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Biologic Effects of Arsenic on Plants and Animals Arsenic has long held a position of ambiguity with regard to its activity in biologic systems. In spite of the recognized toxicity of many forms of arsenic, various arsenicals have been used in the practice of medicine. A specific nutritional role for inorganic arsenic has been uncovered only recently, but animal feeds have been supplemented with "growth-promoting" organic arsenical additives for many years. Another curious feature of arsenic biochemistry is the ability of the element partially to counteract the ill effects of yet another toxic substance, selenium. This chapter summarizes what is known about the detrimental and beneficial effects of arsenic on living systems other than man and discusses in as much detail as appropriate the molecular mechanisms responsible for these effects. Previous reviews have dealt with the toxicology, i09 general biochemistry 8~4 8~5 and pharmacology of arsenic. MICROORGANISMS Toxicity The state of knowledge regarding the effects of arsenic on microor- ganisms was summarized very well in a study by Mandel et al.5~7 on the 117
118 ARSENIC action of arsenic on Bacillus cereus. Trivalent sodium arsenite was found to inhibit growth at a lower concentration (0.4 mM) than penta- valent sodium arsenate (10 rnM). The toxicity of the arsenate could be increased by lowering the phosphate concentration of the growth medium, whereas the inhibitory effect of arsenite was independent of phosphate concentration. This inverse relationship between the toxic- ity of arsenate and the concentration of phosphate might be related to the fact that arsenate can compete with phosphate for transport. However, Da Costai93 found that phosphate could suppress the inhibi- tory effects of arsenite, as well as arsenate, on the growth of fungi. Mandel et al.5~7 showed that neither arsenate nor arsenite produced any specific effects in B. cereus on the incorporation of radioactive precursors into ribonucleic and deoxyribonucleic acids, proteins, or cell wall. Radioarsenite was bound by the microorganism much more strongly than radioarsenate, in agreement with the hypothesis that the toxicity of a particular arsenical was related to the binding of it to the tissues.358 Because no instance of interconversion between As(V) and As(III) could be established, it was concluded that the two compounds inhibited the growth of B. cereus by separate mechanisms. The mecha- nisms of toxicity of arsenicals for this organism seem to be similar to those proposed for mammalian systems, which are discussed later. Adaptation The adaptation of microorganisms to arsenic compounds was of great practical interest during the earlier part of this century, because organic arsenicals were used extensively as trypanocides at that time. The resistance to organic arsenicals was found to depend on the nature of the chemical substituents on the phenyl ring:5 water-attracting groups (such as -OH and -NH2), less hydrophilic groups (such as -CH3 and -NO2), and groups highly ionized at a pH of 7 (such as -COOH). The state of oxidation of the arsenic is of little consequence in inducing resistance to any of these compounds. The mechanism of drug resistance in trypanosomes is usually a decreased permeability to the drug, and the nonarsenical portion of the molecule largely deter- mines the uptake of the drug by the parasite. A decreased permeability to arsenic appears to be a rather wide- spread adaptational mechanism, in that a decreased arsenic uptake was observed in Escherichia cold mutants that were resistant to arsenate70 and in Pseudomonas pseudomallei that had adapted to arsenite.25 In the latter case, no increase in the content of a-ketoglutarate dehydro- genase, total sulfhydryl compounds, or lipoic acid was observed in the
Biologic Effects of Arsenic on Plants and Animals 119 resistant bacteria.72 That the total quantity of free thiol groups may be important in some cases of arsenic tolerance, however, was suggested by the work of Harington,327 who found that resistant strains of the blue tick contained more total sulfhydryl than sensitive strains. Novick and Roth59i showed that the penicillinase plasmids, a series of extrachromosomal resistance factors in Staphylococcus aureus, carry determinants of resistance to several inorganic ions, as well as resistance to penicillin. Among the inorganic ions were arsenite, arse- nate, lead, cadmium, and mercury. Resistance to arsenate was found to be induced in cultures of plasmid-positive strains by prior growth with an uninhibitory concentration of the anion. Dyke et al.225 observed that strains resistant to arsenate, mercury, and cadmium were nearly al- ways resistant to multiple antibiotics and produced large amounts of penicillinase. Although the general genetic and physiologic properties of these ion-resistance markers have been studied, hardly any work has been done on the biochemical mechanisms of this sensitivity and resistance. PLANTS Arsenic occurs in all soils and natural waters; thus, plants have obviously evolved in the presence of arsenic ions. It could therefore well be that arsenic is an essential element for plant growth, but it has not been proved. There are no well-authenticated beneficial effects of arsenic on plants. Arsenic is chemically similar to phosphorus, an essential plant nutrient. That it can substitute for phosphorus in plant nutrition, however, is doubtful; in some soils, the application of phosphate fertilizer increases arsenic toxicity (through the release of fixed arsenic).7~5 Other interactions between arsenic and plant nu- trients are treated later. Biochemical Response to Arsenic Compounds When arsenic in solution penetrates the cuticle and enters the apoplast system (the nonliving cell-wall phase), it bathes the external surface of the plasmolemma of the symplast. This is the location of at least some of the enzymes of the living plant. One of the first symptoms of injury by sodium arsenite is wilting, caused by loss of turgor, and this immediately suggests an alteration in membrane integrity. Reaction of trivalent arsenic with sulfhydryl enzymes could well explain the effects of membrane degradation injury and eventually death.
120 ARSENIC In general, arsenates are less toxic than arsenites. The arsenate symptoms involve chlorosis, but not rapid loss of turgor (at least in the early expression of toxicity), and the contact action of the arsenates is more subtle. Arsenate is known to uncouple phosphorylation. Thus, the coupled phosphorylation of adenosine diphosphate (ADP) iS abolished, the energy of adenosine triphosphate (ATP) iS not available, and the plant must slowly succumb.207 Arsenate has other profound effects on plant systems. For example, Figure 5-1 shows the relative effects of arsenite and arsenate on the activation of the enzyme fumarase. Fumaric acid is a constituent of all plants and is involved in the citric acid cycle. Fumarase carries out the conversion of fumarate to ~-malate. The above examples typify the role played by the organic arsenical herbicides in plant metabolism. When one considers the number of reactions in plants that involve sulfhydryl groups and phosphorus, it is easy to appreciate the ways in which arsenites and arsenates may upset plant metabolism and interfere with normal growth. The ability of arsenate to enter into reactions in place of phosphate is probably the most important way in which arsenic acts as a toxicant. Not only does it substitute for phosphate in a number of ways, but work with labeled arsenates and arsenates has indicated that these compounds are ab- sorbed and translocated much as phosphates are. It is difficult to visualize a more effective way in which an herbicide might kill a plant. Phytotoxicity of Organic Arsenicals Injury symptoms on crop plants resulting from toxic quantities of arsenic in soils were noted in the 1930's, when it was found that young trees planted in old orchard soils grew slowly and were stunted.748 Young apple trees, in addition to being stunted, had leaf symptoms that indicated water-deficiency stress, which implied injury to the roots; pears showed similar symptoms.785 Peach trees planted on these old orchard soils that have accumulated lead arsenate exhibit by midsummer a red or brown discoloration along the leaf margins and then throughout the leaves. The discolored tissues die and drop out, giving the leaves a shot-hole appearance. Defoliation also occurs and may be complete by late summer. The injury appears first in the older leaves; young leaves on shoot tips may remain normal. Yields of fruit may be reduced, and the trees are usually stunted. Thompson and Batjer785 performed experiments aimed at correcting arsenic injury to peach trees. They found correlations between shot- holing and defoliation and between leaf arsenic content and defoliation;
Biologic Effects of Arsenic on Plants and Animals c' o > f 6 7 \ Arser.ate Arsenite 8 pH 9 FIGURE 5-1 Velocity of activation of fumarase by arsenate and arsenite ions as a function of pH. Adapted from Dixon and Webb, 207 (p. 473) 121 arsenic content varied between 1 and 4 ppm. They found that zinc sulfate applied at 10 lb (4.5 kg) per tree reduced defoliation; nitrogen application at 1.4 lb (0.6 kg) per tree also reduced defoliation; a combi- nation of these two treatments reduced defoliation that had been as high as 81% to around 2-3%, and even eliminated it in some orchards. Repeat treatments with zinc in later years had little or no effect, but maintaining a high nitrogen content reduced defoliation at later years. Lindner and Reeves473 explained that arsenic injury was confused with western X-disease, which is caused by a virus. They described the symptoms of the viral disease and arsenic toxicity; both cause shot- holing and defoliation, but arsenic-affected trees have greener leaves, and X-disease causes some chlorosis. Leaves that showed arsenic injury symptoms contained arsenic at 2.1-8.2 ppm; normal leaves, 0.9-1.7 ppm. Viral-diseased trees may produce deformed fruits, which drop prematurely; fruits of arsenic-affected trees are of normal form
122 ARSENIC and remain on the tree for the normal period. Arsenic analyses of leaves provide the most accurate diagnosis. Woolson873 studied the uptake and phytotoxicity of arsenic in six vegetable crops grown in a greenhouse. Sodium arsenate was mixed into three soil types; after moistening, the cultures were left for a month for the arsenic to come to equilibrium. Sensitivity to the arsenic decreased in the following order: green beans, lima beans, spinach, cabbages, tomatoes, and radishes. All crops were fertilized at ra as indicated by standardized soil tests and crop needs. The yields indi- cated that arsenic was generally most phytotoxic in the Lakeland soil; no plants grew when the arsenic concentration was 500 ppm. At 10, 50, and 100 ppm, crops survived; growth was proportional to arsenic concentration. The amount of available arsenic in some treatments continued to change throughout the 19-month experimental period; some of the change may have been the result of the addition of phosphate fertilizers, particularly where available arsenic reached a minimum and then increased. Plant growth at any particular degree of available arsenic in the soil may be affected by the amount of available phosphorus in the soil solution.878 The organoarsenical herbicides are not growth-regulators in the way that plant hormones are; they apparently act through or on enzyme systems to inhibit growth. They kill relatively slowly; the first symptoms are usually chlorosis, cessation of growth, and gradual browning, and dehydration and death follow. Rhizomes and tubers may show browning of the storage tissues; buds fail to sprout, and the whole structure eventually decomposes. Cotton plants treated by directed spraying with MSMA show retarded growth from which they may recover.447 When resprouting of tubers or rhizomes does occur, treatment should be repeated when some of the leaves have reached full size; treatment before then will not result in translocation, because move- ment of the assimilate stream into the underground organs is necessary to carry the toxicant to the proper sites of action. Rumberg et al.692 reported DsMA-induced loss of chlorophyll in crabgrass within 2-3 days of treatment at 75 or 85 F (24 or 29 C), but it was hardly noticeable after 5 days at 60 F (16 C); the results are summarized in Table 5-1. Others have observed symptoms on various annual plants, but few detailed descriptions are noted in the literature. Many annual weeds are ultimately desiccated and become necrotic after treatment. Re- growth from axillary buds is usually chlorotic. Cotton and other plants
Biologic Effects of Arsenic on Plants and Animals TABLE 5-1 Effect of Temperature after Treatment on the Degree of Chlorosis Induced by DSMA in Crabgrassa Chlorosis Ratingb DSMA 60 F (16 C) 75 F (24 C) 85 F (29 C) 123 lb/acre kg/ha 5 Days 10 Days 5 Days 10 Days 5 Days 10 Days 2 2.2 0.2 2.7 3.2 2.2 1.8 2.0 6 6.7 0.2 3.3 6.8 6.0 5.1 7.1 18 20.2 0.3 2.7 7.1 7.1 5.4 7.6 Mean 0.2 2.9 5.7 5.1 4.1 5.6 aDenved from Rumberg et al.693 bA scale of 0-10: 0 = no chlorosis; 10 = complete chlorosis. become deeply pigmented (red) in the stem and petiole and even the leaves, if the arsenicals are applied in sublethal dosages. On perennials, such as Johnsongrass, chlorosis does not develop before necrosis on sprayed foliage, but regrowth usually is chlorotic for some period.4~9 On purple nutsedge (Cyperus rotundus), Holt et al.366 described symptoms as "a chlorotic appearance which starts at the leaf base and progresses toward the leaf tip until the entire leaf is chlorotic" and as "first visible four days after the initial applications of AMA." On hardstem bulrush (Scirpus acutus), an aquatic species, stems became chlorotic; necrosis proceeded from the tip concurrently with development of a tan discoloration over the length of the stem; after a month, the entire stem became brown and collapsed.4~8 Stems regrow- ing from the bulrush rhizome were chlorotic with necrotic tips and usually died. Lange450 studied MSMA toxicity symptoms on stone fruits by spray- ing MSMA at 4 and 16 lb/acre (4.5 and 17.9 kg/ha) on the bottom one-third of the foliage. He observed a spotty chlorosis of the leaf, followed by necrosis of all or part of the leaf and often defoliation. Untreated upper leaves and new growth showed symptoms of injury that indicated movement of the toxic material to the untreated area. Many factors could affect response; they include herbicide applica- tion rate and formulation, surfactant, timing, volume of carrier, quality of diluent, pH, timing of evaluation, ecotypes, senescence, stage of growth, dormant-season disturbance of root systems before treatment, fertility, moisture availability and continuity, plant competition, tem- perature, light intensity, and insect and mechanical wounding of foliage
124 ARSENIC before treatment. Any of these can have a dominant effect on the response. Several researchers who have studied methanearsonates have re- ported temperature-inf~uenced results. Sckerl et al.,7~9 Kempen et al.,4~9 Laurin and Dever,454 Riepma,669 and Bounds90 all mentioned that toxicity was greater at higher temperatures. Toxicity was less on Johnsongrass in regions of California influenced by cool marine air, and higher rates of application were required than in hotter regions. One controlled-environment study of Rumberg et al. 6',3 indicated that chlorosis occurred considerably earlier in crabgrass at higher tempera- ture (24 or 29 C, versus 16 C), and injury, as measured by dry weight after 10 days, was greater at higher temperature. With sodium arsenite and cacodylic acid, temperature had no effect. Kempen4~7 found that relatively high temperature (35 C) and light t2,800 ft-c (30,140 lx)] increased the necrosis of Johnsongrass foliage and the kill of the rhizomes, compared with low temperature (15 C) and light L320 ft-c (3,445 led. He found that only 1 day was required for So-so necrosis of the foliage at the higher temperature and light, but 12 days at the lower temperature and light. McWhorter=~3r, and Kempen et al.4~9 suggested that droughtiness after application increased weed control. In studies on the effects of AMA on food reserves in purple nutsedge, Duble and Holt2'3 found, by tracer experiments and chemical analysis, that starch disappeared and arsenic increased in tubers of plants that were given repeated applications of the herbicide. In general, AMA- treated plants had a higher rate of utilization of the products of photosynthesis than untreated plants. Apparently, carbohydrates were utilized in preference to fats and proteins. From the results of Woolson et al., it is evident that total arsenic in a soil is not correlated with phytotoxicity; correlation between plant growth and available arsenic is better. 876 Some soils can remove arsenic from the soil solution more rapidly and more completely than other soils by fixation on soil colloids. In these experiments Hagerstown soil removed 2-5 times more arsenic than did the other two soils. Woolson et al. concluded that the large amount of available arsenic in the Christiana soil may have resulted from the high phos- phorus content, which prevented formation of insoluble iron arsenates through competition for reaction sites on the surface of soil particles. Also involved is the larger amount of available aluminum in the Hagerstown soil. Woolson, Axley, and Kearney877 have shown that, at high arsenic concentration in some soils, aluminum is more important than iron in removing arsenic from the soil solution.
