Introduction

The recent enactment of a stricter norm of arsenic in drinking water (1) highlights an increasing national concern for the health effects of arsenic. In this context, the large-scale use of organoarsenicals in agriculture should also be carefully evaluated. Roxarsone (3-nitro-4-hydroxyphenylarsonic acid) is utilized in the broiler poultry industry as a feed additive to promote growth and control coccidial intestinal parasites. Approximately 70% of broiler chickens produced in the U.S. are fed roxarsone (2), which is excreted largely unaltered into the manure (3). Approximately 900 metric tons of roxarsone are released annually into the environment in the U.S. (4). The arsenic content of poultry litter ranges from 14 to 48 mg kg^-1 (4-7). The environmental impact of land applying poultry litter is potentially significant when considering that this arsenic-laden material is spread onto relatively small land areas in the direct vicinity of poultry houses. Soil samples from field sites having received broiler poultry litter for years contain significantly higher concentrations of water extractable arsenic, as compared to similar soils with no history of poultry litter addition (8).

Roxarsone is highly water soluble, triggering concerns that it would be mobile in the environment, leading to transport of arsenic to groundwater and surface water (4, 5). Several studies have begun to address changes in arsenic speciation during storage and composting of poultry litter (4, 5) as well as changes occurring after land application to soil (8). Poultry litter contains roxarsone and arsenate (As(V)) with smaller amounts of arsenite (As(III)) and dimethylarsinic acid (DMA(V)) (4-6). Fresh poultry litter was also shown to contain 4-hydroxy-3-aminophenylarsonic acid (HAPA), a reduced biotransformation product of roxarsone, accounting for 12-25% of the species identified (3, 5). As(V) is formed during composting of poultry litter and during incubation of roxarsone with soil (4, 8). Since heat-sterilized and poisoned controls did not convert roxarsone to As(V), the conversions are attributed to biological reactions (4, 8).

The speciation studies so far have been conducted in aerobic environments; however, roxarsone may also occur in anaerobic environments, such as anaerobic zones in compost and anaerobic sediments. The objectives of this study were to evaluate the bioconversion of roxarsone and related N-substituted phenylarsonic acid derivatives under anaerobic conditions.

Materials and Methods

Microorganisms. Anaerobic methanogenic sludge was obtained from an industrial upward-flow anaerobic sludge blanket treatment plant treating recycled paper wastewater (Eerbeek, The Netherlands). The volatile suspended solids (VSS) content of Eerbeek sludge was 12.9%. In one experi ment, aerobic activated sludge was obtained from the Ina Road Municipal Wastewater Treatment Plant in Tucson, AZ.

Biotransformation Assays. Biotransformation of roxarsone, 4-hydroxy-3-aminophenylarsonic acid, and 4-aminophenylarsonic acid (4-APA, also known as 4-arsanilic acid) under different redox conditions was evaluated in short- and long-term incubation periods. Assays were conducted in 165-mL serum flasks supplied with 65 mL of basal mineral medium. The basal medium contained the following components (mg L^-1): NaH₂PO₄·2H₂O (795), K₂HPO₄ (600), NH₄Cl (280), MgCl₂·6H₂O (91.56), and 1 mL L^-1 of a trace element solution. The trace element solution contained (in mg L^-1) FeC1₃·4H₂O (2000), CoCl₂·6H₂O (2000), MnCl₂·4H₂O (50), AlCl₃·6H₂O (90), CuCl₂·2H₂O (30), ZnCl₂ (50), H₃BO₃ (50), (NH₄)₆Mo₇O₂·4H₂O (50), Na₂SeO₃·5H₂O (100), NiCl₂·6H₂O (50), EDTA (1000), HCl 36% (1 mL), and resazurin (200). The final pH of the medium was adjusted to 7 with HCl or NaOH as required. NaHCO₃ (4000 mg L^-1) was also added to the medium as part of the CO₂/NaHCO₃ buffering system. The bioassays were supplied with 1 mM of the corresponding N-substituted phenylarsonic acid and inoculated with 1.5 g VSS L^-1 of anaerobic sludge. When HAPA was used, it was added from a stock solution containing 200 mg L^-1 ascorbic acid via a syringe to the sealed culture bottles that were preincubated overnight to avoid its oxidation by dissolved oxygen. One experiment was inoculated by 10% v/v aerobic activated sludge. Selected treatments were supplemented with either sulfate (1500 mg L^-1) or nitrate (1500 mg L^-1) to create sulfate-reducing or denitrifying conditions, respectively. In experiments in which the effect of different electron donors was investigated, the medium was supplied with lactate (10 mM); acetate (10 mM); glucose (10 mM); a mixture of volatile fatty acids containing acetic (1.00 mM), propionic (4.06 mM), butyric (3.40 mM), valeric (2.94 mM), and hexanoic (2.58 mM) acids; or H₂ (1.2 atm in the headspace). In one experiment, roxarsone (1 mM) was incubated abiotically in the buffered mineral basal medium with either 6 mM FeSO₄ or 3 mM Na₂S in the presence and absence of heat-killed sludge.

