplified by R-As==O, react with enzymes containing sulfhydryl groups. Inhibition of different enzyme systems by these arsenic compounds was shown to be reversed by addition of an excess of a monothiol, e.g. glutathione. Some enzymes contain two thiol groups, which can react with the monosubstituted arsenic compound, thereby yielding a 5-membered ring structure. This is reversed by dithiols, e.g. 2,3-dimercaptopropanol (British antilewisite, BAL), but not by monothiols (WHO, 1981). Mahaffey and Fowler (1977) examined the effects of dietary cadmium and lead on the toxicity of arsenic administered to rats. They showed that the efficiency of food utilization was more reduced by the combination of arsenic and cadmium than by each metal alone. Selenium Selenium exists naturally in several oxidation states and some of its chemical forms are volatile. Selenium has similar chemical and physical properties as sulfur and tellurium. Many selenium analogues of organic sulfur compounds exist in nature. Selenium in the + 6 or selenate state is stable under both alkaline and oxidising conditions. Thus, in alkaline soils it is easily R-As=0
Protein
=
+ 2 R’SH
/
SH
\
SH
Protein
/
= R-As(SR’)s
+
s\ ’
available to plants. Selenium in the +4 state occurs naturally as selenite. In alkaline solution, it tends to oxidise slowly to the +6 state, if oxygen is present. It is readily reduced to elemental Se by a number of reducing agents. Selenium dioxide, an anhydride of selenious acid, sublimes at 317°C. This is important with regard to air pollution through the combustion. Selenite binds tightly to iron and aluminum oxides. Thus, it is quite insoluble in soils. In its -2 state, selenium exists as hydrogen selenide and in a number of metallic selenides. Hydrogen selenide at room temperature is a very toxic gas, a strong reducing agent and a relatively strong acid. In air, it decomposes rapidly to form Se and water. Figure 8 illustrates the biological cycle of selenium (WHO, 1987). In the estuarine environment, it is anticipated that the biomethylation of selenium will be a rapid process, and therefore most of the selenium in the sea is in the form of organoselenium compounds. This microbially mediated process has the potential to create a toxic local situation with respect to coastal marine life, if major anthropogenic inputs of selenium occur in estuaries. Of special interest to the reactivity and movement of the metalloids of arsenic and selenium, is the fact that inorganic species of these elements behave as electrophiles in acidic conditions and as nucleophiles at basic pHs. Figure 9 shows the biomethylation of selenium species by methylcobalamine (vitamin B,,) under acidic conditions (Wood, 1988).
Cl,
+ H,O
AsCH=CHCI
AsCH=CHCl
+
2HCl
s’
Fig. 7. Reaction of arseno compounds with sulfbydryl groups of proteins.
592
S. B. JONNALAGADDA
and P.V.V. PRASADA RAO
Fig. 8. Biological selenium cycle: well-established pathways are indicated by solid lines, those needing further substantiation by dotted lines. Reproduced with permission from WHO (1987).
The intestinal absorption of soluble selenium compounds by rats is highly efficient. It has been shown that these animals absorbed the doses of selenate (92%), selenomethionine (91%) and selenocystine (81%) (Thomson et al., 1975). Only limited information is available regarding the absorption of Se occurring naturally in foods by animals. Poor bioavailability of selenium to animals is reported with certain fish products containing selenium. Studies carried out on human volunteers showed that intestinal absorption of oral doses of selenium was 94% for selenate selenium, 97% for selenomethionine, and 60% for sodium selenite. Only 55% of selenium as it occurs naturally in fish was absorbed, when fish was consumed in diet (Thomson et al., 1986). Hydrogen selenide, which is a volatile compound, is found to be deposited in liver and kidney when inhaled. Sodium selenite and selenium oxychloride
Fig. 9. Synthesis of alkylselenium compounds from selenium salts.
are absorbed through the skin. The distribution of Se appears to be relatively independent of the form and route of administration. The accumulation in human organs was found to be in the following descending order in the tissues: kidney > liver > spleen > pancreas > testes > heart muscle > intestine > lung > brain (Schroeder et al., 1970). Under normal conditions, selenide is methylated to form trimethyl selenonium ion, the main urinary metabolite of selenium. In case of Se toxicity, this pathway is overloaded and dimethyl selenide is produced. These methylation reactions are considered detoxication steps, and the methylated end products are much less toxic for the organism than selenite (Obermeyer et al., 1971). However, both of these methylated selenium derivatives have strong synergistic toxicity with other minerals. Vitamin E deficiency increased the susceptibility of the rats to chronic poisoning by Se as selenite. Selenium poisoning caused by feeding rats seleniferous wheat was decreased by giving sodium arsenite. However, arsenic potentiates the toxicity of the trimethyl selenonium ion. Dietary sulfate partially counteracts the toxicity of selenate, but it has no effect against selenite or organic forms of selenium. The discovery that Se is a component of the enzyme glutathione peroxidase (Rotruck et al., 1973) provided a logical explanation for the nutritional interaction of vitamin E and selenium, a puzzle that perplexed scientists for many years. The estimated safe and adequate range of selenium intake in humans is shown in Table 4 (WHO, 1987).