Biologic Effects of Arsenic on Plants and Animals 125 Arle and Hamilton26 found that topical applications of MSMA affected growth of cotton more than applications of DSMA. There were usually no deleterious effects of single treatments with DSMA; single applications of MSMA later and at higher rates reduced yields. Repeated treatments with MSMA reduced yields more than DSMA treatments. Keeley and Thullen4~4 studied the responses of cotton plants to topical applications of MSMA, DSMA, and MAA at 13, 20, and 3 1 C. DSMA proved to be less injurious to young cotton plants than MSMA. Injury by MSMA was severe at 13 C, intermediate at 20 C, and low at 31 C, and inclusion of 0.4~o surfactant with the MSMA increased the injury. Injury by DSMA was intermediate at 13 C, low at 20 C, and lacking at 31 C, and inclusion of surfactant in the spray solution increased injury only slightly. Cotyledons of cotton seedlings absorbed F14C]MAA and L14C]DSMA. The MSMA solution in these experiments was adjusted to a pH of 6.4, and the DSMA to a pH of 10.4. Autoradiographs of cotton plants treated with ~4C-labeled MSMA, MAA, and DSMA showed evidence of greatest absorption and translocation of MSMA at 13 C, slight translocation at 20 C, and no translocation at 29 C. Very little FI4C]MAA or t~4C]DsMA was translocated. These results are contrary to the generalization for translocation of assimilates and other tracers, which normally penetrate and move more readily at high temperatures. (Rumberg et al. 693 found chlorosis from DSMA treatment and the translocation of DSMA to in- crease with temperature.) Interactions between Arsenicals and Nutrients Several studies have been conducted on interactions between phos- phorus and arsenic in soils and nutrient solutions. Because these two elements have somewhat similar chemical characteristics, substitution of arsenic for phosphorus might occur in plant metabolic products. Rate trials in soil and nutrient solutions, however, have yielded con flicting results, partially because available phosphorus and arsenic concentrations have not generally been determined. Schweizer7~5 showed that high phosphorus content increased the toxicity of DSMA to cotton, but there was considerable variation between the two soil types tested. ~ ittle is known of interactions between arsenic and phosphorus in plants. Everett '39 indicated that phosphorus increased the arsenic content of bluegrass in a turf treated with tricalcium arsenate. How- ever, he found that phosphorus reduced absorption of tricalcium arsenate (measured as arsenic) from nutrient solutions from 246 to 29 . . _
126 ARSENIC ppm. He stated that phosphorus at 100 ppm reduced the soluble arsenic in the nutrient solution from 15.8 ppm to 2.6 ppm. This might account for the lack of increase in arsenic uptake with high phosphorus in nutrient solutions. Everett also indicated that crabgrass absorbed twice as much tricalcium arsenate as did bluegrass; this suggests a species difference. Sckerl,7~6 in his review of the literature, indicated that phosphorus reduced arsenic toxicity. Webb843 suggested that arsenates inhibit various phosphatase en- zymes about as potently as phosphate and probably combine with the enzymes in a similar manner. Sckerl7~6 related that arsenate competes with phosphorus for uptake and transport in the cell. That arsenicals might interact with zinc was indicated by work of Batjer and Bensons5 and Martin (personal communication, 19681. Batjer and Benson showed that toxicity in peaches (but not apples) grown in arsenic-contaminated soils could be reduced by foliar applica- tions of zinc or iron chelates or soil applications of zinc or iron sulfates.55 Zinc chelates worked best and reduced the symptoms and the arsenic content of peach leaves. Martin related that orchardists in the northwestern United States use zinc sulfate at 5 lb (2.3 kg) per tree plus generous amounts of ammonium sulfate when starting peach trees in high-arsenic soils. Burleson and Paged did root studies with flax that indicated that, with absorption of more than optimal phosphorus, phosphorus and zinc reacted together in a manner that reduced either their mobility or their solubility. Sharma et al.722 showed that translocation of zinc to shoots was inhibited by high soil phosphorus. Krantz and Brown438 published a list of zinc- and iron-sensitive plants; there was no obvious correlation between symptoms of defi- ciency and susceptibility to methanearsonate sprays. Mode of Action Considering the overall action of arsenites as herbicides, it seems important that they are able to penetrate the cuticle and enter into the apoplast phase of the plant system. Here, they may move with transpi- ration water and bathe the cells of the foliar organs to- which they have been applied. At low concentration, it seems possible that arsenites are absorbed into the symplast and then translocated for at least short distances. Under most conditions in which these compounds have been used in the field, their concentrations have been such that rapid contact injury has precluded extensive translocation. This is related at least partly to their rapid effect in membrane degradation.
Biologic Effects of Arsenic on Plants and Animals 127 The arsenates, in contrast, have much lower contact toxicity; they are absorbed and translocated, at least in species that have succumbed to treatment, such as Johnsongrass and nutsedge. In susceptible pe- rennial weeds, the great virtue of MSMA and DSMA has been their ability to penetrate into and destroy underground tubers and rhizomes. Thus, with a few repeated applications, these arsenicals have controlled two of the most serious perennial weed species species that have resisted control by any other means. As topical sprays, these compounds are inactivated almost instan- taneously on contact with the soil and therefore may be used with impunity in many row crops; cotton is one of the more important of these. Although arsenicals in ordinary herbicidaI dosages are rapidly rendered unavailable to plants in the soil, and although most soils have a very great capacity to inactivate and hold arsenic, arsenic residues in soils may eventually become troublesome. For this reason, in any weed control activity involving arsenical herbicides, integrated pro- grams of herbicide rotation should be used. If such programs are used, occasional application of the organic arsenicals in the particular roles in which they are highly effective may not result in soil residues of any . ·~ slgn~lcance. As for the chemical mechanisms by which the organic arsenates kill plants, their relatively slow action involving translocation and produc- ing chlorosis as a primary symptom seems to implicate disturbance of phosphorus metabolism. Not only are they absorbed and translocated in plants much as are phosphates, but also they affect many organelles in the cells, including the chloroplasts, in all of which phosphorus plays important roles.35 This interpretation is further strengthened by evidence of Schweizer7~5 that the addition of phosphorus to two silt-loam soils increased the toxicity of DSMA to cotton, possibly by saturating sites in these soils on which both arsenate and phosphate are fixed. As early as 1934, Albert6 reported that residues of calcium arsenate became more toxic to several crops where phosphate fertilizer was applied heavily. There is substantial evidence that phosphates and arsenates tend to replace each other chemically, but that arsenic cannot serve the many essential roles of phosphorus in plants. The uncoupling of oxidative phosphorylation and the formation of complexes with sulfhydryl-containing enzymes may also enter the picture of arsenic phytotoxicity. However, trivalent arsenic is the form commonly associated with these effects, and this would implicate arsenites, rather than arsenates or arsenates.
