.
Ralph A. Zingaro Texas A & M University College Station, Tex. 77843 “In general, a very minor and probably insignificant redistribution of fluorine in the environment occurs due to industrialization, burning of coal, fertilization, fluoridation of water supplies, and so on, but this does not constitute in any way a danger to man.” This was the conclusion presented by Leon Singer at the Symposium on Some Recent Developments 282
Environmental Science & Technology
in the Bio- and Environmental Chemistry of Some Trace Elements. The symposium was scheduled as part of the 34th Southwest Regional Meeting of the American Chemical Society (ACS), held in Corpus Christi, Tex., November 29-December I , 1978. In addition to Dr. Singer’s presentation on fluorine, other speakers included Howard Ganther, University of Wisconsin (selenium), Fred Brinckman, the National Bureau of Standards (NBS, tin), Arthur Martell, Texas A & M University (transition
.
.
metals), and Kurt Irgolic and Ralph Zingaro (arsenic). The most obvious message to emerge from this meeting was the need for accurate and reliable analytical techniques. Hence, the speakers directed a considerable portion of their presentations to recent developments in their laboratories, which involved analytical procedures. The symposium was structured primarily to inform and educate the audience on the current status of the environmental and biochemical status of these trace elements. The emphasis was not placed on
0013-936X/79/0913-0282$01 .OO/O
@ 1979 American Chemical Society
the highly technical aspects of the research by the particular investigators. Fluorine This element, in the form of the fluoride, is most ubiquitous in nature. In some minerals, such as calcium apatites, and in magnesium and iron deposits, the concentrations are high. In soft animal tissues, body fluids and plant materials. fluoride is present in very low concentrations, less than 0.2 PPm. A very minor and probably insignificant redistribution of fluoride has occurred in the environment, because of the industrialization of our nation. This is attributed principally to the production of metals for a variety of uses, and to the burning of coal for energy. A lesser redistribution can be attributed to the extensive fluoridation of municipal water supplies, which often requires, for good oral health, the addition of substantial amounts of fluoride to drinking water. Such drinking water contains less fluoride than optimum ( 1 .O ppm). Other sources are the use of fluoride-containing substances, such as toothpastes and mouth rinses, which, in part, end up in the sewage system for disposal; the extensive use of phosphate fertilizers for improved plant growth; the use of mineral mixes as feed supplements for animals; and the recycling of waste food products, such as bone, in the food chain. Because fluoride has as affinity for bone, it is obvious that products containing bone will, in most instances, increase the fluoride intake. The foregoing lists some of the major reasons why a certain redistribution of fluoride in the environment has taken place. Assertions have been made that the fluoride content of liquid effluent discharged into streams from sewage plants might have a deleterious effect on fish and other life. Thus, Singer, along with W. D. Armstrong, felt that it was important to determine the fate of fluoride in a sewage treatment plant. After all, a large fraction of fluoridated water, which contains approximately one ppm of fluoride, along with the fluoride in the rain and melted snow that enters storm sewers, ultimately reaches the sanitary sewage system. For instance, the liquid effluent from the sewage plant in Minneapolis-St. Paul, Minn., serving the Twin Cities, contained 1.21 ppm fluoride, which was higher than the 1.0 ppm which occurred in the fluoridated water supply of the communities. Discharge of the effluent into the Mississippi River did not greatly in-
crease the fluoride content of the river water. The semi-solid waste from the sewage treatment, which contained over 200 parts per million (ppm) of fluoride, released little fluoride to the atmosphere when burned. However, rain and snow were found to contain detectable quantities of fluoride which did contribute to the fluoride content of sewage. This contaminant fluoride obviously came from environmental sources.