To maintain anaerobic conditions, the medium and headspace were flushed with a mixture of N₂/CO₂ gas (80:20, v/v). In bioassays utilizing H₂, the bottles were flushed first with N₂/CO₂; subsequently, 1.5 atm of H₂/CO₂ (80:20, v/v) was added to the headspace of each bottle. In bottles receiving oxygen, the headspace was flushed with air, and subsequently, 0.2 atm of CO₂ was injected into the headspace. Bottles were incubated in the dark at 30 C. Experiments were incubated statically unless the use of a reciprocal shake table is indicated in the figure captions. Each experiment included several controls. Abiotic controls were prepared without adding microbial inoculum. Killed sludge controls were prepared by adding inoculum and subsequently placing the bottles in an autoclave for two successive treatments of 20 min at 120 C, separated by 1 day. An endogenous substrate control was included in the experiment evaluating different electron donors.

Anlytical Methods. A high performance liquid chromatograph (Hewlett-Packard 1090 HPLC) with a diode array detector (DAD) was used to measure roxarsone, HAPA, and 4-APA. The chromatograph was equipped with an Ion Pac-AS14 analytical Dionex column (4 × 250 mm) and an Ion-Pac-AG14 Dionex precolumn (4 × 50 mm). In the roxarsone and HAPA biodegradation assays, samples were diluted 1:1 with ascorbic acid (3000 mg L^-1) to avoid oxidation of HAPA with air or dissolved oxygen. Samples were centrifuged for 10 min at 10 000 rpm. The eluent utilized was a phosphate buffer (10 mM, pH 7.2), pumped to the system at a flow rate of 2 mL min^-1. Roxarsone and HAPA were detected at 450 and 300 nm, respectively. The injection volume was 5 L.

Inorganic arsenic species (arsenite (As(III)) and arsenate (As(V))) and organic arsenic species (methylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), methylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), roxarsone, HAPA, and 4-APA) in liquid samples were analyzed by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) using a method adapted from Gong et al. (9). The HPLC system consisted of an Agilent 1100 HPLC (Agilent Technologies, Inc., Palo Alto, CA) with a reversed-phase C18 column (Prodigy 3u ODS(3), 150 × 4.60 mm, Phenomenex, Torrance, CA). The mobile phase (pH 5.85) contained 4.7 mM tetrabutylammonium hydroxide, 2 mM malonic acid, and 4% (v/v) methanol at a flow rate of 1.2 mL min^-1. The column temperature was maintained at 50 C. An Agilent 7500a ICP-MS with a Babington nebulizer was used as the detector. The operating parameters were as follows: rf power, 1500 W; plasma gas flow, 15 L min^-1; carrier flow, 1.2 L min^-1; arsenic measured at 75 m/z; and terbium (IS) measured at m/z 159. The injection volume was 10 L. The detection limit for the various arsenic species was 0.1 g L^-1. Total arsenic concentration in liquid samples was determined by direct injection into the ICP-MS. Total arsenic was calibrated with arsenic trioxide (As₂O₃) reductometric standard (Aldrich).