128 LABORATORY ANIMALS Factors That Can Influence the Toxicity of Arsenic Compounds Table 5-2 summarizes information on the toxic and no-effect doses of several arsenic compounds. The variety of systems described in the table suggests some of the factors that can influence the toxicity of arsenic. For example, the studies of Harrison et al.329 illustrated effects that the chemical and physical properties of arsenic trioxide can h Eve on its acute toxicity. The toxicity of arsenic trioxide depended on he purity of the substance used; the crude commercial material was less toxic than the purified material. Although the impure arsenic trioxide was less toxic, it caused much more gastric and intestinal hemorrhage than the purified arsenic trioxide. This study and many of those discussed below were carried out in rats, which have a highly peculiar metabolism of arsenic (see Chapter 41. The reader should be aware of this problem and understand that the rat is an animal to be avoided in arsenic research. In addition to testing the oral toxicity of aqueous solutions of the "crude" and "pure" arsenic trioxide, Harrison et al.329 investigated the acute toxic effects of both preparations when given in the dry state mixed in feed. When arsenic trioxide was administered in this manner, the toxic dosage was almost 10 times as high as when it was given in aqueous solution, regardless of whether the crude or the pure material was tested. These results are in accord with those of Schwartze,7~3 who found that a solution of arsenic trioxide was more toxic than the undissolved compound and that the toxicity of different preparations of solid arsenic trioxide administered orally varied markedly, depending on their coarseness or fineness. The dependence of the toxicity of arsenic trioxide on the physical form in which it is given is probably a result of its rather poor solubility; e.g., Harrison et al.329 commented that heating was required to solubilize arsenic trioxide. The practical consequences of the great variation in toxicity of arsenicals due to their different so'ubilities were recently emphasized by Done and Pear, who criticized government regulations that equated the poison hazard of the highly soluble sodium arsenite with that of the less soluble (and thereby less toxic) arsenic trioxide (although the latter does have a toxic potential). Harrison et al. also demonstrated a species difference in the resis- tance to acute poisoning witl1 arsenic trioxide: mice were less affected by the arsenic compound than were rats. Similar species differences were shown by Kerr cat al.,4'~'' who noted that turkeys and dogs were ARSENIC
Biologic Effects of Arsenic on Plants and Animals 129 more susceptible to the toxic effects of the organic arsenical 3-nitro-4- hydroxyphenylarsonic acid than were chickens and rats. McChesney et al.528 reported that sodium p-N-glycoloylarsanilate was about 20 times as toxic to cats as to mice. The work of Harrison et al.329 revealed that even different strains of mice had very different abilities to tolerate arsenic trioxide. These species and strain differences in the toxicity of arsenic could have important implications regarding the use of laboratory animals as predictive models for human response. The average estimated fatal dose of arsenic trioxide for humans is 125 mg.~5 For a 70-kg man, this is equivalent to about 1.4 mg of arsenic per kilogram of body weight. Thus, a human is much more sensitive to the toxic effects of arsenic on a weight basis than a rat, and it is obviously dangerous to extrapolate results from rodents to humans. Another factor that can influence the toxicity of arsenic is the valence of the element. Direct comparison of the intraperitoneal ~D75 of sodium arsenite and sodium arsenate in the rat shows that the trivalent form of arsenic is about 4 times as toxic as the pentavalent form. 263 This difference due to valence is also seen in tissue-culture studies in which many confounding metabolic effects are avoided.704 Differences related to valence apply to the organic arsenicals, as well as to the inorganic. In fact, the greater toxicity of the trivalent form versus the pentavalent is such a good generalization that a microbiologic assay for distinguish- ing As(III) and As(V) on the basis of their different toxicities has been suggested.489 The toxicity of a number of synthetic aromatic organic arsenicals has been the subject of several investigations, because of the value of these compounds for improving weight gain and feed efficiency in swine and poultry. Generally speaking, the organic forms of arsenic are con- sidered less hazardous than the inorganic forms, and this is shown by the tissue-culture work of Savchuck et al.704 Arsenic concentrations of 84 and 65 ppm in the form of 3-nitro-4-hydroxyphenylarsonic acid and sodium arsanilate, respectively, were needed to cause a 50~o growth inhibition of HeLa cells, whereas a concentration of only 2 ppm in the form of sodium arsenate was required to inhibit growth by the same amount. However, feeding arsenic at 114 ppm in the diet of rats as 3-nitro-4-hydroxyphenylarsonic acid caused an 83% mortality in 4 days,422 whereas arsenic at 125 ppm in the diet as sodium arsenate caused only a mild growth depression after 12 weeks. Furthermore, the intraperitoneal arm of arsenic as 3-nitro-4-hydroxyphenylarsonic acid is 18.8 mg/kg in rats,422 whereas the LD75 of arsenic as sodium arsenate is given as 14-18 mg/kg.263 To put the above studies in the proper perspective, it should be pointed out that the recommended concentra
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132 ARSENIC lion of 3-nitro-4-hydroxyphenylarsonic acid for feed-additive use is only 25-50 ppm in the diet, or arsenic at 7-14 ppm. Arsanilic acid is another aromatic organic arsenical that is widely used as a growth-promoter, and Frost et al. 273 reported that it had no harmful effects on rats over several generations when fed at 500 ppm in the diet. However, Notzold et al.590 noted an incoordinated gait in swine, and Al-Timimi and Sullivani2 saw a growth inhibition in turkeys when arsanilic acid was fed at an arsenic concentration of 400 ppm. Again, to put these studies in perspective, it should be emphasized that the recommended concentration of arsanilic acid for feed-additive use is only 50-100 ppm. Perhaps of more direct concern to consumers is the toxicity of arsenic that occurs naturally in seafood, such as shrimp (such arsenic compounds are commonly referred to as "shrimp arsenical. Public awareness of this potential hazard was heightened by an article in Consumer Reports.275 However, many of the allegations in the article were disputed in a press report.548 The work of Coulson et al.~7i showed that rats (and humans) do indeed absorb shrimp arsenic from the gastrointestinal tract readily, but this form of arsenic is excreted rapidly in the urine. These authors also found no evidence of toxic effects of feeding "shrimp arsenic" at 17.7 ppm in the diet of rats for 52 weeks. Criteria of toxicity included growth, physical appearance, activity, and histologic appearance of the liver, spleen, and kidneys. No toxic symptoms were reported by Morgareidge, 560 who supple- mented rat diets at 16 ppm with protein-bound arsenic derived from the livers of turkeys whose diets had contained 0.56% p-ureido- benzenearsonic acid (carbarsone). Welch and Landau848 observed no toxic reactions in rats fed a diet containing 1% arsenocholine (the arsenic analogue of choline) for a week. The apparent lack of toxicity of this arsenic compound may be of considerable interest, in light of the work of Lunde,493 who discovered in fish oils two arsenolipids with chemical properties that resemble those of phospholipids. Still another class of organic arsenical compounds is the aliphatic arsenicals, such as cacodylic acid and the sodium salts of methanear- sonic acid, which are discussed later. Although these compounds are used widely as herbicides, their toxicity is less than that of inorganic arsenical herbicides. The Problem of Toxic versus "No-Effect" Dosages In addition to the complexities just discussed, there is a factor that confounds the interpretation of toxicologic data namely, the criterion
Biologic Effects of Arsenic on Plants and Animals 133 used to judge whether a given dosage is toxic. For example, arsenic at S ppm in the drinking water as sodium arsenite from weaning until death is not toxic to rats, with respect to growth or life span,7~i and is only slightly toxic to mice.7~0 An identical experiment, however, car- ried out through three generations of mice revealed that the ratio of males to females born increased in mice exposed to arsenic, compared with controls.7~2 It was concluded that exposure to some trace ele- ments in dosages that do not interfere with growth or survival may affect reproduction. Thus, a more sensitive indicator of toxicity showed the detrimental effects of a dosage that had previously been regarded as "safe." With sophisticated assessment techniques, such as biochemical and enzyme measurements, even more subtle effects of poisons can be detected. For example, Bencko and Simane66 found that the respiration rate of liver homogenates prepared from mice that had received arsenic at S ppm as arsenic trioxide in their drinking water was only 61% of that of the normal controls. However, the dosage of arsenic used by Bencko and Simane was 100 times higher than the currently recommended maximal concentration (O.OS ppm) of arsenic in drinking water.578 An even more sensitive method for determining the toxic effect of arsenic compounds was used by Weir and Hine: 846 a conditioned- avoidance technique to assess the deleterious effects of various ions in the aquatic environment of fish. In this study, arsenic (as arsenate) was found to impair conditioned-avoidance behavior of trained goldfish after 48 h of exposure to a concentration of only 0.1 ppm, which is only 1/320 of the lethal concentration for Solo and only 1/lS of the lethal concentration for 15to of the fish. The relevance of this work to mammalian systems is far from clear, but the data suggest at least that similar behavioral experiments should be carried out with animals exposed to various substances suspected of being environmental hazards. Mechanisms of Toxicity of Arsenic Compounds The difference in toxicity between trivalent and pentavalent arsenic compounds can best be understood by considering the biochemical mechanisms of action of these two distinct families of compounds. This aspect of arsenic toxicology has been the subject of numerous re- views.~73976347628~5 The early work of Ehrlich, Voegtlin, and others suggested that organic arsenicals exert their toxic effects in viva by first being metabolized to the trivalent arsenoxide form and then reacting
134 ARSENIC with sulfhydryl groups of tissue proteins and enzymes to form an arylbis torganylthio~arsine: OR' R-As=0+ 2R'SH ~> R-As/ + H2O. SR' Later work showed that several enzyme systems containing thiol groups could be poisoned in this way and that in most cases the activity of the enzyme could be restored by adding an excess of monothiol. An important exception to this generalization, however, proved to be the pyruvate oxidase system, which could not be protected against trivalent arsenicals by even a 200~o excess of monothiol. Such an apparent anomaly was clarified when it was shown that, under some circumstances, arsenicals can complex with two sulfhydryl groups in the same protein molecule, thereby forming a stable ring structure that is not easily ruptured by monothiols. This finding stimulated the testing of various dithiol compounds for their ability to block the action of arsenicals on pyruvate oxidase and led to the discovery of British antilewisite (BAL), which eventually became a widely used antidote for arsenic poisoning. The simultaneous interaction of arsenic with two thiol groups led Peters and associates594 to postulate the existence of a dithiol-containing cofactor in the pyruvate oxidase system. This idea was later verified experimentally when lipoic acid was identified as a component of pyruvate oxidase. The reaction of an arsenoso com- pound with lipoic acid to yield a ring structure that can be cleaved by BAL iS illustrated in Figure 5-2. This reaction summarizes what is currently felt to be the mode of action of trivalent monosubstituted arsenicals in exerting their toxic effects in biologic systems and illus- trates the biochemical rationale for the use of BAL to counteract arsenic . . polsomng. It should be pointed out, however, that the toxic effects of inorganic trivalent arsenic (arsenite) can often be potentiated by BAL in vitro. Fluharty and Sanadi,257 for example, showed that an equimolar mixture of arsenite and BAL uncouples oxidative phosphorylation in rat liver mitochondria and drew the conclusion that arsenite is the true active inhibitory species and that the BAL served only as a vehicle for transporting arsenite to a dithiol enzyme site. Siegal and Albers729 found that addition of equimolar BAL decreased the arsenite concentra- tion necessary to produce 50% inhibition of Electrophorus (electric eel)
Biologic Effects of Arsenic on Plants and Animals COOH ~ I H2 )4 HC-SH + R-As = 0 fH2 H2 C-SH Lip oic acid (6,8~imercaptooctanoic acid) COOH (:H2 )4 H)-S COOH (:H2 )4 HC-S 1 \ CH2 As-R+H2O 1 ' H2 C-S 2~rganyl4~4'<arboxybutyl)- 1 ,3 ,2 ~ithiaarsacyclohexane COOH CH' OH(CH2 )4 fH2 OH 1 -'1 -' + H)-SH~ H:-SH + HC-S. \ I I | \As-R fH2 /As-R H2C-SH fH: H2C-S o H2C-S H2 C-SH 2-Organyl4~4'-carboxybutyl)- British 1,3,2~ithiaarsacyclohexane antilewisite lipoic acid 135 Regenerated 2-Organyl4-hydroxymethyl- 1 ,3 ,2 ~ithiaarsacyclopentane FIGURE 5-2 Reaction of lipoic acid with a trivalent monosubstituted arsenical, and regeneration of lipoic acid by addition of sA~.635 microsomal (Na+/K+~-ATPase from 6 mM to 0.1 mM. The authors suggested that the sA~-arsenite complex reacted directly with the enzyme. Wu882 performed a careful kinetic analysis of the dithiol- dependent inhibition of rat liver glutamine synthetase by arsenite and proposed the scheme shown in Figure 5-3. This scheme was thought to account for several facts regarding the dithiol-dependent inhibition by arsenite, including the ready dissociation of the enzyme-arsenite com- plex and the reversal of the inhibition by cysteine. McDonough529 suggested that the sA~-arsenite complex can act as an inhibitor of germination, inasmuch as lettuce seeds soaked in mixed solutions of sodium arsenite and BAL yielded lower germination ratios than did seeds soaked in either compound alone. The mechanism of action of the toxic effects of inorganic pentavalent arsenicals is less clearly understood than that of the trivalent arsenic compounds. It is possible that pentavalent arsenic is reduced to triva- lent arsenic before exerting its toxic effects,87292 but whether that