Selenium Some dramatic recent developments have contributed greatly to the understanding of the physiological role of selenium a t the molecular level. The nature of these recent discoveries was described by Howard Ganther of the Department of Nutritional Sciences, University of Wisconsin. The element attracted considerable attention during the 1930’s, when it was discovered to be a natural toxicant. These early studies dealt primarily with the poisoning of livestock in South Dakota and adjacent areas, when animals fed on selenium accumulator plants. However, in the 1950’s, pioneering studies, attributed primarily to the late Klaus Schwartz, identified selenium as an essential trace element for animals. A biochemical role for the element was not established until the early 1970’s, as a result of studies on erythrocyte glutathione peroxidase, which culminated in its identification as a selenium-containing protein in 1973. Two additional microbial proteins containing stoichiometric amounts of selenium have also been isolated; they are protein A of the glycine reductase complex, and formate dehydrogenase. Although these proteins were known before 1970, they had not been recognized as selenoproteins. Glutathione peroxidase uses glutathione to reduce hydrogen peroxide and organic hydroperoxides to less harmful products. Thus, a broad role for this enzyme in protecting tissues from oxidative damage is apparent, and its close nutritional interrelationships with vitamin E, sulfur amino acids, and antioxidants are understandable. Glutathione peroxidase could also have an important function in the metabolism of hydroperoxides that are normal intermediates in biosynthetic reactions, such as in the synthesis of various prostaglandin derivatives from arachidonic acid. In view of the great biological activity of prostaglandins and related compounds, a role for selenium in such processes might provide a different approach to interpreting the
role of selenium in nutritional diseases beyond the classic, but poorly focused theory of a role in controlling lipid peroxidation leading to pathological changes. It is also important to note that tissues contain an additional glutathione peroxidase which acts on organic hydroperoxides (but not hydrogen peroxide), and does not contain selenium. This peroxidase activity has been shown to be identical with the glutathione transferases-enzymes well known for their role in catalyzing the conjugation of glutathione with various organic compounds to form thio ethers and other products. Selenocysteine, the selenium analog of cysteine, has been reported to represent the chemical form of selenium in the reduced selenoprotein of the glycine reductase complex, and in glutathione peroxidase. The evidence is largely based on co-chromatography of alkylated derivatives, and has not yet been confirmed through use of more conclusive techniques, such as mass spectrometry. The close relationship which exists between selenium biochemistry and sulfur biochemistry is further apparent in a glutathione-dependent pathway for reduction of selenium to hydrogen selenide (H2Se). This pathway, beginning with inorganic salts such as sodium selenite, proceeds by a reaction with glutathione to form 2-selena, 1,3-dithia derivatives, commonly referred to as “selenotrisulfide” (GSSeSG). This in turn, is reduced enzymatically by N A D P H and glutathione reductase in two steps, forming first the labile selenopersulfide, GSSeH, and followed by further reduction to H2Se. The formation of H2Se and other selenols may explain why heavy metals and selenium sometimes accumulate together at high levels in the liver, apparently in a nontoxic form, as in certain marine mammals, or in cats fed tuna. The dietary selenium intake for most Americans is probably adequate, and no cases of Se deficiency in humans are recorded. However, certain highly purified diets used therapeutically, as in intravenous feeding, are based on pure amino acids rather than proteins or protein hydrolyzates. These diets are exceedingly low in Se, and could conceivably cause problems if they are used as the sole source of nutrients over extended periods of time.
