The Science of the Total Environment, 105 ( 1991 ) 13-28 Elsevier Science Publishers B.V., Amsterdam

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Evolution of trimethylarsine by a Penicillium sp. isolated from agricultural evaporation pond water K. Davis Huysmans and W.T. Frankenberger, Jr* Department of Soil and Environmental Sciences, University of Cal~/'ornia, Riverside, CA 92521, USA (Received Augus! 24th, 1990; accepted October 6th, 1990)

ABSTRACT Arsenicals are used in agriculture a:; pesticides and defoliants, in the Central Valley o1" California, arsenic is present in soil at naturally high concentrations, being derived from marine sedimentary parent material of the Coastal Range. Due to intense agricultural irrigation, soluble arsenic is leached from the soil and accumulates in evaporation ponds where it may pose an environmental threat to the waterfowl and wildlife. A Penicillium sp. isolated from evaporation pond water was found to be capable of methylating and subsequently volatilizing organic arsenic. The major focus of this study was to characterize the environmental conditions. including culture media, arsenic substrates, pH, temperature, and the presence of phosphates, carbohydrates and amino acids on the methylation of arsenic. Trimethylarsine was monitored by gas chr,m~atography(GC)-flame ionization detection and identified by GC-mass spectrometry. The conditions or additions for optimum trimethylarsine production were: a minimal medium in which 100mgl ~ methylarsonic acid served as the arsenic source, pH 5-6, temperature of incubation 20°C, and phosphate concentration of 0.1-50mM (KH.,PO4). The addition of carbohydrates and sugar acids to the minimal medium suppressed trimethylarsine production. The .amino acids phenylalanine, isoleucine, and glutamine protnoted trimethylarsine production with an enhancement ranging from 10.2- to 11.6-fold over the control without amino acid supplementation. The information obtained from this study may be useful in developing a bioremediation approach in trapping the arsenic gas evolved from soil or water as a mitigation alternative in the cleanup of arsenic contamination.

INTRODUCTION

The Central Valley of California is one of the most productive agricultural regions in the world because of its climatic conditions. However, the soils on the west side of the valley contain high amounts of soluble salts and trace *Author to whom correspondence should be addressed.

0048-9697/91/$03.50

(i) 1991 ..... Elsevier Science Publishers B.V.

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K.D. HUYSMANS AND W.T. FRANKENBERGER JR

elements, being derived from marine sedimentary parent material of the Coastal Ravage. The trace elements of concern include selenium, arsenic, boron, chromium, mercury, and molybdenum (Letey et al., 1986). Recently there has been some concern with high levels of arsenic occurring in the Tulare Lake Basin occupying approximately one-third of the southern Central Valley. Elevated concentrations of arsenic in evaporation pond water in the Tulare Lake Drainage District South Basin may pose a threat to wildlife. Levels of arsenic in these drainage evaporation ponds have been reported to be as high as 2400 #gl -j (R. Fujii, 1988. Water quality and sediment chemistry data of drain water and evaporation ponds from Tulare Lake Drainage District, Kings County, California, U.S. Geological Survey, 19pp.). The ubiquity of arsenic, its biological toxicity, and its redistribution in the environment are factors evoking public concern (Tamaki and Frankenberger, 1991). Microbial transformations of arsenic in soil and water involve oxidation, reduction and methylation reactions. Methylation of arsenic involves the conversion of inorganic and organic arsenic into volatile organic methylated forms such as dimethylarsine and trimethylarsine. Inorganic arsenic methylation is coupled to the methane biosynthetic pathway of methanogenic bacteria and may be a mechanism for arsenic detoxification (McBride et ai., 1978). The pathway proceeds by reduction of arsenate to arsenite followed by methylation in the presence of coenzyme M (CoM), a low molecular weight cofactor found in all methanogenic bacteria. Anaerobic biomethylation of arsenic by bacteria proceeds only to dimethylarsine, which is unstable in the presence of oxygen. The importance of fungal metabolism of arsenic dates back to the Nineteenth Century when a number of poisoning incidents in Germany and England were caused by a volatile methylarsine gas. The victims lived in musty rooms with a characteristic garlic-like odor. Trimethylarsine was identified as the toxic compound (Challenger, 1945). Moulds growing on wallpaper decorated with arsenical pigments (Scheele's green and Schweinfiirter green) produced the toxic trimethylarsine gas. Since then, several species of fungi have been isolated capable of volatilizing arsenic (Cox and Alexander, 1973a). The fungus Penicillium brevicaule (Scopulariopsis brevicaulis) produces trimethylarsine when grown on bread crumbs containing either methylarsonic acid (MAA) or dimethylarsinic acid (DMA). The biochemical pathway for trimethylarsine production has been proposed by Challenger (1945). In more recent studies, three diffe~oent fungal species, Candida humicola, Gliocladium roseum, and Penicillium sp., were found to be capable of converting MAA and DMA into trimethylarsine (Cox and Alexander, 1973a, b). The fungal methylation pathway for the formation of trimethylarsine is: MAA ~ DMA --, trimethylarsine oxide ~ trimethylarsine