136 ARSENIC CH2 OH CH2 OH HC-S HC-S ~ ~AsO- ~ Enz-SH - ~ >As-S-Enz +OH HC-S HC-S H H BA -arsenide complex (inhibitor) Enzyme-inhibitor complex FIGURE 5-3 Reaction of glutamine synthetase with sA~-arsenite complex. happens in viva is controversial.709~868 Unlike the trivalent arsenicals, "the pentavalent forms do not appear to react directly with the active sites of enzymes.397 Rather, arsenate can compete with inorganic phosphate in phosphorylation reactions to form unstable arsenyl es- ters, which then decompose spontaneously. Arsenate has also been shown to uncouple oxidative phosphorylation,~82 presumably by com- peting with inorganic phosphate at one of the energy-conserving steps. Chan et al.~42 have isolated an arsenylated component of rat liver mitochondria that they feel may represent the arsenic analogue of a low-molecular- weight pho sphorylated mitoch ondrial constituent that plays a role in oxidative phosphorylation. A nonhydrolytic mode of action of arsenate in inhibiting mitochondrial energy-linked functions has recently been proposed.549 Adaptation to Toxicity of Arsenic Compounds Most early investigators reported that animals were unable to adapt to the toxic effects of inorganic arsenic compounds,7~3 although adapta- tion to some organic arsenicals was readily achieved.44i In spite of the early failures to demonstrate adaptation to inorganic arsenic, Bencko and Symon68 have recently shown that the arm for arsenic as arsenic trioxide administered subcutaneously could be increased from 10.96 to 13.98 mg/kg in hairless mice as a result of giving arsenic at 50 ppm as arsenic trioxide in the drinking water for 3 months. Additional evidence that suggested an adaptive response to arsenic was the finding that the decreased metabolic oxygen consumption observed with the liver homogenates from mice given arsenic at 50 ppm in the water for 32 days returned to normal after 64 days in the experiment.63 However, no
Biologic Effects of Arsenic on Plants and Animals 137 such adaptation was seen in mice given arsenic at either 5 or 250 ppm in the water. Moreover, this experiment seems somewhat inconsistent with an earlier report from the same laboratory, which showed that liver homogenates from mice given arsenic at 50 ppm in the water for 256 days exhibited a decreased consumption of oxygen.66 Apparently, the range of experimental conditions under which adaptation to arsenic can be obtained is quite limited. The mechanisms of these adaptive responses to arsenic are not known, but Bencko and Symon67 found that mice given arsenic at 50-250 ppm in the water accumulated arsenic in the liver and skin until the sixteenth day of the experiment, after which retention decreased. The authors suggested that either decreased absorption or increased excretion of arsenic could account for their results. Studies with t74AsUarsenate revealed that mice previously exposed to arsenic at 50 ppm as arsenite in the water for 64 days displayed a significant decrease in the retention of a later dose of radioarsenate administered parenterally.65 Although the authors interpreted their results as evi- dence of an increase in capacity of the excretory mechanism for arsenic due to arsenic exposure, this experiment could also perhaps be ex- plained by a saturation of the tissue binding sites for arsenic by previous arsenic intake, which could then cause an "apparent" in- crease in the excretion of the element. Clearly, more research is needed to determine whether animals are able to adapt to the toxic effects of inorganic arsenicals. Experimental Inhalation Toxicity Air pollution due to arsenic is a particular problem in some parts of Czechoslovakia, because of the high arsenic content of some coal burned in power plants there. Consequently, Bencko and associates of the Institute of Hygiene in Prague have carried out a number of studies concerned with the experimental inhalation toxicity of arsenic.6269 These workers pioneered the use of the hairless mouse for such investigations, because other animal models had several disadvantages for research in arsenic toxicology.68 The rat, whose arsenic metabolism is peculiar (arsenic tends to accumulate in the blood), was ruled out as a test animal. The rabbit and guinea pig were also considered unsuit- able for these studies, because in some of the work arsenic was to be administered via the drinking water, and the variable consumption of fresh vegetables by the animals would contribute to an irregular water intake. However, the hairless mouse had a number of experimental advantages for inhalation toxicity determination. First, it could not put
138 ARSENIC its nose into its fur to ''filter" the air being breathed. Moreover, there fiasco hair to trap the arsenic-containing dust; such dust could otherwise be ingested later as a result of cleaning or grooming. Finally, the lack of hair on the mouse enabled the investigators more readily to determine any dermatologic changes caused by the arsenic. Hairless mice were exposed to fly ash whose particle size was less than 10,um and that contained 1% arsenic in the form of arsenic trioxide. The exposures were carried out on 5 days/week for 6 in/day in dust chambers specially designed for the application of solid aerosols. The mean arsenic concentration in the dust chamber was 179.4 + 35.6 ,ug/m3 of air, which was about 3 times higher than the maximal concentration of arsenic found in the vicinity of the offending power plants. During the first 2 weeks of the experiment, there was a considerable increase in the concentration of arsenic in the livers, kidneys, or skin of the exposed mice. However, there was a significant decrease in the arsenic content of the liver and kidney samples during the fourth week of exposure, although no such decrease was observed in the skin. This decline in the arsenic content of tissues was similar to that seen in animals given arsenic orally and suggested an adaptation to arsenic. Unfortunately, these workers did not carry out physiologic measurements to evaluate effects of the arsenic exposure on the metabolism of the experimental animals. Zharkova890 studied the effect of continuous 24-h exposures to arsenic trioxide at 25-37 ,ug/m3 of air on various physiologic charac- teristics in rats. He found that such treatment resulted in a lag in weight gain, disordered chronaxy ratios of antagonist muscles, suppression of cholinesterase activity, a reduction in concentration of sulfhydryl groups in blood proteins, an increase in the number of reticulocytes, a decrease in blood hemoglobin, porphyrinuria, a reduction in ascorbic acid in all organs and tissues, and accumulation of arsenic in the organs and tissues. Few experimental details were presented in the translated paper, so it is difficult to assess the biologic significance of these results. Although the physiologic importance of disordered chronaxy ratios, suppressed enzyme activities, and reduction in sulfhydryl groups in proteins might be questioned, weight lag, anemia, and porphyrinuria are more difficult to ignore. Moreover, the concentra- tions of arsenic used in the investigation were very low, although they still exceeded the occupational exposure standard for inorganic arsenic recently recommended by the National Institute for Occupational Safety and Health (NIOSH).809 Rozenshtein688 investigated the effect of continuous exposure to arsenic trioxide aerosols on albino rats. He found that round-the-clock
Biologic Effects of Arsenic on Plants and Animals 139 exposure to arsenic trioxide at 60.7 ,ug/m3 produced inhibition in the central nervous system, reduced the content of sulfLydryl groups, inhibited cholinesterase activity, and raised the concentration of pyru- vate in the blood. Similar continuous exposure to a concentration of 4.9 ,ug/m3 caused disturbances of conditioned reflexes and of the chronaxy ratio of antagonistic muscles and a reduction in the content of sulfLy- dryl groups in the blood. Exposure to both concentrations resulted in a marked accumulation of arsenic in the body and morphologic altera- tions in the organs and tissues. Inasmuch as no functional, biochemi- cal, or morphologic alterations were observed when the animals were exposed to arsenic trioxide at 1.3 ,ug/m3, Rozenshtein recommended 1 ,ug/m3 as the maximal mean diurnal permissible concentration of this compound in the atmosphere.688 Again, the physiologic implications of these results are not clear. The main drawback in the research of both Zharkova and Rozen- shtein was that the test animal used was the rat, which has a peculiar arsenic metabolism. Also, the rats had considerable body hair, which could trap the arsenic aerosol. The arsenic trapped in this way could then be ingested by the animal as a result of cleaning and grooming. Finally, the rats used in both studies were exposed to arsenic trioxide on a continuous 24-in/day basis, whereas the NIOSH standard was meant to apply only in situations of intermittent exposure. Nonetheless, further research should be carried out to evaluate both experiments.806 Arsenals and Resistance to Infection Gainer and Pry278 showed that virus-infected mice treated with large doses of arsenicals had higher mortality rates than untreated controls. Viral diseases so affected by arsenic included pseudorabies, en- cephalomyocarditis, and St. Louis encephalitis. Although several ex- perimental protocols were used, arsenical treatment generally con- sisted of injecting subacute doses of sodium arsenite at the time of inoculation with virus or administering sodium arsenite or 3-nitro-4- hydroxyphenylarsonic acid at rather high concentrations of arsenic (75-150 ppm) in the drinking water for various periods before or after inoculation. In one case (western encephalitis virus), mortality was significantly reduced if the mice were given sodium arsenite at the time of inoculation with virus, but mice treated with 3-nitro-4- hydroxyphenylarsonic acid in the drinking water after inoculation had higher mortality than did controls. British antilewisite did not inhibit, but appeared to stimulate, the mortality-increasing activity of sodium arsenite in pseudorabies infection. This observation is consistent with
140 ARSENIC other reports that under some circumstances BAL may potentiate the toxicity of arsenicals. The protective effect of a synthetic double-stranded homopolynu- cleotide complex of polyinosinic acid and polycytidylic acid Holy I/poly C) against mortality in mice due to western encephalitis virus was inhibited by sodium arsenite treatment. Because the protective role of poly I/poly C against viral disease has been associated with the action of interferon, Gainer and Pry278 hypothesized that the arsenical stimu- lation of mortality after inoculation with viruses was at least partially explainable by interferon dysfunction. Indeed, a later paper by Gainer277 showed that the induction of interferon by poly I/poly C in rabbit kidney cell cultures could be inhibited by sodium arsenite. It was found somewhat unexpectedly, however, that, although high concen- trations of arsenite inhibited the action of exogenous mouse interferon added to cultures of mouse embryo cells, low concentrations of arse- nite increased the antiviral activity of low concentrations of interferon. The research of Gainer and Pry278 seems to have two major ecologic consequences. First, exposure to large doses of arsenic clearly impairs a mouse's ability to resist viral disease. However, the doses of arsenic used in these studies were such that any relevance of these data to human pollution problems would have to be limited to outbreaks of massive arsenical toxicosis, such as the Morinaga dry milk incident575 and the Ube soy sauce episode.7~ In this regard, the follow-up study revealed that children poisoned in the Morinaga incident, among other problems, also had a decreased resistance to infection. The mecha- nism of action of arsenic in decreasing resistance to infection is not known with precision, but the results of Gainer indicate that decreased interferon production or action may be involved. A nonspecific effect of heavy-metal poisoning cannot be ruled out, inasmuch as Hemphill et al.343 have shown that mice treated with lead had a greater susceptibil- ity to challenge with Salmonella typhimurium than controls that re- ceived no lead. A decrease in resistance to infection might very well be expected in any group of animals additionally stressed by exposure to a metabolic poison. Although Koller and Kovacic433 recently found that exposure to lead decreased antibody formation in mice, Gainer and Pry were able to rule out alterations in antibody formation or action as the primary factors accounting for the stimulating effects of arsenicals on the mortality of their virus-infected mice. A second ecologic consequence of Gainer's experiments is related to the remarkable observation that low concentrations of arsenic ap- peared to increase the ability of exogenously added mouse interferon to block the infection of cultured mouse embryo cells with vesicular