Toxicity, bioavailability and metal speciation Table 4. Estimated safe and adequate range of selenium intake. Reproduced with permission from WHCl (1987) Group Infants Children
Adults
O-Q.5 0.5-I
Daily selenium intake ocg) IO-40 20-60
1-3 46 7+
20-80 30-120 S&200
Age (a)
5&200
a = years.
The ecological evidence strongly favours a relationship between low selenium status and the incidence of Keshan disease (an endemic cardiomyopathy) in children in China (Yang et al., 1984), and Kashin-Beck disease (an endemic osteoarthropathy) in children in China and Eastern Siberia (Sokoloff, 1985). When selenium and arsenic interact, arsenic has a protective effect against the toxicity of a variety of forms of selenium in several species. The most logical hypothesis is that arsenic combines with selenium by reacting with selonol (-SeH) groups to form a detoxification conjugate that passes readily into bile, enhancing the biliary excretion of selenium (Levander, 1977). A study carried out on mice demonstrated that arsenic(II1) was more efficient in protecting against selenium toxicity than arsenic(V). Mercury Various toxic effects of mercury compounds can be best understood by considering the speciation and the ways in which they are handled by the body. For this purpose the mercurial compounds may be divided into the following main groups: elemental mercury vapour, divalent inorganic mercury salts, short-chain alkyl and other organic compounds; and aryl and alkyl mercury compounds. Global transport, bioaccumulation, and transformation of inorganic
593
mercury arise from the conversion of mercury compounds into methylmercury as well as the exposure to methylmercury through sea-food and other foods. There is a well recognized global cycle for mercury, whereby emitted mercury vapour is converted to soluble forms and deposited by rain onto soil and water. Figure 10 illustrates the global cycle of mercury. Metallic mercury is oxidised in water to the divalent mercuric ion Hg’+. Much of this precipitated as insoluble mercuric sulphide, especially in anaerobic environments, becomes oxidised to sulfate, releasing mercuric ion again. Despite the uncertainties concerning speciation, the global cycle of mercury is believed to involve the inorganic forms almost exclusively. Mercuric salts and organic mercury are readily taken up by organisms in water. The change in speciation of mercury from inorganic to methylated forms is the first step in the aquatic bioaccumulation process. Methylation can occur non-enzymatically or through microbial action. Aquatic organisms and fish take up the metal and retain it in the tissues, as methylmercury, although most of the environmental mercury to which they are exposed is inorganic. Table 5 summarises the methylmercury levels in the muscle tissue of various species of fish in different regions (WHO, 1990). Environmental levels of methylmercury depend upon the balance between bacterial methylation and demethylation. There is little information that fish themselves either methylate or demethylate mercury. Methylmercury is efficiently absorbed by all living organisms due to its great affinity for sulfhydryl groups. Most of it becomes protein bound and very little is excreted. As a result of food-chain biomagnification, highest levels are found in the tissues of such predatory species as fresh water trout, pike, bass and ocean tuna, swordfish, and shark. The bioconcentration factor, i.e. the ratio of the concentration of
‘-4s
BOTTOM SEDIMENT (20x lo-6g/kgI
Fig. IO. The global cycle of mercury. Reproduced with permission from WHO (1990).