Tin Man has been involved with tin since ancient times. Tin bronzes were exploited by ancient Sumerians for tools and weapons 2000 years before Volume 13, Number 3, March 1979
283
properties as catalysts, polymer stabilizers, and biocides, now find ubiquitous use and worldwide distribution in many important materials. These incorporants, usually present in small concentrations, can be expected to exhibit some of the tendencies of organotins for ready uptake in plants and animals, depending upon specific end-uses of these.materials, and their refractory behavior in the environment. Such trends for both metallurgical and organotins, coupled with heightened awareness of environmental consequences of man’s activities, makes this topic timely and challenging. Underlying changing perceptions of tin’s active role in biological processes is the greater overall recognition that many heavy metals, hitherto regarded as either toxic or inert, can participate in numerous biotransformations. During the past three decades, arsenic, selenium, tellurium, lead, and thallium, as well as tin, have been implicated in environmental biomethylations, to produce lipophilic or volatile metabolites. Clearly, a new outline for biogeochemical cycles of so-called “non-essential” elements is emerging
Egyptian pewterers were busily fabricating household objects of this metal in 1500 B.C. The metal is widely dispersed and diluted in the lithosphere, typically about 2-10 ppm in rocks and soils. Local accretions of its familiar oxide ores provided then, as now, access to the ductile, low-melting metal, by simple thermal reduction techniques. In the United States, where the per-capita consumption of beverage cans exceeds 190/year, tin-plate is used for foodstuff preservation in over half of the cans produced. Conventional experience suggests that tin in its insoluble or metallic forms, such as that found in rocks or food cans, offers very limited bioavailability, and that such widespread direct human contact results in little adverse uptake. However, it is currently recognized that urban sewage sludges-representing gross “indicators” of heavy metal pollution and biocycling-contain unusually high tin concentrations (about 1 1 1-492 ppm, dry weight). Information is lacking on the chemical forms, or bioavailability, of these anthropogenic sources. Organotins displaying many useful FIGURE 1
How biological and chemical transformation8 form methylmetal(loid)s Men M(o)
(Figure 1). The studies of Schwarz in 1970 gave researchers a clue to the essentiality of tin in mammalian metabolism. Other clues to possible floral or animal uptake pathways involving bi-elemental synergisms, including that between tin and mercury in aquatic plants, have also been discussed.
Organotin uses It is important to recognize that organotins offer great potential for specific biological applications in the materials preservation, agriculture, and health fields. Their biocidal properties have been long known: toxic responses of microbiota, insects, or animals are well documented. For a given R group, mammalian toxicity increases in the order RSn3+ < R2Sn2+ < R4Sn R3Sn+, with tetraorganotins’ toxicity dependent mainly upon rates of metabolic conversion to R3Sn+. In contrast, for a given structure, such as R3Sn+, animals show decreasing toxic responses to R = ethyl > methyl propyl > butyl > phenyl, although dosages are found to be species-dependent. Also, iso-alkyl derivatives are more lethal than their normal-alkyl isomers. Nearly an inverse order of lethality for R3Sn+ agents is observed for microbiota, such as fungi and bacteria. Typically, trace amounts of these organotins dissolved in aqueous saline cellular media process ionic properties. Their toxicities are relatively unaffected by presence of different labile anionic ligands, such as chloride or hydroxide. In addition, the aquatic organotins [R,Sn(OH2),](4-n)+, are highly solvated (n m = 5 or 6) and involatile, and they show strong lipophilic properties. Dramatic recent successes in synthetic organotin chemistry imply that a very great range of highly organism-specific biological applications could be available through appropriate tailoring of steric and constitutional features of commercial organotin biocides. Evidence to date also suggests that residues from such applications should also be localized in use, and non-refractory to eventual Sn-C cleavages, in order to provide environmentally compatible materials. Molecular design and use of specifically active organotin biocides are presently limited by several factors, such as: development of storage and release matrices for controlled release of organotin biocides into environmental media at very low concentrations development of ultra-trace (ppb or less) speciation techniques per-
-
+
?--/if
/
M q - , M’
t
MeCl
\ \
“M%M+ Particulates
Me,E‘
Water column
1
Me,_, M’T Me,,,-,E
/ P
MhE
T
MeCl
’
0 Gases
O0
O
0
0
0
For example, M might be Sn(lV), and E might be Hg(ll), or other metals, such as Pb(IV), and TI(III), respectively
284
Environmental Science & Technology
mitting accurate definition of amounts and molecular features of released agents development of bioassay techniques, in order to provide options for improved molecular tailoring and increased environmental regulation. Much progress has been made, lately, by chemically incorporating selected trialkyltin moieties into both organic and inorganic polymers. Controlled release systems are thereby produced, which can be blended into surface coatings, to give prolonged resistance to marine fouling or biodeterioration. These release systems can also act as specific long-term inhibitors to reproduction of aquatic snails which host the dread Schistosomiasis disease which afflicts perhaps 350 million people in the Earth’s equatorial belt. The latter application represents a very cheap and simple mode of field application in those areas of the world incapable of the primary science, but greatly in need of the product. Future advances are expected to result from new understanding of the coordination chemistry of di- and trialkyltin functions of biologically critical sites, particularly those protein or enzyme components bearing nitrogen or sulfur donor atoms.