EVOLUTION OF 1RIMF.1HYLARSINE BY A P E N I C I L L l l M SP.

!5

It has been demonstrated that various soils have the potential to produce alkylarsines (Woolson, 1977). Soils amended with inorganic and organic arsenic herbicides produce dimethylarsine and trimethylarsine (Woolson and Kearney, 1973; Woolson, 1977; Baker et al., 1983; Hassler et al., 1984). The organisms responsible for volatilization of arsenic are from diverse environments, suggesting that a number of different species have the capacity to produce alkylarsines. It was demonstrated that mixed communities of microorganisms in soil produce dimethylarsine and trimethylarsine in the headspace trapped in bell jars over soil and law~: treated with methylarsenic~,.ls (Braman and Foreback, 1973). The major focus of this study was to characterize the environmental conditions which affect biomethylation and subsequent volatilization of arsenic by a Penicillium sp. isolated from evaporation pond water. The parameters considered in monitoring volatilization of arsenic included various culture media, arsenic substrates, pH, temperature, and the presence of phosphate, carbohydrates and amino acids. MATERIALS AND METHODS

Reagents Sodium arsenite (NaAsO2) and sodium arsenate (Na2HAsO4" 7H,O) were obtained from Fisher Scientific (Pittsburg, PA), and MAA (disodium salt)and DMA were purchased from Pfaltz and Bauer, Inc. (Stamford, Coan.). Trimethylarsine was obtained from Morton Thiokol, Inc. (Danvers, Mass.). The following carbohydr~tes were obtained from Sigma Chemical Co. (St. Louis, MO): arabinose, galacturonic acid, glucose, glucuronic acid, maltose, ral-finose, rhamnose, sucrose and xylose. The following L-amino acids were also obtained from Sigma Chemical Co. (St. Louis, MO): arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tr)ptophan, tyrosine and valine.

Em'ichmeat Water samples were collected from an evaporation pond consisting of a reservoir of agricultural drainage water at the Martin Ranch (Tulare County, CA). The water was collected in Nalgene bottles, transported on blue ice, and stored at 5°C. A l-ml sample was taken from the evaporation pond water a~,d a series of dilutions were made in sterile tap water. Aliquots (100#i) were plated out onto an isolation medium consisting of the following per liter:

16

K.D. HUYSMANS AND W.T. FRANKENBERGER JR

CaC12"2H20, 0.15g; MgSt34.7H20, 9.25g; casamino acids, 1.0g; peptone, 1.0g; agar, 15.0g, and supplemented with 1000rag of arsenic as MAA per liter. The inoculated plates were incubated at reom temperature (23°C) for 3 days. Individual colonies were "~,lected from the plates for further culturing to determine if they produced trimethylarsine (see Method of assay). The most active methylating organism, isolated from the Martin Ranch water, was identified as a Penicillium sp. The inoculum was prepared by harvesting spores from fungal lawns into sterile tap water and diluting to yield 175 colonyforming units (CFUs) per 250/~1 on Difco R2A medium and 140CFUs per 250/~l on a minimal medium (composition is described below).