Biologic Effects of Arsenic on Plants and Animals 141 stomatitis virus.277 Although the author himself had some reservations concerning the proper interpretation of his data, the stimulation of interferon action by arsenic seemed to be real. Gainer suggested that an increased antiviral activity of interferon could provide a rationale for the beneficial "growth-promoting" effects of arsenical feed additives in livestock through reduction in disease incidence or severity.277 Any conclusions regarding the possible effects of low concentrations of arsenicals in the environment on the ability of humans to resist disease must await further research. Arsenic as Antagonist to Selenium Poisoning Moxon565 first demonstrated the protective effect of arsenic against selenium poisoning when he found that arsenic at 5 ppm as sodium arsenite in the drinking water largely prevented liver damage in rats whose diet contained selenium at 15 ppm as seleniferous wheat. Moxon and DuBois566 then showed that arsenic was unique in its ability to prevent selenium toxicity; all other elements tested were unable to protect against all manifestations of chronic selenosis. Sodium arsenite and sodium arsenate were equally effective against seleniferous grain, but the arsenic sulfides were ineffective. Arsanilic acid and 3-nitro- 4-hydroxyphenylarsonic acid, two organic arsenicals used as '~growth-promoters" for livestock, also exhibited a beneficial action against selenium poisoning in rats when given in the drinking water.344 There is evidence that it would be practical to use these two agents to protect swine and poultry in high-selenium regions.~30834 Amor and Pringle~3 even suggested the use of an arsenic-containing tonic as a prophylactic agent against selenium poisoning in exposed industrial workers. The metabolic basis for the beneficial effect of arsenic in selenium poisoning remained confused for some time, because arsenic was known to block the biosynthesis of dimethylselenide, a detoxification product in animals that received subacute doses of selenium by injec- tion. 599 Moreover, the protective effect of arsenic against dietary selenium was not seen if the arsenic was given in the diet, instead of the drinking water,280 and Frosted has shown that the toxicities of arsenic and selenium are additive if both elements are given in the drinking water. These results agree with those of Obermeyer et al.,593 who recently observed an additive toxicity between arsenite and trimethylselenonium chloride or dimethylselenide. Ganther and Baumann279 studied the influence of arsenic on the metabolism of selenium when both elements are injected in subacute
42 ARSENIC doses and found that the excretion of selenium into the gastrointestinal tract was markedly stimulated by arsenic. Levander and Baumann463 observed an inverse relationship in arsenic-treated rats between the amount of selenium retained in the liver and the amount excreted into the gut; and they concluded that the bile might be the route by which selenium was appearing in the gastrointestinal tract. This hypothesis proved correct when it was discovered that in 3 h over 40~o of the selenium injected could be recovered in the bile of rats that a] ;o received arsenic, whereas only 4% of the selenium was excreted in ;o the bile of rats not given arsenic.464 This effect of arsenic on the biliary excretion of selenium was not confined to subacute-toxicity experi- ments: A response of selenium to arsenic was seen at dosages ap- proaching a rat's daily intake of selenium when fed some crude commercial diets. Sodium arsenite was the most effective form of arsenic in enhancing the biliary excretion of selenium, but arsenate and 3-nitro-4-hydroxyphenylarsonate were also active to some extent. In experiments with radioactive arsenic, it was found that selenium stimulated the biliary excretion of arsenic, just as arsenic stimulated the excretion of selenium. Initial attempts to characterize the forms of selenium in rat bile suggested that the element is probably present in several forms, including some macromolecularly bound selenium. Although these studies provide an understanding on a physiologic basis of how arsenic counteracts selenium toxicity, the chemical mech- anism of the process is still far from clear. The most logical hypothesis to account for the arsenic-selenium antagonism from the molecular point of view assumes that arsenic combines with seleniu~perhaps, in analogy with sulfur chemistry, by reacting with selenol (-SeH) groups to form a detoxification conjugate that passes readily into the bile. Nutritional Essentiality of Arsenic A number of older reports suggested that arsenic in small amounts may play a useful metabolic role in tissues, rather than being merely an accidental contaminant. Underwood8°' has cited several of these early references that claimed beneficial effects of arsenic, including stimula- tion of growth in tissue cultures and enhancement of growth and metamorphosis of tadpoles. More recently, Askerov et al.36 have found that spraying leaves with a 0.002Yo arsenic solution leads to a logo increase in viability of silkworm caterpillars and a 29~o increase in cocoon yield. Despite several subjective reports on the effects of arsenic on the
Biologic Effects of Arsenic on Plants and Animals 143 appearance of the haircoat or in the prevention or cure of anemia, numerous attempts to induce an experimental dietary deficiency have failed, probably because of the ubiquity of the element. Hove et al.37 fed rats a milk diet fortified with iron, copper, and manganese and found that arsenic caused a slight initial delay in the decrease of hemoglobin when the minerals were withdrawn from the milk. How- ever, these authors concluded that, if arsenic is essential for rats, the requirement must be somewhere below the 2,ug daily that was pro- vided by the milk alone. Schroeder and Balassa709 reported that rats and mice grew well and survived normally when they received only 0.26 ,ug of arsenic per 100 g of body weight per day in food. Skinner and McHargue736 found that rats responded to arsenic supplements with increased hemoglobin when fed a ration composed mainly of skim milk powder and sucrose and adequately supplemented with iron and copper. Sharpless and Metzger723 have presented some evidence that arse- nate can act as a mild goitrogen when fed in the diet at 5 ppm. To get a significant goitrogenic effect, however, such high concentrations of arsenic were needed that these workers concluded that there was only a remote possibility that arsenic could act as a positive goitrogenic agent in man. A report by Muth et al.570 suggested that arsenic may have some activity in preventing selenium-deficiency diseases, inasmuch as addi- tion of arsenic at 1 ppm as sodium arsenate to selenium-deficient ration significantly reduced the incidence of myopathy in lambs. This obser- vation has not been confirmed, however (Westwig and Whanger, unpublished data). Attempts to demonstrate a beneficial effect of arsenic in other selenium deficiency diseases, such as liver necrosis in rats7~4 and exudative diathesis in chickens, 62 i have been unsuccessful. Using purely theoretical arguments based on the tissue distribution of various trace elements, Liebscher and Smith469 decided that arsenic behaves more like an environmental contaminant than a nutritionally essential mineral. However, a recent preliminary communication has presented evidence of a requirement for arsenic by the rat.s86 To demonstrate an arsenic deficiency, the experimental animals had to be housed in plastic cages placed in laminar flow racks. The rats were fed a specially formulated purified diet that contained arsenic at only 30 ppb. The deficiency signs were most striking in male rats and included rough haircoat, low growth rate, splenomegaly, decreased hematocrit, and increased osmotic fragility of red cells. These preliminary results appear to have been verified by recent work describing an arsenic deficiency in goats and minipigs fed semisynthetic rations containing
144 ARSENIC arsenic at less than 50 ppb. ~9 Deficiency signs included impaired reproductive performance, decreased birth weights, increased neonatal mortality, and lower weight gains in second-generation animals. None of these deficiency signs were observed in control animals fed the semisynthetic diet supplemented with arsenic at 350 ppb. DOMESTIC ANIMALS Inorganic and Aliphatic Organic Arsenicals Arsenic appears to be second only to lead in importance as a toxicant in farm and household animals.~5335 Toxicoses caused by inorganic and aliphatic organic arsenicals are generally manifested by a syndrome entirely different from that caused by the phenylarsonic feed additives and therapeutic agents; therefore, the phenylarsonic compounds will be discussed separately. Some of the more common sources of arsenic poisoning include grass clippings from lawns that have been treated with arsenical crabgrass-control preparations; grass, weeds, shrubbery, and other foliage that have been sprayed with arsenical herbicides;~3~4 dipping of animals in vats that years before had been charged with arsenic trioxide; and soils heavily contaminated with arsenic, either through the burning of arsenic formulations in rubbish piles or through the application of arsenical pesticides to orchards and truck gardens. ~53 656 The more common sources of arsenic for small animals, especially cats, include ant and snail baits, which usually contain 1-2% arsenic. in Man and all lower animals are susceptible to inorganic arsenic poisoning, but poisoning is most often encountered in the bovine and feline species and results from the contamination of their food supply. The incidence of arsenic poisoning in these two species is closely followed in other forage-eating animals, such as sheep and horses.564 Poisoning by inorganic arsenicals occurs only occasionally in dogs and rarely in swine and poultry. The toxicity of inorganic arsenicals varies with the species of animal exposed, the formulation (e.g., trivalent arsenicals are more toxic than pentavalent), the solubility, the route of exposure, the rate of absorp- tion from the gastrointestinal tract, and the rates of metabolism and excretion.~53 656 In practice, the most dangerous arsenic preparations are dips, herbicides, and defoliants in which the arsenic is in a highly soluble form. Unfortunately, animals often seek out and eat such materials as insulation, rodent baits, and dirt and foliage that have been contaminated with an inorganic arsenical.
Biologic Effects of Arsenic on Plants and Animals 145 Because so many factors influence the toxicity of arsenic, there is little point in attempting to state its toxicity in terms of milligrams per kilogram of body weight. The lethal oral dose for most species, however, appears to be 1-25 mg/kg of body weight as sodium arsenite, and 3-10 times that range as arsenic trioxide. That the toxicity of an arsenical is greatly influenced by its solubility and particle size and thus by the extent of its absorption from the intestinal tract or skin is illustrated by an experiment conducted with swine.~5 Sodium arsenite was given in the feed at up to 500 ppm continuously for 2 weeks. The pigs readily ate the contaminated feed, but manifested no signs of acute arsenic poisoning. When the concen- tration was increased to 1,000 ppm, the pigs refused to eat the feed. When sodium arsenite was added to their drinking water at 500 ppm, severe poisoning and death occurred within a few hours. It was concluded that the lethal dose of sodium arsenite via drinking water was 100-200 mg/kg of body weight. Experience with field cases of arsenic poisoning has indicated that animals that are weak, debilitated, and dehydrated are much more susceptible to arsenic poisoning than normal animals, probably be- cause renal excretion is reduced. Arsenic poisoning in most animals is usually manifested by an acute or subacute syndrome. Chronic poisoning, although it has been re- ported, is seldom seen and has not been clearly documented. Arsenic affects tissues that are rich in oxidative systems, primarily the alimentary tract, kidneys, liver, lungs, and epidermis. It is a potent capillary poison; although all capillary beds may be involved, the splanchnic area is the most commonly affected. Capillary damage and dilatation result in transudation of plasma into the intestinal tract and sharply reduced blood volume. Blood pressure usually falls to the point of shock, and cardiac muscle becomes weakened; this contributes to circulatory failure. The capillary transudation of plasma results in the formation of vesicles and edema of the gastrointestinal mucosa, which eventually lead to epithelial sloughing and discharge of the plasma into the gastrointestinal tract.656 Toxic arsenic nephrosis is more commonly seen in small animals and man than in farm animals.~5 ~48 Glomerular capillaries dilate, allowing the escape of plasma; this results in swelling and tubular degeneration. The anhydremia that results from the loss of fluid through other capillary beds and the low blood pressure contribute to the oliguria that is characteristic of arsenic poisoning. The urine usually contains pro- tein, red blood cells, and casts.~5 After percutaneous exposure, capillary dilatation and degeneration
46 ARSENIC may result in blistering and edema, after which the skin may become dry and papery. The skin may then crack and bleed, providing a choice site for secondary invaders.656 Most textbooks report that arsenic is accumulated in the tissues and slowly excreted, but this appears to be true only in rats. Most species of livestock and pet animals apparently excrete arsenic rapidly. This phenomenon is very important when one considers arsenic content of tissues as a means of confirming suspected poisoning. Experience with field cases in the Veterinary Diagnostic Laboratory, Iowa State Uni- versity, has indicated that, if an animal lives several days after consum- ing a "toxic" amount of arsenic, the liver and kidney tissues may contain less arsenic than is ordinarily considered diagnostic of arsenic poisoning.~0 ii2 Other authors have reported similar findings.564 Signs and Lesions of Toxicosis Peracute and acute episodes of poisoning by inorganic and aliphatic organic arsenicals are usually explosive, with high morbidity and mortality over a 2- to 3-day period. The poisoning produces intense abdominal pain, staggering gait, extreme weakness, trembling, saliva- tion, vomiting (in dogs, cats, pigs, and perhaps even cattle), diarrhea, fast feeble pulse, prostration, ruminal atony, normal to subnormal temperature, collapse, and death.~5656 In subacute arsenic poisoning, animals may live for several days and show depression, anorexia, watery diarrhea, increased urination fol- lowed by anuria, dehydration, thirst, partial paralysis of the rear limbs, trembling, stupor, coldness of extremities, and subnormal tempera- ture. The stools may contain shreds of intestinal mucosa and blood. Convulsive seizures are not usual. Poisoning resulting from arsenical dips usually results in some of the signs noted previously, in addition to blistering and edema of the skin, followed by cracking and bleeding with associated secondary infection.656 Characteristic gross lesions associated with inorganic and aliphatic organic arsenic poisoning include localized or general reddening of the gastric mucosa (abomasum in ruminants), reddening of the small intestinal mucosa (often limited to the first few feet of the duodenum), fluid gastrointestinal contents (sometimes foul-smelling), a soft yellow liver, and red edematous lungs. Occasionally, in peracute poisoning, no gross changes are noted postmortem. The inflammation is usually followed by edema, rupture of the blood vessels, and necrosis of the mucosa and submucosa. The necrosis sometimes progresses to perfo- ration of the stomach or intestine. The gastrointestinal contents may