S. B. JONNALAGADDA
594
and P. V. V. PRASADA
Uo
Table5.The range of published average values of methylmercury (mg mercury/kg wet weight) in the muscle tissue of various species of fish. Reproduced with permission from WHO (1990) Species Non-predators Mackerel Sardine Unspecified Number of edible species Predators Tuna Swordfish Shark, ray, do&h
Atlantic ocean
Pacific Ocean
Indian Ocean
Mediterranean Ocean
0.07-0.2 0.03-0.06
0.16-0.25 0.03
0.005 0.006
0.24 0.15
0.08-0.27
0.07-0.09
0.02-0.16
0.1-0.3
0.03-0.8 0.8-1.3
0.3 1.6
0.064-6.4 -
1.2 1.8
1.0
0.7-I. 1
0.094-1.5
1.8
methylmercury in fish tissue to that in water, is usually between 10,000 and 100,000 (U.S. EPA, 1990). There is no evidence in the literature for the synthesis of organomercury compounds in human and mammalian tissues (WHO, 1976). The conversion of methylmercury to inorganic mercury is considered a key step in the process of excretion of mercury after exposure to methylmercury (WHO, 1990). Mercury vapour is more soluble in plasma, whole blood and haemoglobin than in distilled water (Hursh, 1985) and it readily crosses the blood-brain barrier. The oxidation of metallic mercury vapour to divalent ionic mercury is not fast enough to prevent the passage of elemental mercury through the bloodbrain barrier, the placenta, and other tissues. The oxidation serves as a trap to hold the mercury and leads to accumulation in brain and foetal tissues (WHO, 1976). The neurotoxic effect seen after exposure to metallic mercury vapour is attributable to the divalent mercury ion formed through oxidation in the brain tissue. Results of both human and animal studies indicate that about 80% of inhaled metallic mercury vapour is retained by the body, whereas liquid mercury is poorly absorbed via the gastrointestinal tract. Inhaled inorganic mercury aerosols are deposited in the respiratory tract and absorbed, the rate depending on the particle size (Hursh et al., 1976). The reduction of divalent mercury to metallic mercury has been demonstrated both in animals (mice and rats) and humans (Sugata et al., 1979). Inorganic mercury compounds are probably absorbed from the human gastrointestinai tract to a
level of less than 10%. The kidney is the main depository of mercury after the administration of elemental mercury vapour or inorganic mercury compounds (SO-90% of the body burden of animals). In humans, methylmercury in the diet is almost completely absorbed into the blood stream and is distributed to all tissues in about 4 days (Kershaw et al., 1980). ~ethylmercury is converted to inorganic mercury, assumed to be Hg2+ in mammals. The nervous system is the principal target tissue for the effects of methylmercury on adult human beings. Renal damage is one of the most frequently described non-neural effects of methylmercury. The kidney is the critical organ following the ingestion of inorganic bivalent mercury salts. It has been estimated that humans have a daily intake of about 2.4pg methylmercury from all sources. The total daily intake of all forms of mercury from ail sources has been estimated to be about 6.7 pg. The average daily intake and retention of total mercury and mercury compounds in the general population not occupationally exposed to mercury is shown in Table 6. Metals have been used by mankind for thousands of years, but many of the most useful metals are toxic, and the price of their utility has often been high. The increased demand for metals of all kinds that followed the Industrial Revolution was accompanied by the appearance of metal-induced occupational diseases on a large scale. Even the so called essential elements are also characterized by a surprisingly narrow range of optimal activity. A scant 4-5 fold change in concentration is su~cient to convert overt
Table 6. Estimated average daily intake and retention (pg/day) of total mercury and mercury compounds in the general population not occupationally exposed to mercury. Reproduced with permission from WHO (1990) Exposure Air Food Fish Non-fish Drinking water Dental amalgams Total
Elemental mercury vapour
Inorganic mercury com~unds
Methylme~ury
0.030 (0.024)
0.002 (0.001)
0.008 (0.0064)
0 0
0.600 (0.042) 3.6 (0.25) 0.050 (0.0035) 0 4.3 (0.3)
2.4 (2.3) 0 0 0 2.41 (2.31)
3.8%ZP(317) 3.9-21 (3.1-17)
Figures in parentheses represent the estimated amount retained in the body of an adult.
Toxicity, , ,I 1Swvival $!eficiency
,IToxicity
Optimal
bioavailability ;Letholity
i
(lJ---~ ESSENTIAL
)1g/d -10 “9
/d -.5
ELEMENT.
DOSE
-50
-Se-200-103-10~ -
-2
-
- F -
P
10 -20
-100
Fig. 11.Dose-response relationship for an essential element. Estimates for specific daily requirements are in pg/day for selenium and in mg/day for fluorine.