Method of assay Routinely, trimethylarsine production was monitored as follows: 5 ml of a minimal medium was added to a 10-ml screw cap Erlenmeyer flask. The minimal medium was composed of the following per liter: phosphate stock solution, 10ml; trace element stock solution. 10ml; yeast extract, 0.1 g; ammonium nitrate, I g; and agar, 15 g (Huysmans and Frankenberger, 1990). The phosphate solution contained the following per liter: KH, PO4, 1.36g; Na2HPO4"7H20, 2.13g; and MgSO4"7H20, 0.2g. The trace element stock solution contained the following per liter: CaCI.,.2H.,O, 530 mg; FeSO4.7H.,O, 200mg; CuSO4"5H20, 40mg; MnSO4.5H, O, 20mg; ZnSO4.7H.,O, 20mg; H3BO~, 3rag; CoC1,., 4mg; Na, MoO4"2H,O, 4mg; and concentrated sulfuric acid, I ml. The minimal medium was supplemented with the following arsenic species: sodium arsenite (NaAsO,.), sodium arsenate (Na.,HAsO4.7H,O), MAA and DMA. Each of the arsenic compounds was dissolved in deionized water and filter sterilized (0.22/~m pore size, Millipore, TypeGS, Bedford, Mass.) using stainless steel pressure filter holders (Fisher Scientific, Pittsburgh, PA) and added to the sterile medium to achieve the desired arsenic concentration per liter. The flasks were inoculated with 250 td of the spore suspension and sealed with gas-tight Mininert valves (Dynatech, Baton Rouge, LA) disinfected with 70% ethanol. To help prevent gas leakage the caps were sealed with Teflon tape. The flasks were incubated at room temperature (23°C) for 7 days and then sampled for trimethylarsine. A l-ml gas sample was taken from the headspace of the 10-ml Erlenmeyer flask with a gas-tight series 2 Pressure-Lok gas syringe (Alltech, Deerfield, IL) and directly injected into a gas chromatograph. The analysis was performed on a Shimadzu GC-Mini 2 (Kyoto, Japan), connected to a Hewlett-Packard (Avondale, PA) 3390A integrator. Operational conditions were as follows: detector, flame ionization: stainless steel column, Chromosorb 101 (mesh 80/100) (6foot by l/8inch o.d.); column temperature, 135°C; injector and

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LUTION OF TRIMETHYLARSINE

BY A PFNICILLIUM SP.

17

detector temperature, 180°C; N2, 40mlmin- ~" H, 60mlmin- ~" air, 450ml min -~. Calibration of trimethylarsine was performed with a standard for quantification.

Gas chromatography~mass spectrometry The methylated gas was identified by gas chromatography/mass spectrometry (GC/MS). The gas chromatograph (Hewlett-Packard Model 5890) was equipped with a 30m x 0.32mm DB-5 capillary column (J&W Scientific, Folsom, CA) and connected to a Hewlett-Packard Model 5970 MSD mass spectrometer. The operating conditions were as follows: injector temperature, 220°C; column temperature, 50°C rising to 180°C after 4 min at 8°C per min; mass range, 40-270; scan rate, l s-~; threshold, 600; electron impact, 70eV; ionizing source temperature, 250°C. A I-ml sample was taken from the headspace using a gas-tight syringe and directly injected. The mass spectrum was interpreted on the basis of molecular weight and fragmentation pattern of trimethylarsine as a reference standard.

Method of data analysis All experiments were conducted in replicates of five. Trimethylarsine released in the headspace is reported as nanograms per milliliter. Error bars in each figure depict the standard error of the mean. RESULTS AND DISCUSSION The evolved arsenic gas from the Penicillium sp. was unequivocally identified as trimethylarsine by GC/MS. l'he identity of this gas was based upon its molecular weight and characteristic fragmentation pattern in relation to a standard spectrum of trimethylarsine. The gas exhibited a base peak at 120m/z with fragmentation occurring at m/zl03, 89 and 77. Cox and Alexander (1973a) also confirmed the identification of trimethylarsine from fungi isolated from sewage with an apparent peak at 120 m/z.

Factors affecting volatilization of arsenic Media Various culture media were tested as a growth matrix for this Penicillium sp. and its subsequent production of trimethylarsine (Fig. 1). Growth upon the minimal medium supplemented with 100mgl -~ MAA resulted in the highest yield of trimethylarsine. Second to the minimal medium was potato dextrose followed by R2A and Sabourauds dextrose agar supplemented with

K.D. H U Y S M A N S A N D W.T. F R A N K E N B E R G E R JR

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MAA (100mgl-I). Difco R2A agar is a low nutrient-based medium containing yeast extract, peptone, casamino acids, dextrose, starch, and pyruvate. Both potato dextrose and Sabourauds dextrose media are frequently used for cultivating yeast, fungi, and aciduric microorganisms. The minimal medium was most effective in promoting volatilization of arsenic, most likely because it lacked a carbon source other than MAA, which forces preferential utilization of the organic arsenic. Preliminary experiments indicated that the yeast extract added to the minimal medium did not serve as the carbon source for growth. Th~ other three media contained peptone, glucose and/or potato as an organic carbon source. Apparently, Penicillium sp. will utilize other carbon sources over MAA when supplied in the culture medium.