Biologic Effects of Arsenic on Plants and Animals 147 include blood and shreds of mucosa. There may occasionally be hemorrhages on all surfaces of the heart and on the peritoneum.~53 Histopathologic changes include edema of the gastrointestinal mu- cosa and submucosa, necrosis and sloughing of mucosal epithelium, renal tubular degeneration, hepatic fatty changes and necrosis, and capillary degeneration in vascular beds of the gastrointestinal tract, skin, and other organs. In cases involving cutaneous exposure, a dry, cracked, leathery, peeling skin may be prominent.656 Diagnostic Criteria In peracute, acute, and subacute poisoning, arsenic tends to be concen- trated in the liver and kidneys. Normal animals usually have a concen- tration of arsenic in these tissues of less than O.S ppm (wet-weight basis). In animals that are dying of acute or subacute arsenic poisoning, the concentration may be 2-100 ppm in these organs, usually higher in the kidneys than the liver. A concentration above 10 ppm would, confirm arsenic poisoning.~5 The urine of poisoned animals often contains protein, red blood cells, and casts. The arsenic content of the urine varies with the form of arsenic, the route of exposure, and the species and usually ranges from 2 to 100 mg/liter.~5 Whenever an episode of illness is characterized by rapid onset and gastroenteritis, with only minor signs of central nervous system in- volvement, and results in weakness, prostration, and rapid death, inorganic or aliphatic organic arsenic poisoning should be considered. The diagnosis is substantiated by the finding of excessive fluid in the gastrointestinal tract with inflammation and necrosis of the gastroin- testinal mucosa. Liver, kidney, stomach and intestinal contents, and urine should be obtained for arsenic analysis. A modified Gutzeit method has worked well in one laboratory; it involves the diges- tion of S g of wet tissue in nitric-perchloric-sulfuric acid or air oxidation in the presence of magnesium oxide in a muffle oven and the use of an arsine generator and a silver diethyldithiocarbamate arsenic- sensitive color reagent. In acute poisoning, renal tissue and often hepatic tissue may contain arsenic at more than 10 ppm (wet-weight basis). If several days have elapsed since exposure, however, the liver tissue may contain only 2-4 ppm, whereas the kidney tissue may have a diagnostically signifi- cant concentration. in 335 The concentration of arsenic in gastrointesti- nal contents and urine will also aid in determining the route and degree of exposure. Diseases often confused with arsenic poisoning, especially in rumi
148 ARSENIC nants, include hypomagnesemia (grass tetany), urea poisoning, organo- phosphorus-insecticide poisoning, bovine viral diarrhea (mucosal disease complex), and poisoning from plants containing nitrates, cyanide, oxalates, selenium, or alkaloids. Lead poisoning in bovines sometimes results in sudden death and could be confused with arsenic poisoning. However, central nervous system signs such as blindness, circling, depression, and convulsive seizures are more prominent in lead poisoning.1l5 Conditions that may be easily confused with arsenic poisoning in dogs and cats include heavy-metal intoxications (thallium, mercury, and lead) and ethylene glycol poisoning. Arsenic poisoning is con- siderably more acute than the syndromes associated with heavy met- als. Enteric infections that cause vomiting, diarrhea, and collapse can also resemble arsenic poisoning. Therapeutic Measures The key to successful treatment of inorganic and aliphatic organic arsenic poisoning is early diagnosis. Even so, the prognosis should be heavily guarded. In ruminants and horses, which do not vomit readily, large doses of saline purgative may be given in an attempt to remove the unabsorbed material from the gastrointestinal tract. Demulcents may be given to coat the irritated gastrointestinal mucous membrane. Sodium thiosul- fate should be given orally and intravenously: adult horses and cattle, 20-30 g orally in approximately 300 ml of water and 8-10 g in the form of a 10-20~o solution intravenously; and sheep and goats, about one-fourth of those amounts. British antilewisite is a sulfhydryl- containing specific antidote for trivalent arsenic. Its value as a therapeutic agent for arsenic poisoning in large animals is questionable. Therapeutic results with this compound in large animals have been disappointing, perhaps because veterinarians have not repeated the treatment every 4 h for the first 2 days, four times on the third day, and twice a day for the next 10 days until recovery is complete, as has been recommended. Five-percent BAL iS added to a loo solution of benzyl- benzoate in arachis oil and given at 3 mg/kg of body weight.~53 It is important to give supportive therapy, such as electrolytes to replace body fluids, and to provide plenty of drinking water. In small animals, if there is an opportunity for early treatment, the stomach should be emptied before the arsenic can pass into the intestine and be absorbed. Gastric ravage with warm water or a lo solution of sodium bicarbonate is preferred, although such emetics as
Biologic Effects of Arsenic on Plants and Animals 149 apomorphine may be used early in the treatment. When signs of arsenic poisoning are already present, gastric ravages or emetics should not be used. BAL should be given intramuscularly at 6-7 mg/kg of body weight three times a day until recovery. Fluids should be administered par- enterally to rehydrate animals that have been vomiting or have had diarrhea. If uremia has developed, lactated Ringer's solution should be used; B-complex vitamins may be added to the Ringer's solution. After rehydration, 10% dextrose solution should be administered at 20 ml/kg of body weight; this should result in diuresis. The urinary bladder should be catheterized to determine the rate of urine flow. If flow increases considerably after the administration of logo dextrose and the urine contains considerable sugar, the uremia may be controlled by administering lactated Ringer's solution and 5-lO~o dextrose alter- nately. If acidosis is present, 50~o sodium lactate may be added to the lactated Ringer's solution at 2.5-5.0 ml/liter. Protein hydrolysates may be added to supply amino acids, but they must be given slowly to avoid inducing more vomiting. B-complex vitamins should be injected daily, and whole blood should be transfused if indicated by the occurrence of anemia or shock. There should be no effort to administer drugs or food orally during the period when the animal is vomiting. When emesis has stopped, kaolin-pectin preparations can be given orally to aid in controlling diarrhea. Antibiotics are indicated to prevent secondary infections, and meperidine should be given as needed to lessen abdom- inal pain. As improvement occurs, a high-protein low-residue diet should be fed, and other supportive therapy discontinued.487 Phenylarsoruc Feed Additives Organic arsenical formulations have been used as feed additives for disease control and improvement of weight gain in swine and poultry since the mid-1940's. These compounds are phenylarsonic acids arsanilic acid, 3-nitro-4-hydroxyphenylarsonic acid, 4-nitrophenylar- sonic acid, and 4-ureidophenylarsonic acid and their salts. The most widely used compounds are arsanilic acid; its sodium salt, sodium arsanilate; and 3-nitro-4-hydroxyphenylarsonic acid.5l 587 The additives are considered to improve weight gain and feed efficiency and to aid in the prevention and control of some enteric diseases of swine and p 0 u 1 t r y . 7 6 , 1 1 4 , 2 6 8 , 5 5 8 , 5 5 ~ There is still considerable discussion regarding the mode of action of the organic arsenicals. However, it seems certain that the phenylar- sonic compounds have an action different from that of inorganic and aliphatic organic arsenicals. The arsenic incorporated in the additives
150 ARSENIC is in the pentavalent form, and it is likely that they have their primary action as pentavalent arsenicals, which may account for their charac- teristic rapid renal excretion. There have been several theories as to the possible therapeutic and nutritional effects of phenylarsonic feed additives. First, it is known that some organisms cause a thickening of the intestinal wall; thus, the additives may inhibit these organisms by interfering with their enzyme systems, which would result in a thinner intestinal wall and better nutrient absorption. A second theory is that the additives, by inter er- ing with the development of the bacterial cell wall or by inhibiting normal cellular production of proteins and nucleic acids, lower the harmful bacterial population. A third possibility is that these com- pounds have a sparing action on one or more of the nutrients required by growing animals.5~ Some workers have suggested that both the toxicity and the efficacy of these compounds are due to their degradation and reduction to inorganic trivalent forms 227,333,334,825 Eagle and Doak227 reported that arsenoso compounds have direct activity, whereas arsenic acid com- pounds become active when they are converted to arsenoso com- pounds. Other research, however, clearly established that arsanilic acid and acetylarsanilic acid (4-acetylaminophenylarsonic acid) were excreted unchanged by chickens and that there is no evidence that these compounds are changed to any others or converted to inorganic arsenic.~835556~36~46~6 Similar results were obtained in studies with 3-nitro4-hydroxyphenylarsonic acid and 4-nitrophenylarsonic acid in chickens. Similar experiments by other workers with rats, rabbits, and swine indicated that the phenylarsonic acids for the most part are excreted unchanged by the kidneys, although some apparently undergo a limited amount of biotransformation.556 557 Because pentavalent arsenic compounds do not react readily with sulfhydryl groups and the phenylarsonic acids are apparently excreted unchanged, one must conclude that the mechanism of their action is something other than interaction with sulfhydryl-containing enzymes and proteins. The predominant lesions produced by these compounds in swine and poultry are peripheral nerve demyelination and gliosis, and it has been postulated that the phenylarsonic acids act to produce a vitamin B-complex deficiency, such as a deficiency of vitamin B6 or B,.~5 This postulation has not been studied experimentally. When the phenylarsonic compounds are injected parenterally, they are mostly excreted in the urine within 24-48 h. When they are given orally, a considerable percentage is excreted in the feces. This indi- cates that they are poorly absorbed by the intestinal tract. The propor
Biologic Effects of Arsenic on Plants and Animals 151 lion that is absorbed, however, apparently is excreted rapidly by the kidneys.5l 555-557 614 Recommended Uses and Factors Affecting Toxicity The registered uses of the phenylarsonic acid feed additives are listed in Table 5-3. Arsanilic acid and sodium arsanilate are recommended at 50-100 ppm (0.005-0.01%) in swine and poultry feeds for improving weight gain and feed efficiency and for other uses. They are recom- mended at 250-400 ppm (0.025-0.0456) in swine feed for 5-6 days for the control of dysentery. The margin of safety for arsan~lic acid and its TABLE 5-3 Arsenic Compounds Used as Feed Additivesa Concentration in Compound Complete Feed, Go Species Purpose Arsanilic acid or O.OOS-O.O1 Chickens (broilers) Increase growth rate sodium arsanilate and feed efficiency Growing turkeys Laying hens Improve pigmen- tation Improve egg production Swine Increase growth rate and feed efficiency; prevent dysentery 0.025-0.04 Chickens (up to Prevent coccidiosis 8 days) Swine (S-6 days) Control dysentery 3-Nitro4-hydroxy- 0.0025-0.005 Chickens and Increase growth rate phenylarsonic acid turkeys and feed efficiency 0.0025-0.0075 Swine Increase growth rate and feed efficiency 0.02 Swine (S-6 days) Control dysentery 4-Nitrophenyl- 0.01875 Growing turkeys Prevent blackhead arsonic acid and chickens 4-Ureidophenyl- 0.0375 Growing turkeys Prevent blackhead arsenic acid and increase growth rate aDenved from the Feed Additive Compendium, 1975.587
152 ARSENIC salt is wide in normal animals.269 However, the effective concentration and the chronic-toxicity concentration may impinge on one another under some conditions. The health of the exposed animals and man- agement practices, especially those involving availability of water, are important contributing factors for adverse reactions to organic arseni- cal feed additives. Animals with diarrhea are usually dehydrated and thus are excreting very little urine. Because these arsenicals are excreted via the kidneys, their toxicity is greatly increased when they are given to animals with diarrhea. The morbidity is usually high, and the mortality very low. Experimentally, clinical signs appear after 3-10 days of exposure to high concentrations in the feed (e.g., 1,000 ppm) and within 3-6 weeks at lower concentrations (e.g., 250 ppm).~5 The maximal safe dietary concentration of arsanilic acid for young turkeys (up to 28 days old) was reported to be between 300 and 400 ppm (0.03 and 0.04%~.~2 Roxarsone, 3-nitro-4-hydroxyphenylarsonic acid, is recommended at 25-SO ppm (0.0025-0.005%) for chickens and turkeys and at 25-75 ppm (0.0025-0.0075%) for swine for improving weight gain and feed efficiency. It is also recommended at 200 ppm (0.02%) for S-6 days for the control of dysentery.587 Swine may exhibit clinical signs after consuming 250 ppm in the feed for 3- 10 days and have been chronically poisoned by a concentration of 100 ppm for 2 months. ~4 4-Nitrophenylarsonic acid has been recommended for chickens and turkeys at 188 ppm for prevention of blackhead. It has not been recommended for ducks or geese and has only limited use as a feed additive. Carbarsone p-ureidophenylarsonic acid) is recommended for the prevention of blackhead in turkeys and increased growth rate at 375 ppm in the feed. Arsanilic acid and sodium arsanilate are most commonly used as swine feed additives. Toxicoses may occur, however, with any of the phenylarsonics in any of the species. The circumstances usually as- sociated with toxicoses related to the organic arsenicals used as feed additives include: · Purposeful incorporation of excessive amounts in feed or water.458 · Mistaken feed formulation resulting in excessive amounts in feed. · Prolonged and excessive administration in combination with other drugs. · Treatment of animals with severe diarrhea and debilitation, which have increased susceptibility because of reduced renal excretion of the arsenical.