All liquid samples were centrifuged and membrane-filtered (0.45 m) immediately after sampling and stored in polypropylene vials (2 mL). The filtered samples were then stored at -20C until the analysis was performed in order to reduce changes in arsenic speciation.

Volatile arsenic species were determined by flushing bottles from the biological assays with N₂ gas for 12 h. The gas was bubbled through 20 mL of 2 M nitric acid. Samples of the scrubbing fluid were analyzed for total arsenic. The arsenic content in the sludge was measured by extracting a sample in 10 mL of HCl (6.75 N) in a MDS 2100 microwave digestion system (CEM Corporation Matthews, NC) for 35 min. Soluble ferrous iron was determined colorimetrically by the phenanthroline method.

Chemicals. Roxarsone, HAPA, and 4-APA were obtained from Aldrich Chemical Co., Inc (Milwaukee, WI), Pfaltz & Bauer, Inc. (Watebury, CT), and Avocado Research Chemicals (Heysham, England), respectively.

Results

Reduction of Roxarsone. Figure 1 shows the results of an experiment in which roxarsone was incubated with anaerobic sludge under methanogenic, sulfate-reducing, and denitrifying conditions as well as methanogenic conditions supplemented with lactate. Under denitrifying conditions, only negligible removal of roxarsone occurred during the 21-day experiment. On the other hand, under methanogenic and sulfate-reducing conditions, rapid decreases in the roxarsone concentration were observed. Roxarsone was removed by 96% after 16 days. Supplementation of lactate increased the rate of roxarsone elimination, corresponding to 95% removal after 12 days. Biologically mediated reactions are implicated, since roxarsone was not removed in the control lacking added sludge. The slow removal of roxarsone in the heat-killed sludge controls indicated that there are also some abiotic mechanisms of roxarsone removal due to components present in the sludge. HAPA was identified as a major biotransformation product of roxarsone degradation, indicating that the main reaction was a reductive transformation of the nitro group to an amino group. HAPA was recovered with a molar yield of ~75%.

Figure 1 Anaerobic biotransformation of roxarsone by methanogenic sludge under different redox conditions. Experiments were incubated on a reciprocal shake table. A. Roxarsone concentrations determined by HPLC-DAD. B. HAPA concentrations determined by HPLC-DAD. Legend: Abiotic control, - - - -; heat-killed sludge control, - - - -; methanogenic, -·-; sulfate reducing conditions, --; denitrifying conditions, --; and methanogenic conditions with 10 mM lactate added, --.

Roxarsone was also incubated for 18 days with aerobic activated sludge. No bioconversion of roxarsone occurred under aerobic conditions or under denitrifying conditions. However, 41-46% removal of roxarsone occurred under anaerobic conditions without and with sulfate addition, respectively. The removal was increased to 71% in the treatment receiving lactate (results not shown). HAPA was recovered as the major biotranformation product with a molar yield of 38-50%.

An experiment was conducted to evaluate the impact of different electron-donating substrates on roxarsone biotransformation by anaerobic sludge incubated under methanogenic conditions, as shown in Figure 2. The endogenous-substrate control and the acetate-supplemented treatments had the lowest rates of roxarsone reduction. All other electron-donating substrates significantly stimulated the roxarsone biotransformation rates. Hydrogen, glucose, and lactate significantly increased the rates of roxarsone biotransformation by 2.8-, 1.9, and 1.8-fold, respectively, as compared to the endogenous substrate control. The molar yields of HAPA in the biologically active treatments ranged from 67 to 92% of the roxarsone removed.

Figure 2 Effect of electron donors on the anaerobic biotransformation of roxarsone by methanogenic sludge under methanogenic redox conditions. Experiments were incubated on a reciprocal shake table. A. Roxarsone concentrations determined by HPLC-DAD. B. HAPA concentrations determined by HPLC-DAD. Legend: abiotic control, - - - -; heat-killed sludge control, - - - -; no added electron donor, -· -; acetate, 10 mM, - - - -; volatile fatty acid mixture, --; glucose, 10 mM, --; H2, 1.2 atm, -* -; and lactate, 10 mM, --. The average zero order fits (± standard deviation) were -0.222 (± 0.007), -0.151 (± 0.015), -0.143 (± 0.003), -0.107 (± 0.006), -0.101 (± 0.018), and -0.081 (± 0.003) mM d-1; respectively.