EVOLUTION OF TRIMETHYLARSINE BY A PE.%'I('II.LII'.~I SP.

19

Arsenic substrates Tnnethylarsine production by the Peniciilium sp. was monitored in the presence of various concentrations of arsenate, arsenite, MAA and DMA. When cultivated on Difco R2A agar containing either arsenite or arsenate, there was no trimethylarsine released into the headspace. Difco R2A agar was used because a carbon source was needed to maintain the Penicillium sp. for growth and proliferation. This indicates that the Penicillium sp. could not convert inorganic arsenic into a volatile gaseous form. Trimethylarsine gas was only detected when MAA and DMA were incorporated into the minimal medium. The highest yield of trimethylarsine (158 ng trimethylarsine m l - ' ) was found upon incubation with 100mgl -~ of MAA (Fig. 2). Concentrations as high as > 500mgl -~ MAA inhibited production of trimethylarsine and 200

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20

K.D. HUYSMANS AND W.T. FRANKENBERGER JR

may be toxic to the Penicillium sp. Although trimethylarsine was also detected with the addition of DMA to the minimal medium, considerably less was detected (9.9 ng trimethylarsine ml-t). The optimum concentration of DMA added to the minimal medium in promoting trimethylarsine production was 1000mgl -I (Fig. 3). The addition of 100mgl -I of MAA to the minimal medium resulted in 16-fold greater production of trimethylarsine than the most optimum concentration of DMA added (1000mgl-I). The amount of arsenic recovered within the headspace as trimethylarsine was proportional to the increasing concentration of DMA added to the minimal medium. Cox and Alexander (1973a) found that Gliocladium roseum also produced greater amounts of trimethylarsine when cultivated in the presence of MIrA compared with DMA. In support of our work, Cox and Alexander (1973a)

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21

EVOLUTION OF TRIMETHYLARSINE BY A PE,~'I('II.LIt'M SP.

did not detect trimethylarsine by gas chromatography when this organism was grown in the presence of either sodium arsenite or sodium arsenate at concentrations ranging from 10 to 1000 mg l- ~. Similar observations were noted upon work with a Penicillium sp. isolated from sewage (Cox and Alexander, 1973a).

pH The minimal medium supplemented with 100 mg 1- ~of MAA was adjusted at various pH from 5.0 to 8.5. Penicillium sp. was inoculated onto the medium and trimethylarsine gas was assayed within the headspace of each flask. The results indicate that the optimum pH for trimethylarsine production occurs between pH 5.0 and 6.0 (Fig. 4). Increasing pH ( > 6.5-8.5) showed decreasing 400

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22

K.D. H U Y S M A N S A N D W.T. F R A N K E N B E R G E R

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yields of trimethylarsine. The trimethylarsine yield at pH 6.0 was approximately four-fold greater than that detected at pH 8.5. Cox and Alexander (1973b) found that the optimum pH for trimethylarsinc production by resting cells of Candida humicola was pH 5 with the use of a citrate buffer.

Temperature Arsenic volatilization increased with increasing temperature up to 20°C and thereafter decreased proportionally up to 35°C (Fig. 5). Trimethylarsine was not detected when incubated at 0, 5 and 40°C. At 20°C there was approximately a 3.7-fold enhancement in trimethylarsine production over that detected at 10°C. Evaporation pond water typically ranges between 8 and 34°C. 300

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23

EVOLUTION OF TRIMEIHYLARSINE BY A PE.'~'iCILLll'.~I SI'

Phosphates Studies were conducted to determine the optimum phosphate concentration in production of trimethylarsine. The optimum yield was found between 0.1 and 50mM KH2PO4 (Fig. 6). This range of phosphate was required for adequate growth of the organism. The highest concentration used (1000 mM KH2 PO4) reduced trimethylarsine production from MAA.