signs of deficiency to overt signs of toxicity. A further change of ltss than an order of magnitude in either direction is sufficient to cause death. Figure 11 illustrates the dose-response relationship of essential elements, selenium and fluorine. In general, toxicity is usually caused by a few specific chemical species. The effect is further complicated by other metals and ligands in the environment, requiring a multi-variable approach. One must consider the entire exposure process, including other constituents that accompany the element of interest, which can interact with the element and alter its bioavailability or toxicity. Further, the oriental approach to the treatment of illness employing the natural products, i.e. the concoctions and decoction containing various species, inclusive of certain toxic trace elements, provokes thoughts towards the importance of the speciation and interactions of various elements; and their bioavailability and toxicity. REFERENCES
Casarett L. J. (1980) The Basic Science of Poisons In Casarett and Doull’s Toxicology. (Edited by Doull J., Klassen C. D. and Amdur M. 0.). 2nd Edn., Chap. 3, p. 39. Macmillan Pub. Co., Inc., New York. Chaney R. L. (1988) Metal Speciation: Theory, Analysis and Applicafion (Edited by Kramer J. R. and Allen H. E.), pp. 219-260. Lewis Publishers, Inc., Chelsea, U.S.A. Eisler R. (1985) Cadmium hazards to fish, wildlife and invertebrates: A synoptic review, Washington, DC, U.S. Dept of the Interior, Fish and Wildlife Service, Biological Report No. 85. Flanagan P. R., McLellan J. S., Haist J., Cherian G., Chamberlain M. J., and Valberg L. S. (1978). Increased Dietary Cadmium Absorption in Mice and Human Subjects with Iron Deficiency. Gastroenterology 74, 841-846. Hursh J. B., Clarkson T. W., Cherian M. G., Vostal J. V., and Mallie R. V. (1976) Clearance of mercury (Hg-197, Hg-203) vapor inhaled by human subjects. Archs. envir. Health. 31, 302-309. Hursh J. B. (1985) Particle coefficients of mercury (203 Hg) vapour between air and biological fluids. J. appl. Toxic. 5, 327-332. Kershew K. G., Clarkson T. W. and Dhahir P. H. (1980) The relationship between blood levels and dose
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595
of methylmercury in man. Archs. envir. Healih 35, 28-36. Lamphere D. N., Dorn C. R., Reddy C. S. and Meyer A. W. (1984) Reduced Cadmium body burden in cadmiumexposed calves fed supplemental zinc. Envir. Res 33, 119-124. Levander 0. A. (1977) Metabolic interrelationships between arsenic and selenium. Envir. Health Perspect. 19, 159-164. McBride B. C., Merilees H., Cullen W. R. and Pickett W. (1978) Anaerobic and aerobic alkylation of arsenic. In Organometals and Organomeralloids (Edited by Brinckman F. E. and Bellama J. M.), pp. 94-115, ACS symp. Ser. 82. Am. Chem. Sot., Washington, DC. Mahaffey K. R. and Fowler B. A. (1977) Effects of concurrent administration of lead, cadmium and arsenic in the rat. Envir. Health Perspect. 19, 165-171. Nordberg M., Nuottaniemi I., Cherian M. G., Nordberg G. F., Kjellstrom T. and Garvey J. S. (1986) Characterization studies on the cadmium binding proteins form two species of New Zealand Oysters. Envir. Health Perspect. 65, 57-62. Obermeyer B. D., Palmer I. S., Olson 0. E. and Halverson A. W. (1971) Toxicity of trimethylselenonium chloride in the rat with and without arsenite. Toxic. appl. Pharmac 20, 135-146. Rotruck J. T., Pope A. L., Ganther H. E., Swanson A. B., Hafeman D. G. and Hoekstra W. G. (1973) Selenium: biological role as a component of glutathione peroxidase. Science 179, 588-590. Schroeder H. A., Frost D. V. and Balassa J. J. (1970) Essential trace metals in man: Selenium. J. chron. Dis. 23, 227-243. Sokoloff I. (1985) Endemic form of osteoarthritis. Clin. rheum. Dis. 11, 187-202. Sugata Y. and Clarkson T. W. (1979) Exhalation of mercury-Further evidence for an oxidation-reduction cycle in mammalian tissues. Biochem. Pharmac 28, 3474-3476. Thomson C. D., Robinson B. A., Stewart R. D. H. and Robinson M. F. (1975) Metabolic studies of 75Se selenocystine and ‘?3e selenomethionine in the rat. Br. J. Nutr. 34, 501-509. Thomson C. D. and Robinson M. F. (1986) Urinary and faecal excretions and absorption of a large supplement of selenium as selenite or as selenate. Am. J. C/in. Nutr. 42, 1015-1022. U.S. EPA (1990) Ambient Water Quality Criteria for Mercury. Criteria and Standards Division (EPA-600/479049), Washington, DC, U.S.A. WHO (World Health Organization) (1976) Mercury (Environmental Health Criteria l), p. 131. WHO (World Health Organization) (1981) Arsenic (Environmental Health Criteria 18) Geneva. WHO (World Health Organization) (1987) Selenium (Environmental Health Criteria 58). WHO (World Health Organization) (1990) Methylmercury (Environmental Health Criteria 101). WHO (World Health Organization) (1992) CadmiumEnvironmenfal aspects (Environmental Health Criteria 135). Wood J. M. (1988) Transport, bioaccumulation and toxicity of metals and metalloids in microorganisms under environmental stress. In Metal Speciation, Theory, Analysis and Application (Edited by Kramer J. R. and Allen H. E.), pp. 295-314. Lewis Publishers, Chelsea, Michigan. Yang G., Chen J., Wen Z., Ge K., Zhu L., and Chen X. (1984) The role of selenium in Keshan disease. In Advances in Nufritional Research (Edited by Draper H. H.), pp. 203-231. Plenum Press, New York.