Ferrous iron (Fe²⁺) and sulfides are inorganic reducing agents present in anaerobic environments; therefore, the ability of stoichiometric amounts of FeSO₄ (6 mM) and Na₂S (3 mM) to reduce roxarsone (1 mM) abiotically in the presence of heat-killed sludge and the basal mineral medium was evaluated (Figure 3). Under these conditions, Fe²⁺ and, to a lesser extent, S^2- supported the abiotic reduction of roxarsone. On day 5, the molar yield of HAPA was 72 and 22% of roxarsone eliminated by Fe²⁺ and S^2-, respectively. The abiotic reactions followed first-order kinetics (dashed lines); whereas the biologically catalyzed reaction with H₂ as electron donor followed zero-order kinetics (solid line). The abiotic reactions were catalyzed by undefined components in the basal mineral nutrients or autoclaved sludge, as evidenced by the negligible rates in the absence of mineral basal nutrients or the absence of autoclaved sludge in the case of Fe²⁺ or S^2-, respectively (results not shown).

Figure 3 Effect of reducing agents on the reduction of roxarsone. Legend: roxarsone incubated in basal medium in the absence of heat-killed sludge, ; roxarsone incubated in basal medium in the presence of heat-killed sludge, ; roxarsone incubated in basal medium with 6 mM FeSO4 in the presence of heat-killed sludge, ; roxarsone incubated in basal medium with 3 mM Na2S in the presence of heat-killed sludge, ; and roxarsone incubated in basal medium with living sludge and H2, ·. The dashed lines are first-order fits of the abiotic reactions (k = -0.014, -0.153, and -0.067 d-1 and r2 = 0.8483, 0.9733, and 0.9880 for no addition, FeSO4, and Na2S, respectively). The solid line is a zero-order fit to the biological reaction (k = -0.113 mM d-1, r2 = 0.9975).

Long-Term Biodegradability of Roxarsone. An experi ment was conducted to determine the long-term biodegrad ability of roxarsone. The results after 10 days were in agreement with the findings of the first experiment (Figure 1) in that roxarsone was only partially converted to HAPA in the heat-killed sludge treatment (29%) but completely converted to HAPA in the biological active treatments amended with lactate (99%). The molar yield of HAPA was 86% of roxarsone removed. After 120 days, roxarsone was also largely converted in the heat-killed sludge treatment to HAPA due to a sustained slow abiotic reaction (Table 1). The HAPA, which accumulated, was recovered at a molar yield of 83%.

In the two biologically active treatments, the HAPA intermediate was subjected to further biodegradation, as evidenced by the dramatically lower HAPA levels on day 120 as compared to levels that had previously accumulated on day 10. The HPLC-ICP-MS data revealed that part of the HAPA that was eliminated had accumulated as As(III) and As(V) (Table 1). As(III) was the most important inorganic arsenic compound, and it had significantly accumulated up to 18% of the arsenic supplied. The soluble inorganic arsenicals (corrected for background) detected in the endogenous substrate and lactate-fed treatments correspond to a molar yield of 18 and 23% of the phenylarsonic acid compounds removed, respectively. The data of arsenic speciation was compared with the total arsenic measured in liquid samples (Table 1). In the biologically active treatments, the sum of identified arsenic species ( sp) accounted for about one-third of the arsenic. Most of the unidentified arsenic was present in the liquid.

Long-Term Biodegradability of HAPA. Under methanogenic and sulfate reducing conditions, HAPA was significantly removed, as compared to the abiotic and heat-killed sludge controls (Figure 4). Lactate supplementation had no significant effect on the rates of degradation under methanogenic conditions. The most important biodegradation product observed was As(III). On days 113-134, there was some temporal accumulation of As(V). The molar yield of soluble inorganic arsenicals (corrected for background inorganic arsenic) in the methanogenic and sulfate-reducing treatments ranged from 15 to 21% of HAPA removed on day 132 and ranged from 19 to 28% of HAPA removed on day 229. No significant removal of HAPA occurred in uninoculated or heat-killed sludge controls. Likewise, there was no significant production of inorganic arsenicals in these controls. These observations confirm that the removal in the methanogenic and sulfate reducing treatments was due to biological mechanisms.