Carbohydrates Among the carbon sources tested, it is apparent that monosaccharides as well as sugar acids do not promote trimethylarsine production by this Peniciilium sp. When 100mgl -~ of MAA was added to the minimal medium 200

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K.D. HUYSMANS AND W.T. FRANKENBERGER JR

24 2O0

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Carbohydrates Fig. 7. Influence of carbohydrates on trimethylarsin¢ production by Penicillium sp.

containing 2 g of carbon as carbohydrates per liter, less trimethylarsine was produced compared with the control. Figure 7 shows that the control lacking a supplemental carbon source (other than MAA) yielded 3.4-fold greater production of trimethylarsine than glucuronic acid plus MAA. Trimethylarsine yield was 46- and 336-fold greater without added carbohydrates when contpared with adding glucose and maltose, respectively. This study indicates tha'~ the Penicillium sp. will preferentially use monosaccharides as well as sugar acids as a carbon source over MAA.

EVOLUTION OF TRIMETHYLARSINE BY A P E N I C I L L I U M SP.

25

Amino acids Various amino acids were tested to assess their effect upon trimethylarsine production by this Penicillium sp. Amino acids were added at a 1 g carbon lconcentration, in the presence of 100 mg 1- ~of MAA, to the minimal medium. The control was used as a reference point to which amino acids either inhibited or stimulated trimethylarsine production (Fig. 8). The following amino acids suppressed trimethylarsine production in the presence of MAA in the minimal medium (ranked in order): lysine, serine, proliae, methionine, cysteine, glutamic acid, arginine, threonine, asparagine, aspartic acid, histidine, and glycine. These amino acids were apparently preferred as a carbon source over M A A by the Penic'/llium sp. Amino acids added to the minimal medium which stimulated trimethylarsine production included (listed 2000

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I n f l u e n c e o f a m i n o a c i d s o n t r i m e t h y l a r s i n e p r o d u c t i o n b y Penicilli~.,,m sp.

26

K.D. HUYSMANS AND W.T. FRANKENBERGER JR

in increasing order): tryptophan, leucine, valine, phenylalanine, isoleucine and glutamine. The addition of glutamine promoted trimethylarsine production as much as 11.6-fold greater than the control. Further work is needed to characterize the metabolism of these amino acids in fungi to formulate some hypotheses about the metabolic process involved in arsenic methylation. Volatilization of other elements This Peniciilium sp. was tested for its ability to methylate other elements including selenium and tellurium added to Difco R2A agar. Difco R2A agar was selected because it serves as a good medium for selenium volatilization. Selenium was added at 10 mg 1-~ as selenite-Se and selenate-Se. Tellurium was added as tellurite-Te and tellurate-Te at a concentration of 10mgl -~. The method of detection was by capillary gas chromatography coupled with fluorine-induced chemiluminescence detection (conducted by R. Fall and co-workers, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO). Upon the addition of selenite, only dimethylselenide (CH3SeCH3) was detected, but in the presence of selenate, dimethylselenide, dimethyldiselenide (CH.~SeSeCH3) and dimethylselenenylsulfide (CH3 SeSCH~) were produced. Dimethylselelfide and dimethyldiselenide production was confirmed by the method described by Frankenberger and Karlson (1989). When tellurite and tellurate were added to the medium, both dimethyltelluride (CH3TeCH3) and dimethylditelluride (£H3TeTeCH3) were detected. The results of this study indicate that this fungus is capable of methylating and volatilizing arsenic, selenium and tellurium. These transtbrmations may possibly be involved in a detoxification mechanism, allowing the organism to grow and proliferate while removing toxic concentrations of trace elements within its surrounding environment. CONCLUSIONS

Environmental conditions wl"ich promoted the production of trimethylarsine gas by the. Penicillium sp. isolated from evaporation pond water included: a minimal basal salt medium containing 100mgl -~ of MAA; pH 5-6; temperature of incubation of 20°C; and a phosphate concentration of 0.1-50 mM KH,PO4. Incorporation of carbohydrates and sugar acids into the medium suppressed trimethylarsine production by the Penicillium sp. The addition of specific amino acids, including lysine, serine, proline, methionine, cysteine, glutamic acid, arginine, threonine, asparagine, aspartic acid, histidine, and glycine, also suppressed trimethylarsine production. However, the addition of tryptophan, leucine, valine, phenylalanine, isoleucine and glutamine stimulated trimethylarsine production considerably in comparison with a

EVOLUTION O F T R I M E I " H Y L A R S I N E BY A P E N I C I L L l l ' M SP.