Experiments at NBS Brinckman described some of these activities a t the National Bureau of Standards (NBS) laboratories. Studies there have focused on developing speciation methods (Figure 2) for trace bioactive organometallic molecules in environmental media; establishing a descriptive aquatic organometallic chemistry with emphasis on reaction mechanisms; and providing a qualitative survey of the extent and kinds of metal transformations occurring in microbiota, which involve organometallic species. Several years ago, these laboratories reported on the biomethylation of Sn (IV) by a prevalent marine species of Pseudomonas, which had earlier been found to reduce mercuric ion, exclusively, to gaseous Hgo. In separate studies, this group observed that the hydrated trimethyltin cation was capable of abiotically transferring CH3 to aqueous Hg2+ a t a rate competitive with biomethylation of tin. These observations led to critical experiments in which it was shown that Pseudomonas sp., metabolizing under dual stress of both Sn4+and Hg2+, yielded not only HgO, but CH3Hgf as well. This reaction required intermediacy of the biogenic CH3-Sn metabolite. The prospect of several bioactive
metal ions interacting both biologically and abiotically to generate organometallic metabolites cannot be regarded as an unusual process, although so far, only methylation appears to be involved. Several exo-cellular metabolites capable of methylating metal and metalloid ions have been suggested, but the best known is methylcobalamin (a vitamin B-12 derivative), in which the methylcobalt bond readily methylates a number of main-group and transition metal ions, including tin. The ubiquity of such microbiological processes in the biosphere suggests that under some circumstances, even inorganic or metallurgical forms of tin may be available to a methylation “pool,” for example, through solubilization by biogenic methylcorrinoids. It would appear equally likely that anthropogenic organotins would also find entry into this “pool,” although many abiotic degradative reactions might intervene. More detailed experiments are needed to reveal, convincingly, whether or not biomethylation of tin is an important environmental pathway. Brinckman reported on the workof
R. Braman at the University of South Florida, which describes widespread distribution of methyltins at extremely low concentrations in diverse environmental media. Braman finds that the former range about 2.5-13.5 ng/L in seawater to rain water, and comprise slightly less than half of the total tin detected. These observations are very important for assessing the overall biogeochemical tin cycle, particularly with the goal of establishing baseline data permitting differentiation between natural and anthropogenic fluxes. Perhaps, the thrust of environmental chemistry in the Seventies might be summarized as a combination of advances in trace element detection with “clean room” protocols. These accomplishments would go hand-in-hand with a broadening vista of aqueous organometallic chemistry coupled with microbiology, to focus on questions of bioavailability, transformations, and flux of industrial metals. A central problem remains to ensure that a data base of sufficient scope and reliability emerges to allow examination of those environmental models which will difVolume 13, Number 3,March 1979
285
ferentiate between planetary and man-made stresses and alterations of bioactive metals. This is especially true of tin, because of its wide distribution in nature upon which man’s technology places ever-increasing burdens.
Arsenic The conversion of inorganic and certain organic arsenic compounds by organisms, such as Pseudomonas brevicaule and methanobacterium, to organic arsines of the general formula R,AsH3., (n = 1, 2, 3) is now a wellestablished fact. Although some of these conversions have been known for almost one hundred years, detailed mechanisms for these reactions are still not available. Arsenic has a complicated chemistry. It forms a great variety of organic and inorganic compounds whose effects on living organisms vary greatly. It is, therefore, not surprising that organic arsenic compounds, other than simple arsines, have been detected in various organisms. For example, Lunde found organic arsenic derivatives in the lipids of marine and limnetic organisms. Acid hydrolysis of the arsenic-containing lipid fractions produced water soluble, organic arsenic compounds. This is documented by the isolation of arsenobetaine from rock lobsters, as reported in 1977. These findings clearly demonstrate that transformations, much more complex than simple methylation, are undergone by arsenic compounds in biological systems. In order to identify the organic arsenic compounds, which are known to occur in many marine organisms, the
marine alga Tetraselmis chuii, a green flagellate (Chlorophyta) was grown on a large scale in “Instant Ocean”” medium, in the presence of 10 ppm As (arsenate). Extraction of the harvested cells with chloroform/methanol revealed that arsenic occurs both in the extract and in the extraction residue. The arsenic in the extraction residue, together with proteinaceous water, was dissolved in water. Addition of methanol precipitated an arsenic-protein complex. Exchange with 74As-arsenic with non-radioactive arsenate occurred only after an excess of arsenate had been added. The nature of the arsenic-protein complex is not known at this time. The lipid-containing chloroform layer was freed from green pigments by chromatography. A phospholipid fraction with an arsenic content of OS%, which contained two arsenic compounds, was isolated.