Figure 4 Anaerobic biotransformation of HAPA during a long-term incubation with methanogenic sludge. A. HAPA concentrations. B. As(V) concentrations. C. As(III) concentrations. All species shown were determined by HPLC-ICP-MS. Legend: abiotic control, - - - -; heat-killed sludge control, - - - -; methanogenic, -·-; sulfate reducing conditions, --; denitrifying conditions, --; and methanogenic conditions with 10 mM lactate added, --.

The heat-killed control and the methanogenic treatments were incubated up to day 310, at which time HAPA was removed by 98.9%, and As(III) corrected for background levels accounted for 26% of the HAPA removed in the biologically active treatment. By comparison, HAPA was removed by only 38.8% in the heat-killed treatment with negligible production of As(III) after the extended incubation (results not shown).

Biodegradation under denitrifying conditions was delayed and slower. By the end of the experiment (day 229), there was still no significant decrease of HAPA beyond the levels in the controls.

The fraction of total arsenic lost from the liquid is indicated by the difference between total arsenic in the abiotic control and that of the biologically active treatments (the "not in liquid" fraction in Table 2). Up to day 132, the loss of total arsenic was minor, ranging from 10 to 19% in the methanogenic and sulfate reducing treatments; however, by the end of the experiment on day 229, the loss of total arsenic was quite substantial, accounting for 41-53% of the arsenic in the abiotic control. The data of arsenic speciation was compared with the total arsenic measured in liquid samples (Table 2). In the methanogenic and sulfate reducing treat ments, the sum of identified species ( sp) accounted for 59-63% of the arsenic on day 132 and for 30-42% of the arsenic on day 229.

Long-Term Biodegradability of 4-APA. 4-APA is structurally related to HAPA and is also used as a feed additive in poultry. An initial experiment was conducted to evaluate the biodegradabilty of 4-APA by anaerobic sludge under several redox conditions. In this experiment, the HPLC-DAD was used to monitor the 4-APA concentration. 4-APA was subject to immediate anaerobic biodegradation in the methanogenic and sulfate-reducing treatments. At the end of the experiment (day 68), 4-APA degradation reached 71.3-82.8% (results not shown). Partial elimination of 4-APA occurred under denitrifying conditions after a lag phase of 41 days. No significant removal of 4-APA occurred in the uninoculated or heat-killed sludge controls, indicating that the removal in the biologically active treatments was due to biodegradation.

A second experiment was conducted with the methanogenic treatment and controls to obtain data about the release of inorganic arsenicals. 4-APA was also reliably biodegraded under methanogenic conditions in the second experiment (Figure 5). 4-APA biodegradation was accompanied by significant production of As(III) as well as a lower production of As(V). These inorganic arsenicals (corrected for background) were produced at a molar yield of 24%, as compared to 4-APA eliminated on day 174. Total As measurements indicate that about one-half of the arsenic was removed from the liquid at the end of the experiment (results not shown).

Figure 5 Anaerobic biotransformation of 4-APA during a long-term incubation with methanogenic sludge. A. 4-APA concentrations determined by HPLC-DAD. B. As(V) concentrations determined by HPLC-ICP-MS. C. As(III) concentrations determined by HPLC-ICP-MS. Legend: abiotic control, - - - -; heat-killed sludge control, - - - -; and methanogenic condition, - ·-.

Discussion

The results from this study demonstrate that the commonly utilized feed additive of broiler chickens, roxarsone, is rapidly transformed under anaerobic conditions to the correspond ing aromatic amine, HAPA. The formation of HAPA is attributable to the facile reduction of the nitro group and occurred only under methanogenic or sulfate-reducing conditions. During the period of HAPA formation, no significant release of inorganic arsenicals from the phenylarsonic acid structure was observed; however, HAPA and the closely related 4-APA were slowly degraded under anaerobic conditions. This degradation was associated with the partial release of freely soluble inorganic arsenicals in molar yields ranging from 19 to 28% of the amino-substituted phenylarsonic acids removed. The predominant inorganic arsenic species was arsenite, most likely due to reduction of released arsenate.