Biologic Effects of Arsenic on Plants and Animals 153 · Limiting of the water available to animals being exposed to therapeutic concentrations of organic arsenicals.~5830 Poisoning by organic arsenicals in swine is not uncommon and prob- ably is second in frequency only to water-deprivation-sodium-ion- toxicosis syndrome.458 Signs and Lesions of Toxicosis Acute clinical signs may appear after 3-S days of exposure to high concentrations of phenylarsonic compounds in the feed. Signs include incoordination, inability to control body and limb movements, and ataxia. After a few days, swine and poultry may become paralyzed, but will continue to eat and drink (Figure 5-41. Arsanilic acid and sodium arsanilate may produce blindness, but this is rarely seen with 3-nitro- 4-hydroxyphenylarsonic acid toxicosis. Erythema of the skin, espe- cially in white animals, and sensitivity to sunlight may also be ob- served. The clinical signs are reversible up to a point. Removing the excess arsenical will result in recovery within a few days, unless the FIGURE 54 Pig with quadriplegic after 18 days of feeding arsanilic acid at 900 goon (992 g/tonne). Reprinted by courtesy of Marcel Dekker, Inc., from Ledet et al.409 (P- 443)
154 ARSENIC clinical signs have progressed to partial or complete paralysis resulting from irreversible peripheral nerve degeneration. ~5 598 Chronic poisoning occurs in swine and poultry when excessive but lower concentrations of phenylarsonic compounds are given in the feed or water for more than a few weeks. Animals will continue to eat and drink and remain alert while progressively developing blindness and partial paralysis of the extremities. The onset of signs is usually insidious and therefore not alarming to the herdsman. Goose-stepping, knuckling of the hock joints, and other manifestations of abnormal locomotion occur. Such animals usually have poor weight gain and feed efficiency. Poultry usually become incoordinated and ataxic after consuming excessive concentrations of 3-nitro-4-hydroxyphenylarsonic acid, but more commonly exhibit ruffled feathers, anorexia, depression, coma, and death when exposed to excessive concentrations of arsanilic acid or sodium arsanilate. in ~4 539 Postmortem findings in swine and poultry affected by organic arseni- cals include no gross changes, except skin erythema in white pigs and muscle atrophy in chronic cases.~2~4326 Harding et al.326 reported abnormal distention of the urinary bladders in pigs poisoned by ar- sanilic acid. Detectable histopathologic changes in swine are confined to the optic tracts, optic nerves, and peripheral nerves. Major lesions noted are necrosis of myelin-supporting cells, degeneration of myelin sheaths and axons, and gliosis of affected tracts (Figures 5-5 through 5-71. Damage is first seen after about 6-10 days of feeding on excessive arsenical and is characterized by fragmentation of the myelin into granules and globules and, several days later, by breaking up of the axons. There is an obvious increase in the severity of the lesions with the progression of the toxic syndrome. No microscopic changes are seen in the brain, cord, kidneys, liver, or other organ systems.326 458 459 Excretion and Recommended Withdrawal Time In general, phenylarsonic compounds are rapidly excreted by the urinary system in domestic animals and poultry. Once they are ab- sorbed from the gastrointestinal tract, 50-75% of the material is excreted within 24 h. Excretion of the remaining 25% is much slower and may take 8-10 days. i25 458 555-557 Although nervous tissue tends to accumulate relatively small amounts of the phenylarsonic compounds, their excretion rate from this tissue appears to be relatively low, less than Silo excretion 11 days after withdrawal.458
Biologic Effects of Arsenic on Plants and Animals 155 Ledet et al.459 measured arsenic contents of various organs from swine after their consumption of arsanilic acid at 1,000 ppm in the diet (10 times the recommended concentration for continuous feeding for improving weight gain and feed efficiency) for 19 days. The results are presented in Table 5-4. Evans and Bandemer237 measured the arsenic content of eggs from hens fed diets containing arsanilic acid at 100 and 200 ppm for 10 weeks and found concentrations below the tolerance, established by the FDA, of 0.5 ppm. Barony reported on the accumulation and depletion of arsenic in tissues of chickens fed a ration containing 3-nitro-4-hydroxyphenyl- arsonic acid at 50 ppm (0.005%~. Medication was started when the chickens were 4 weeks old, and the birds were killed at l, 2, 3, 4, S. 7, 9, 11, 14, 28, 56, and 70 days of medication and on day 1-14 after withdrawal of medication. Five birds of each sex from both the medicated and nonmedicated groups were killed on the days indicated. The arsenic concentrations found in kidneys, liver, muscle, and skin are presented in Table S-S. FDA regulations require that all labels of feeds containing any of the phenylarsonic compounds include a warning that such feed must be withdrawn from swine and poultry S days before slaughter. Elemental TABLE 5-4 Arsenic in Swine Organs after Consumption of Arsanilic Acida Elemental Arsenic (wet wt), ppm after withdrawals Tissue Control O Days 3 Days 6 Days 1 1 Days Kidney <0.02 8.33 2.90 2.24 i.90 Liver <0.02 9.67 3.10 1.65 1.75 Muscle ' 0.92 0.29 0.29 0.31 Blood <0.02 1.94 0.25 0.19 0.45 Rib ' 0.46 0.18 0.24 0.08 Peripheral nerve <0.02 1.57 1.17 1.06 0.61 Spinal cord <0.02 0.74 0.76 0.80 0.25 Brain stem <0.02 1.04 0.90 0.91 0.62 Cerebellum <0.02 1.23 1.58 1.10 0.85 Cerebrum ' 0.82 1.09 0.84 0.51 aDenved from Ledet et al.459 bControl, mean of three animals; O days, mean of three animals; 3, 6, and I I days, mean of two animals. CNegative to test.
156 ARSENIC TABLE 5-5 Arsenic in Chickens Fed Ration Containing 3-Nitro~- Hydroxyphenylarsonic Acid at 50 ppma Arsenic Concentration (wet wt), ppm Medicated Chickens On Medication Off Medication Nonmedi cated Tissue Day 1 Day 7 Day 56 Day 70 Day 71 Day 75 Day 80 Day 84 Chickens Kidney 0.93 0.76 0.52 0.64 0.22 0.10 0.09 0.08 0.05 Liver 1.31 2.43 1.26 1.26 0.69 0.43 0.32 0.19 0.08 Muscle 0.03 0.07 0.05 0.04 0.03 0.01 0.02 0.02 0.02 Skin 0.05 0.11 0.06 0.05 0.08 0.02 0.03 0.03 0.02 aDenved from Baron.51 arsenic tolerances of 2.0 ppm for uncooked swine liver and kidney tissues and 0.5 ppm for uncooked pork muscle and edible chicken and turkey tissue and eggs have been set. Diagnosis of Toxicosis Produced by the Phenylarsonic Acids Organic arsenical poisoning in swine and poultry can be diagnosed tentatively on the basis of the characteristic signs of wobbly, incoordi- nated gait and ataxia. Animals and birds that have paralysis of the extremities without central nervous system involvement, that undergo high morbidity with low mortality, that continue to eat and drink if food and water are made available (especially in the case of swine), and that show little or no gross change on postmortem examination should be suspected of having been exposed to excessive concentrations of phenylarsonic compounds.5~ (Figure 5-4 shows the appearance of a pig suffering from arsenic paralysis.) Concentrations of arsenic in tissue are rarely diagnostic, because the organic arsenicals are excreted without being metabolized by the kidneys. If the animal has not been eating for 3-5 days, the arsenical will for the most part have been excreted from the body and will not be of diagnostic value. If liver and kidney specimens are obtained from animals that have been on feed containing excessive organic arsenical, an arsenic concentration of 3-10 ppm (wet-weight basis) would have diagnostic significance. Blood concentrations of 1-2 ppm would also be diagnostically significant. More important diagnostically is the concentration of organic arsenical in the feed. Arsanilic acid and
Biologic Effects of Arsenic on Plants and Animals 157 3-nitro-4-hydroxyphenylarsonic acid concentrations of 250 and 100 ppm, respectively, should be viewed as significant if such other factors as diarrhea and limited water intake are evident in swine and poultry. Microscopic examination of longitudinal sections of peripheral and cranial nerves is important in confirming a diagnosis of organic arseni- cal toxicity in swine and poultry. It should be kept in mind, however, that demyelination and gliosis will not be evident in the optic tract earlier than 10 days after the beginning of exposure, nor will these lesions be evident in sciatic and brachial nerves earlier than 2 weeks after the beginning of exposure326 458 (see Figures 5-5-5-71. AQUATIC ORGANISMS Because arsenic compounds are poisonous to microorganisms and lower aquatic organisms, they have been used in wood preservatives and paints and in pesticides. Arsenates have a limited use in power-plant cooling towers to control various fungi that attack and cause deterioration of structural wood. However, they are rarely used for this purpose, because of their relatively high toxicity; instead, a "preservative" (which may contain arsenate) is used. It has been suggested that some of the "preserva- tive" may enter the aquatic environment. FIGURE 5-5 Sciatic nerve from control pig. An axon appears as a faint gray line between arrows 1. Note darkly stained neurokeratin network, arrow 2. Harris hematoxylin and eosin Y stain. Reprinted by courtesy of Marcel Dekker, Inc., from Ledet et al. 459 (Pa 447)
ARSENIC FIGURE 5-6 Sciatic nerve from pig fed arsanilic acid at 900 g/ton (992 g/tonne) for 16 days. Note contraction of myelin around intact axon, arrow 1; myelin fragment, arrow 2; and myelin ovoid, arrow 3. Harris hematoxylin and eosin Y stain. Reprinted by courtesy of Marcel Dekker, Inc., from Ledet et al. 459 (Pi 447) FIGURE 5-7 Sciatic nerve from pig fed arsanilic acid at 900 g/ton (992 g/tonne) for 27 days. Animal had developed quadriplegic. An estimated 60~o of the nerve fibers were damaged. Note axon with myelin contracting around it, arrow 1; fragment of myelin, arrow 2; and myelin-containing fragment of axon, arrow 3. Fragmented axon is stained darker than myelin. Harris hematoxylin and eosin Y stain. Reprinted by courtesy of Marcel Dekker, Inc., from Ledet et al. 459 (P' 448)
Biologic Effects of Arsenic on Plants and Animals 159 Arsenic has been found to be quite toxic to invertebrates and has therefore found application in the control of the shipworm Bankia setacia and other wood-borers. It tends to be accumulated by mollusks and may have chronic effects on them. In addition to the acute toxicity of chemical compounds under controlled laboratory conditions, there is a need to examine pollutants for chronic toxicity. The long-term effects of exposure to sublethal concentrations may be as important as direct lethality, in that such exposure may limit development, growth, reproduction, metabolism, or other physiologic processes. For those who are charged with re- sponsibility for managing the aquatic environment and its renewable resources, it may be important to know the sublethal concentrations of arsenic at which long-term chronic effects become manifest. In es- tuaries, for example, where migrating anadromous fish tend to linger in order to become acclimatized to changing salinity, the sublethal con- centrations of a pollutant could have serious consequences. Although the fish may not be killed, the stress of sublethal concentrations of pollutants may have serious biochemical, physiologic, and behavioral implications. Adult fish migrating upstream may be unable to reach their spawning grounds or may be unable to reproduce for other reasons. Effects of long-term exposure to low concentrations of arsenic singly or in combination with other metals are generally unknown. Pollutants are rarely found in the environment in isolation. Most laboratory bioassays are conducted on single chemicals under con- trolled conditions. This provides a simpler toxicologic experiment than do mixtures. It is known, however, that some substances can act synergistically or antagonistically. Arsenic renders selenium less toxic and has been experimentally added to feeds for cattle and poultry in areas high in selenium. The two elements appear to have an antagonis- tic effect on each other, causing a reduced toxicity. Copper and mercuric salts, however, are each more toxic when in the presence of the other. Although considerable information has been published on the effects of arsenic on aquatic organisms, most of the research has concentrated on freshwater organisms;~73 348 very little is known about effects on marine organisms. Again, however, most of the data collected have been related to lethality, not sublethal physiologic stress. In compiling data on the effects of arsenic on aquatic organisms, considerable use was made of two publications, Water Quality Criteria03i and Toxicity of Power Plant Chemicals to Aquatic Life. Although it was not possible to review all the original articles covered in those two documents, the information provided can be used to
160 ARSENIC delineate the effects of arsenic on aquatic organisms, particularly fish and shellfish. Toxicity data on arsenic for fish and shellfish are com- piled in Table 5-6. The toxicity data given for a particular compound can be highly variable, not only because of different responses by different aquatic organisms, but also because of such other factors as water quality. Among the characteristics of water that can influence the results of bioassays are temperature, pH, dissolved oxygen, con- ductivity, hardness, oxidation-reduction potential, dissolved chlorides, turbidity, and the presence of potentially toxic ions. With respect to the lower forms of aquatic life, arsenic concentra- tions of 3-14 ppm have not harmed mayfly nymphs, and concentra- tions of 10-20 ppm have been harmless to dragonflies and dam- selflies.692 Surber and Meehan774 carried out a comprehensive study of the toxicity of arsenic trioxide to many different fish food organisms, and their results indicated that those organisms could tolerate a concentra- tion of 2.0 ppm. According to Jones,400 sodium arsenate is not highly toxic to fish. He found that sodium arsenate at 234 ppm, as arsenic, was lethal to minnows at 16-20 C. Sodium arsenite has been used extensively as an herbicide for the control of mixed submerged aquatic vegetation in freshwater ponds and lakes. Commercial sodium arsenite contains various amounts of other arsenic compounds and impurities and is labeled in terms of equivalent arsenic trioxide. For the control of submerged vegetation in ponds and lakes, applications of 2-5 ppm as arsenic trioxide (1.5-3.8 ppm as arsenic) have been found effective.455 772 773 These concentra- tions are generally considered to be safe for fish. WILDLIFE The association of arsenic with murder and suicide has made its agricultural and industrial uses particularly controversial. It has often been claimed, for example, that widespread kills of domestic animals, songbirds, and other wildlife were caused by extensive use of arseni- cals as pesticides. It must be borne in mind that the biochemical characteristics of this family of compounds vary considerably. Previous chapters have shown that the term "arsenicals" does not imply a homogeneous group of compounds, but rather a heterogeneous group that have highly indi- vidualistic properties and, in particular, greatly varying toxicity. Arse