A new technique The detection, identification, and quantification of these organic arsenic compounds are being greatly simplified by a new analytical technique which combines high-pressure liquid chromatography (HPLC) with a Hitachi-Graphite Furnace-Zeeman Atomic Absorption Spectrophotometer as an element-specificdetector. The technique, being developed at Texas A & M University, does not require a chemical modification of the arsenic compounds for detection and quantification. The excellent resolving power of HPLC, the wide choices in column materials and mobile phases, and the great sensitivity of the Zeeman atomic
Detection. This apparatus is being used to determine arsenic compounds 286
Environmental Science 8 Technology
absorption detector produce a signal only when an arsenic compound is eluted. The fractions containing arsenic compounds need not be pure for their detection. They may contain other, arsenic-free materials, which in most cases will not affect the performance of the arsenic-specific detector. Such an analysis and detection system is ideal for the speciation of arsenic compounds, and other compounds containing elements that can be detected bv atomic absomtion smctrometry. The HPLC-graphite furnace atomic absorption spectrophotometer system has been automated through the development of an electronic interface. This system has been used to detect organic arsenic compounds in phospholipids, to effect the separation of inorganic arsenic, arsenobetaine and arsenocholine, and to detect, simultaneously, arsenate, arsenite, methylarsonic acid and dimethylarsinic acid.
Transition metals Arthur Martell, who has a long and distinguished career in transition metal chemistry, addressed the subject of essential transition metals. He pointed out that all living systems require the elements H, C, N, 0, Mg, P, S , Ca, Na, K, CI, Mn, Fe, Cu, Zn, and Mo. The number of species requiring the remainder of the elements thought to be essential ranges widely; however, barium, for example, has been shown to be required only by one species (the rhizopod Zenophyophora). The first eleven elements referred to are present in living systems in relatively large quantities, and are designated as major elements. The remainder come under the category of trace elements, with iron representing a borderline case. The recent demonstration of the essentiality of nickel means that all of the first-row transition metals, except for Ti, are considered essential. The latter has been found to be stimulatory, and its essential nature may possibly be demonstrated in the future. The closely related non-transition metals Zn and Cd, as well as Mo, a second-row transition metal, are also essential trace metals. Since the nutritional requirement of an essential metal must be based on vital biological functions, demonstration of trace metal requirements should be followed up by studies of the molecular basis for biological activity. The functions of many transition metals in biological systems have been known for some time, and are well established; for example, the activation of various enzymes by Ma, Fe, Co, Cu,
TABLE 1
Biological functions and toxicities of essential trace metals TranMlon
metal
V
Cr Mn
Fe
co Ni
cu Zn a
Biological ?unction
(Oxidation-reduction enzyme) (Glucose tolerance factor) Pyruvate oxidase Xanthine oxidase Cytochrome c Ferridoxin O2 transport and storage Vitamin BI2 coenzyme Urease Phenol oxidase Cytochrome oxidase Alkaline phosphatase Carboxypeptidase Carbonic anhydrase
Toxlc level effects
I,B
C,I
ID C,I
CD.1 C,I DJ,M C,I
Aldolase Alcohol dehydrogenase MO
Xanthine oxidase
Cd a
I,B
C,D,I,B,M
Not a transition metal. C = carcinogenic; B = blocking of essential enzymes: I = interference with regulatory mechanism: vital organ OT metabolic function; D = interaction wlth DNA, RNA polymeraSe. and so on; M = interference with membrane function.
and Zn, and the role of Fe in oxygen transport and electron transfer.