Reductive Biotransformation of Nitro Group. The facile reduction of the nitro group in roxarsone to an aromatic amine is in accordance with the rapid reduction of nitroaromatics in anaerobic mixed cultures from bioreactor sludge (10), sewage effluent (11), and aquatic sediments (12). Many anaerobic microorganisms are also known to readily reduce a variety of nitroaromatic compounds (13, 14). The best studied are clostridia, sulfate-reducing bacteria, and methanogens. The reduction of a nitroaromatic group requires the donation of six electrons. Anaerobic microorganisms typically catalyze the reactions with nonspecific nitroreductases converting the nitro group via nitroso- and hydroxylamine intermediates to aromatic amines by successive addition of electron pairs from the metabolism of an electron-donating substrate.

Some insights are available on the enzymatic basis of aryl-nitro reduction in anaerobes. Nitro group reduction in the genus Clostridium is catalyzed by hydrogenases and dehydrogenases (15). In most cases, the nitroreductases are dependent on cofactors such as ferredoxin (16) or flavin adenine dinucleotide (FAD) (17). Reduced ferredoxin catalyzed the same reaction as the nitroreductase, suggesting that the role of nonspecific reductases is to catalyze the formation of reduced cofactors that, in turn, are responsible for the chemical reduction of the nitro group (16). Four distinct NAD(P)H-dependent nitroreductases were purified from Bacteroides fragilis with varying degrees of substrate specificity (18). Little is known about the biochemical basis of nitroaromatic reduction in sulfate-reducing bacteria and methanogens.

Abiotic Reduction of the Nitro Group. In this study, heat-killed sludge was also found to reduce roxarsone to HAPA at rates that were 5-fold slower than the biologically active treatments. Nitroaromatics are known to be reduced by chemical reducing agents such as ferrous iron (19) and sulfides (13, 20) that are potentially present in anaerobic environments. The measured freely dissolved Fe²⁺ in the assays ranged from 0.018 to 0.072 mM, which would be sufficient only to reduce 1.2% of roxarsone added. Significant chemical reduction of roxarsone to HAPA was observed in this study when FeSO₄ was supplied at a stoichiometric concentration. Iron may have a role at substoichiometric concentrations in biologically active treatments if it is regenerated by iron reducing bacteria. The total Fe and total S content of the sludge utilized in the experiments of this study was 56.6 and 46.6 mg g^-1 VSS, respectively (21). The assay sludge concentration of 1.5 g VSS L^-1 could, thus, potentially introduce up to 1.5 e meq L^-1 of Fe(II) and 4.4 e meq L^-1 of S^2-, sufficient to reduce about 1 mM of roxarsone to HAPA.

Electron Donors. The electrons to support the biological nitro group reduction are released during the anaerobic degradation of substrates. Remarkably, substantial rates of roxarsone reduction to HAPA were evident in treatments without any added electron donor, indicating that endogenous decay of sludge biomass was probably supplying the electrons. The endogenous substrate level in the sludge was estimated from the methane production after incubating the sludge with inorganic basal medium for 30 days. The methane yield corresponds in value to 18.8 e meq L^-1. This concentration of endogenous substrate was in excess of the 6 meq e L^-1 required to reduce 1 mM roxarsone to HAPA.

Addition of the electron donating substrates increased the rates of roxarsone reduction beyond the endogenous rate. The substrates hydrogen, glucose, lactate, and a mixture of volatile fatty acids significantly increased rates. The best results were obtained with hydrogen. On the other hand, acetate had no effect on improving the rate. These observa tions are consistent with studies evaluating the effects of electron donors on nitrophenol reduction (13, 22). Either hydrogen or substrates supplying interspecies hydrogen are required for rate enhancement, whereas acetate had no effect because it is not commonly a source of interspecies hydrogen.