Biologic Effects of Arsenic on Plants and Animals 161 nic is a ubiquitous substance that is commonly found in animal tissues, even when pollution is not suspected. By and large, the organic forms of arsenic are less toxic than the inorganic forms, and the pentavalent compounds are usually less hazardous than trivalent.475 Unfortunately, most published reports refer to total arsenic concentrations, and not to specific forms of arsenic. Toxic oral doses of several arsenicals in some common wild species are listed in Table 5-7. Early work on this subject was concerned with the effects of the use of arsenicals in various insecticides. Chappellier and Raucourti44 reported on extensive studies in France with domestic rabbits, wild rabbits (Sylvilagus), hares (Lepus), and the gray partridge (Perdix perdix) to evaluate the potential hazard of using lead arsenate, copper acetoarsenite, and calcium arsenate to control the potato beetle (Doryphorus). The arsenicals were incorporated into starch pellets and given orally to the experimental subjects. The authors noted that the wild hares and domestic rabbits succumbed to similar amounts of the arsenicals, but the former were able to tolerate doses for slightly longer periods e.g., arsenic at 40 mg/kg as lead arsenate killed the domestic rabbit in about 24 h, compared with 60 h for the hare. These studies showed that the mammals were generally more susceptible to the arsenicals studied than the partridges, which in turn were significantly more sensitive than domestic fowl. Rabbits generally appeared to consider foliage treated with the three arsenicals repugnant and con- sumed other food if given the choice. The authors concluded that the practice of using these arsenicals to control the potato beetle did not pose a great threat to wildlife. In a series of toxicity studies, Heath et al.34i and Hill et al.353 found that the mallard (Anas platyrhynchos) is even more tolerant to arseni- cals than gallinaceans, such as quail and pheasants (Table 5-71. These observations are based on 8-day Ecus determinations; 2- to 3-week-old animals were fed arsenicals in the diet for 5 days and then given an arsenic-free diet for the remaining 3 days. The order of sensitivity among the several species of birds observed by these workers is bobwhite (Colinus virginianus) > Japanese quail (Coturnix coturnix japonica) >ring-necked pheasant (Phasianus colchicus)> mallard. Earlier reports have shown the mallard to be tolerant to an arsenic dosage of 8 mg/day as sodium arsenite for a period that provided a total dose of 973 mg/kg. Early work by Chorley and McChleryt49 and VanZyl820 showed that arsenic-poisoned grasshoppers could be fed to domestic fowl without lethal effect and, in fact, were recommended as a supplement to poultry feed in Rhodesia. These studies were conducted with arsenic
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66 ARSENIC trioxide-poisoned grasshoppers containing arsenic at up to 910 ppm as a dry meal. Lilly470 fed grasshopper bait containing sodium arsenite or freshly poisoned grasshoppers to ring-necked pheasants without ap- parent ill effect. One bird consumed over 2,500 poisoned grasshoppers (approximately 8 mg of arsenic) in a 20-day period and, after a 7-day rest period, was sacrificed and analyzed for arsenic (Table 5-81. Little accumulation of arsenic was observed. The author noted that pheas- ants were reluctant to consume arsenic-containing baits, but readily consumed poisoned grasshoppers. The results indicate that there was little danger to this species from the grasshopper-poisoning activities. Helminen342 also noted that spraying a potato field with an arsenical insecticide had little effect on pheasants penned on this field. Several studies conducted by Whitehead854 (Table 5-9) are ques- tionable, although noteworthy because they are among the few that involved songbirds. Arsenic trioxide was used to poison western grass- hoppers (Melanophis bivittatus, M. femus-rubrum, M. bispinosus), which were fed to bobwhite quail, mockingbirds (Mimus polyglottos), robins (Turdus migratorious), meadowlarks (Sturnella magna), redwing blackbirds (Agelaius phoeniceus), brown thrashers (Toxos- toma radium), dickcissels (Spiza americana), orchard orioles (Icterus spurius), scissortails (Muscivora forficata), and English sparrows (Passer domesticus). The author noted that, when quail consumed the maximal amount (25 g) of grasshoppers, less than lOYo of a toxic dose was ingested; therefore, no detrimental effects were noted. Poisoned grasshoppers were force-fed to the various nestling birds in the wild. Many uncontrollable variables consequently also affected the outcome of these experiments. The results showed that fairly large numbers of poisoned grasshoppers (up to 134, containing a total of about 40 mg of arsenic) could be fed to nestling songbirds without any noticeable toxic effect. About 49~o of all the birds fed poisoned grasshoppers in this experiment matured, compared with about 60~o of those fed un- poisoned grasshoppers. Because there was great variability in these data, no significant detrimental effects were attributed to the arsenic consumption. Generally, the data indicated that songbirds experienced little danger from the ingestion of this pesticide in the form of poisoned grasshoppers. Work undertaken at the Patuxent Wildlife Research Center698 to evaluate the possible dangers of widespread use of copper acetoarse- nite for mosquito control, particularly in southern marshes, indicated that there is little hazard when this compound is applied at the recommended rate of 0.75 lb/acre (0.84 kg/ha). Male cowbirds (Mola- thrus ater) were poisoned only when fed copper acetoarsenite at about
Biologic Effects of Arsenic on Plants and Animals 167 225 ppm (arsenic at 100 ppm) in the diet (Table 5-7) for 3 months; similar diets containing copper acetoarsenite at 25 and 75 ppm (arsenic at 11 and 33 ppm) appeared to have no effect on mortality. Arsenic residues (Table 5-8) were determined in birds that had died from consuming diets containing arsenic at 100 ppm (W. H. Stickle, per- sonal communication). Whole-body concentration reached a peak of about 1.7 ppm (dry weight) in yearling male cowbirds after about 6 months of feeding arsenic at 11 ppm in the diet and thereafter appeared to level off. Birds given arsenic at 33 ppm reached a maximal whole- body concentration of about 6.6 ppm for the same period. Whole-body arsenic content continued to rise at this dose, reaching 8.6 ppm at 7 months. The latter concentration approaches toxicity. The herbicidal properties of arsenic made its use as a tree-debarker an important factor in the northeastern U.S. wood pulp industry in the 1940's and 1950's. Cooki62 reported two cases in New York in which about 10 white-tailed deer (Odocoileus virginianus) consumed fetal amounts of sodium arsenite that was used to debark pulp trees. Field studies by Boyce and Verme93 showed that 923-2,770 mg of arsenic (as sodium arsenite) was lethal to deer when licked from the bark of treated trees. No body weights were given, but, if we assume that one of the poisoned deer (a yearling doe) weighed about 27 kg, then we can calculate a minimal lethal dose of about 34 mg/kg. In a study to determine the palatability or acceptability of sodium arsenite, potas- sium arsenite, and ammonium arsenite to deer, the authors recorded the number of licks that the test deer made on trees coated with solutions of these compounds. They observed that sodium arsenite was as palatable as sodium chloride, whereas potassium arsenite was significantly less palatable, and ammonium arsenite was the least acceptable to the deer. The authors also report that wildlife kills from arsenic poisoning in Michigan's upper peninsula in 1952 amounted to five deer, four porcupines, and one rabbit on about 200 acres (81 ha) of commercially treated trees. In 1953 and 1954, apparently, only one wildlife mortality was found in over 5,000 acres, or 2,023 ha (which contained over 500 acres, or 202 ha, of treated trees), in lower Michi- gan. The practice of debarking trees with arsenicals for commercial use has been almost completely replaced by mechanical debarking equip- ment. A well-documented report of a wildlife killoff attributable to arsenic was made by Swiggart et al.776 They reported the poisoning of 23 white-tailed deer in Tennessee by the apparent misuse of arsenic acid as an herbicide to control Johnsongrass. The herbicide (USDA Reg. 295-6) was labeled for use in controlling crabgrass and Dallisgrass on
68 ARSENIC Bermuda-grass lawns and was applied to a 600-acre (243-ha) field in preparation for planting soybeans. A 5-gal mixture containing 0.5 gal of arsenic acid was applied per acre (0.05 m3 of mixture containing 0.005 m3 of arsenic acid per hectare). The dead deer were all found on the 600-acre field and appeared to have died on the same day. Appar- ently, the toxicity of this herbicide dissipated in a few days, inasmuch as no further mortalities were recorded. Autopsies showed that the deer died of massive hemorrhagic gastroenteritis. Analyses performed by the EPA Toxicology Laboratory showed that surface soil samples contained arsenic at up to 2.4 ppm, whereas water samples from the area averaged 0.42 ppm. Arsenic concentrations in the dead deer are shown in Table 5-8. It is interesting that the farmer and cropduster using the pesticide in this case were both taken to court by the Tennessee Health Department and sued for damages. In an apparently unprecedented decision, the defendants were made to pay for the poisoned deer $109/head, the amount needed to transport new deer into the area (W. D. Turner, personal communication). In March 1974, another deer kill involving at least two white-tailed deer was discovered in southwest Memphis and Shelby County, Ten- nessee, by R. C. Swiggart and W. D. Turner (personal communica- tion). Although these deer were found in an area that had been treated with a cotton defoliant in the fall of 1973, the probable cause of death was determined to be the contamination of the water in a runoff ditch with MSMA. Several empty MSMA drums were found in the ditch, and mixing apparatus was nearby. Tissue analyses of these deer (Table 5-8) showed what are considered toxic concentrations of arsenic. This is in agreement with the studies of Dickinson,~76 who showed that the concen- trations of arsenic in the livers of cattle fed toxic doses of MSMA were less than half those found in these dead deer. Analyses for chlorinated pesticides and other heavy metals were performed, but none were present in apparently toxic concentrations. As a point of perspective, these deer were found on land cultivated by the same farmer referred to by Swiggart et al.776 Although both reports can be related to misuse of arsenicals, the Swiggart et al. report appears to refer to an instance in which wildlife died from consuming contaminated herbage. It is there- fore in contrast with other reports cited here that indicated little hazard from the extensive use of some arsenic compounds. This is probably because of the varying toxicity of the numerous arsenicals, as well as the saturation effects of spraying huge acreages with arsenicals and leaving little untreated foliage for the local wildlife population to consume.
Biologic Effects of Arsenic on Plants and Animals 169 Little information is available on background or environmental con- centrations of arsenic in various wild species. Bencko et al.,64 how- ever, stated that rabbits reproduced normally when exposed to air from a plant that discharged large amounts of arsenic for up to 12 months. They observed significant although apparently nontoxic accumulations of arsenic in the kidneys, hair, and nails of rabbits exposed for 9 and 12 months. Martin and Nickerson524 monitored starlings (Sturnus vul- garis) from 50 sites in the United States during 1971; except for one sample in Michigan, all contained arsenic (whole body) at 0.04 ppm or less (Table 5-81. Similarly, Stickle (personal communication) found that trapped yearling male cowbirds not exposed to arsenic had brain, liver, kidney, muscle, and feather-skin concentrations of less than 0.78, less than 0.41, less than 1.48, less than 0.19, and 0.13 ppm, respectively. Andren et al. }7 have monitored the ecosystem of the Walker Branch watershed in Tennessee for a number of trace elements, including arsenic. Unfortunately, arsenic was determined by spark-source mass spectrometry with a method that is only about 50~o accurate. The quantitative validity of these data are therefore questionable, but the trends observed are worth noting. Such animals as earthworms (19 ppm) and cryptozoa (100 ppm), which are close to the soil surface and tree roots (11 ppm), contained high concentrations of arsenic. Simi- larly, tree-canopy insects also had high arsenic contents (10 ppm). Arse- nic was the only element studied, however, that showed any decline in concentration with higher trophic levels. For example, fieldmice con- tained arsenic at 1 ppm of the whole body, but owls, which consume large quantities of mice, contained only 0.05 ppm (Table 5-81. It is noteworthy that the hawk sample contained 8 times as much arsenic as the owl, although one would expect that they consumed similar diets. In 1969, a large dieoff of common auks guillemots (Uria allge), razorbills (Alca torda), and puffins (Fratercula arctica) was observed in the Irish Sea. Because the populations of these species of seabirds have been declining in recent years, an extensive study360 of this dieoff was undertaken by the Natural Environment Research Council of Britain. These birds congregate in large groups in open water and spend most of the year at sea, except during March and April, when they gather at breeding grounds. It is significant that the killoff oc- curred in the late summer and early fall, when the birds are flightless because of molting. The dieoff period began in late July and ended in mid-October, when a total count of over 12,000 dead birds was re- corded. Most of the dead and dying birds were washed ashore by storms, and nearly all the birds were severely emaciated. Although
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172 ARSENIC TABLE 5-9 Results of Feeding Arsenic Trioxide-Poisoned Grass- hoppers to Wild Birdsa Species Arsenic Total Dose, mg Comment Bobwhite quail 141 No effect observed Mockingbird (nestling) 27.2 Matured Robin (nestling) 40 Matured Meadowlark (nestling) 17.2 Matured Redwing blackbird (nestling) 13.8 Matured Brown thrasher (nestling) 5.7 Died Dickcissel (nestling) 10 Matured Orchard oriole (nestling) 10.9 Matured Scissortail (nestling) 12.2 Matured English sparrow (nestling) 10 Matured aDerived from Whitehead.854 extensive pathologic, microbiologic, and chemical testing was con- ducted, no conclusive explanation for the deaths was determined. The arsenic content of livers from 36 guillemots ranged from less than 0.1 to 41 ppm (dry basis), with an average of 7.1 ppm. Of the 36 samples, only five contained arsenic at more than 10 ppm. Furthermore, apparently healthy birds shot in the same area were found to contain arsenic at 0.7-20 ppm (average, 5.6 ppm) in their livers. These data are similar to those obtained on the birds in the dieoff and do not indicate that arsenic was directly involved in this instance.