When it’s good or bad Examples of the biological functions of essential transition metals, and indications of their toxic effects are given in Table 1. Specific enzyme systems are cited in cases in which metalloenzymes and metal-activated enzyme systems have been identified. In the case of V and Cr, enzyme functions have been reported, but specific enzymes have yet. to be isolated. Cd is frequently listed as essential, but has not yet been assigned an essential function. Of the metals listed in Table 1, Cr, Zn, and Cu display the widest separations between beneficial and toxic levels. In the case of Mn, there are efficient detoxification mechanisms which prevent toxic accumulations. A very low spread between toxic and beneficial dosages is observed for Cd and V. Ni is extremely toxic and carcinogenic at high levels, but is rendered relatively harmless in the diet because of low absorption through the gut, as the result of the lack of efficient transport mechanisms. Threshold vs. “one-hit” Of the ten essential metals listed in Table 1, six are known carcinogens when present in mammalian systems in sufficiently large amounts. In some cases, such as Cd and Ni, only relatively small doses are carcinogenic. This dual behavior, in which a trace
element is beneficial or essential for biological function a t low levels and carcinogenic a t higher concentration, is a phenomenon common to many other (non-metal) carcinogens. Such behavior strongly supports the threshold concept of chemical carcinogenic activity, and directly counters the “single-event’’ or “one-hit’’ hypothesis that has been translated into law, in the form of the Delaney amendment to the Food and Drug Act. The Delaney amendment continues to be greatly controversial. Nitrilotriacetic acid (NTA) is another example of a compound of extremely low toxicity, which seems to cause tumors when massive doses are administered to experimental animals over long periods of time under conditions such that essential physiological functions are extensively altered. One of the earlier concerns about this ligand and its potential large-scale use in detergents was that it would find its way through sewage effluents into drinking water, and rearrange trace metal levels in the environment and in the body. Although its use as a detergent builder has been prevented by a Federally-sponsored “voluntary” ban in the U.S., it is used extensively for that purpose in Canada and elsewhere, so that evaluation of its effect on the environment is now possible. As it turns out, the high biodegradability of N T A lowers its level to a few parts per billion in some drinking water, and to undetectable levels in other waters (such as Lake Ontario).
Model calculations of N T A speciation in natural waters and in test-animal feed experiments have been done. The results show that N T A is always present in the environment in the form of its metal chelates. At normal environmental levels, as in Canadian waters, N T A occurs mainly as chelates of strongly coordinating transition metals. At abnormally higher ( 1 OOX) levels it is primarily bound to alkaline earth ions. In animal feeding experiments, however, competition with phosphates and natural chelating ligands in the feed result in nearly all the N T A being in the free state (alkali metal salts). Since the properties and biological effects of ligands are highly dependent on the metal ions to which they are bound, these results demonstrate the futility of testing environmental NTA chelates for carcinogenicity by the animal feeding experiments of the type generally employed.
Acknowledgment The financial assistance in support of this Symposium furnished by the Magcobar Division; Dresser Industries, Houston, Texas, is gratefully acknowledged.
Additional reading Occurrence and Fate of Organometals and Organometalloids in the Environment, American Chemical Society Symposium Series 82, Washington, D.C. (1978). Proceedings of the 5th International Symposium on Controlled Release of Bioactive Materials, Nat. Bur. Stand., Gaithersburg, Md., August, 1978. Trace Element Metabolism in Animals, W. R. Hoekstra, J. W. Suttie, H. E. Ganther, and W. Mertz, Eds., University Park Press, Baltimore, Md., 1974. Addison, A. W., Cullen, W. R., Dolphin, D., and James, B. R., Biological Aspects of Inorganic Chemistry, Wiley-Interscience, New York, N.Y., 1977.
Ralph A. Zingaro is professor of Chemistry of Texas A & M Unicersity and remains actiue in all aspects of selenium and arsenic chemistry, an area in which he has been inuolced f o r 25 years. He is a member of the subcommittee on arsenic of the Committee on Medical and Biologic E f fects of Environmental Pollutants of the National Academy of Sciences. Coordinated by J J Volume 13, Number 3, March 1979
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