Biodegradation and Biotransformation of Aromatic Amines. Aromatic amines tend to accumulate in anaerobic environments. The simplest aromatic amine, aniline, is persistent in methanogenic consortia (23, 24); however, there is ample evidence that aminophenols, aminobenzoates, and aminosalicylates are mineralized under methanogenic conditions to CH₄ and NH₃ (22, 25-27). The anaerobic degrada tion of aromatic compounds generally follows a common pathway. Aromatic compounds are first metabolized to benzoic acids, which are ligased with coenzyme A (CoA) and simplified to the common intermediate, benzoyl-CoA, that is subsequently metabolized further by ring reduction (28, 29). Anaerobic aniline degradation has been described in a sulfate-reducing strain (30) and in a facultative denitrifying strain (31). In both cases, degradation is initiated by carboxylation to form 4-aminobenzoic acids. This intermediate is subsequently ligased with CoA, forming 4-aminobenzyl-CoA. The amino group is reductively deaminated to yield benzyl-CoA (30). Citrobacter freundii strain WA1, isolated from a methanogenic consortium, was shown to reductively deaminate 5-aminosalicylate, in addition to reducing the benzyl group to yield 2-hydroxybenzyl alcohol (27).

In this study, HAPA and 4-APA were slowly biologically eliminated under anaerobic conditions. The metabolism of these two aminophenylarsonic acids was associated with the partial release of inorganic arsenic. This constitutes the first report of a biologically catalyzed rupture of the phenylarsonic group under anaerobic conditions. If the phenylarsonic group were hydrolyzed, arsenate would be the most likely species of inorganic arsenic released, and arsenate was, indeed, detected in this study; however, arsenite was the predominant inorganic species recovered, most likely due to subsequent biological reduction of arsenate to arsenite in methanogenic sludge (32).

Losses in Dissolved Arsenic. Total arsenic measurements made by direct injections of liquid samples in the ICP-MS indicated that most of the arsenic added was recovered in the liquid during incubations of ~130 days or less; however, the recoveries of total arsenic in the liquid phase of the HAPA and 4-APA assays after extended incubations of 174 and 229 days, respectively, were low (47 to 59%). These losses can only be clarified by volatilization or by accumulation in the sludge. The contribution due to volatilization was measured after incubating HAPA for 310 days under methanogenic conditions, and the volatiles accounted for 0.03% of the arsenic supplied initially as HAPA. These findings are in agreement with negligible recoveries of volatiles in experi ments conducted under similar conditions with arsenite (33). Therefore, accumulation in sludge would be a more likely explanation. The sludge was extracted with HCl, but the quantity of arsenic recovered accounted for only 10% of the missing arsenic. The arsenic residuals in the sludge are, therefore, present in forms that are not amendable to HCl extraction. Arsenic is poorly extracted from sulfides by HCl (34), which would be consistent with the possibility that arsenite formed from aminophenylarsonic acid degradation was becoming sequestered by background level sulfides in the sludge, possibly forming orpriment (As₂S₃) (35). Arsenite supplied at 24 M was observed to become sequestered by 84% in 10 days when incubated with the same sludge used in this study (33). Another possibility is that arsenic was present as bound residue in sludge organic matter. Covalent bonds between aromatic amines and soil organic matter are known to be formed under anaerobic conditions as a result of experiments evaluating the fate of reduced ¹⁵N-trinitrotoluene utilizing ¹⁵N NMR spectroscopy (36).

Environmental Significance. Nitrogen-substituted phenylarsonic compounds utilized as poultry food additives can be transformed under anaerobic conditions to eventually produce the toxic arsenic species, arsenite (33). Thus, in anaerobic environments, such as sediments and subsurafce soil, such compounds, if present, would lead to potential damage to the ecosystem.

Acknowledgment

The work presented here was funded by a USGS, National Institute for Water Resources 104G grant (2002AZ9G), and by a seed grant from the NIEHS-supported Superfund Basic Research Program (NIH ES-04940). Participation of R. Sierra-Alvarez in this study was partly funded by the National Science Foundation under grant 0137368. The use of trade, product, or firm names in this report is for descriptive purposes only and does not constitute endorsement by the U.S. Geological Survey.