27

control without amino acid supplementation. The amino acids phenylalanine, isoleucine and glutamine were particularly effccti+,~ in promoting trimethylarsine production, ranging from 10.2- to l l.6-fold over the control. This Penicillium sp. was also capable of methylating selenium and tellurium. The knowledge obtained from this investigation may be useful in implementing a bioremediation technique to remove arsenic from water or soil. The chemical species of arsenic in marine water, freshwater, soil and sediments is often in organic form (Sanders, 1980). Although we do not propose that this microbial transformation be optimized to release trimethylarsine into the atmosphere, we do believe that the alkylarsine gas generated can be captured and concentrated on an effective trap (e.g., activated carbon). Further studies are needed to evaluate other environmental parameters which may affect arsenic removal directly from evaporation pond water as well as from sediments. ACKNOWLEDGEM ENTS

This investigation was supported by a grant funded by the University of California Salinity & Drainage Task Force. We are grateful to Ray Fall, University of Colorado, for detecting methylated gases of Se, Te and S by GC-fluorine induced chemiluminescence and Centrum Analytical Laboratories (Redlands, CA) for GC-MS analysis. REFERENCES Baker, M.D., W.E. Inniss, C.i, Mayfield, P.T.S. Wong and Y.K. Chau, 1983. Effect ofpH on the methylation of mercury and arsenic by sediment microorganisms. Environ. Technol. Lett., 4: 89-100. Braman, R.S. and C.C. Foreback, 1073. Methylated forms of arsenic in the enviromllent. Science, ! 82: ! 247-1249. Challenger, F., 1945. Biological methylation. Chem. Rcv., 36: 315-3(~1. Cox, D.P. and M. Alexander, 1973a. Production of trimethylarsine ga+,; fi'om various arsenic compounds by three sewage fungi. Bull. Environ. Contam. Toxicoi., 9: 84-88. Cox, D.P. and M. Alexander, 1973b. Effect of phosphate and other anions on trimethylarsine formation by Candida humh'ola. Appl. Microbiol., 25: 408-413. Cox, D.P. and M. Alexander, 1974. Factors affecting trimethylarsine and dimethylselenide formation by CLmdida humicoh:. J. Microb. Ecol., I: 136-144. Frankenberger, W.T. Jr and U. Karlson, 1989. Environmental factors affecting microbial production of dimethylselenide in a selenium-contaminated sediment. Soil Sci. Soc. Am. J., 53: 1435-1442. Hassler, R.A., D.A. Klein and R.R. Meglen, 1984. Microbial contribution to soluble and volatile arsenic dynamics in retorted oil shale. J. Environ. Qual., 13: 466-470. Huysmans, K.D. and W.T. Frankenberger Jr, 1990. Arsenic resistant microorganisms isolated from agricultural drainage water and evaporation pond sediments. Water, Air, Soil Pollut., 53: 159-168.

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Letey, J., C. Roberts, M. Penberth and C. Vasek, 1986. An agricultural dilemma: drainage water and toxics disposal in San Joaquin Valley. Division of Agricultural and Natural Resources, University of California Publication 3319. McBride, B.C., H. Merilees, W.R. Cuilen and W. Pickett, 1978. Anaerobic and aerobic alkylation of arsenic. Ix: F.E. Brickman and J.M. Bellama (Eds), Organometals and Organometalloids Occurrence a,,,,d Fate in the Environment. Am. Chem. Soc. Symp. Ser., 82:94-115. Sanders, J.G., 1980. Arsenic cycling in marine systems. Mar. Environ. Res., 3: 257-266. Tamaki, S. and W.T. Frankenberger Jr, 1991. Environmental biochemistry of arsenic. Rev. Environ. Contam. Toxicol., in press. Woolson, E.A., 1977. Generation of alkylarsine from soil. Weed Sci., 25: 412-416. Woolson, E.A. and P.C. kearney, 1973. Persistence and reactions of ~4C-cacodylic acid in soils. Environ. Sci. Technol., 7: 47-50.

Evolution of trimethylarsine by a Penicillium sp. isolated from agricultural evaporation pond water.

Arsenicals are used in agriculture as pesticides and defoliants. In the Central Valley of California, arsenic is present in soil at naturally high con...
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