Van de Wiele et al. IVG method was previously validated against in vivo data for As bioaccessibility (Rodriguez and Basta 1999), whereas the SHIME has been validated against in vivo data for microbial community composition and metabolic activity toward drugs and phytoestrogens (Molly et al. 1994; Possemiers et al. 2006). Soils were incubated in the gastric and intestine solution (30 mL) of the IVG pro­ tocol at a liquid­to­soil (L/S) ratio of 150 (Rodriguez and Basta 1999). These intesti­ nal digests from the IVG protocol were sub­ jected to colon conditions by adding 30 mL of the colon suspension sampled from the dis­ tal colon compartment of the SHIME reac­ tor, resulting in an L/S ratio of 300 for the soil digests. The vessels containing the colon digests were capped with butyl rubber stop­ pers and subsequently flushed with N2 for 30 min to obtain anaerobic conditions and incubated on a shaker at 150 rpm at 37°C for 18 hr. See Supplemental Material, Figure 1 (doi:10.1289/ehp.0901794) for a schematic of the experimental setup. Sample treatment. To preserve the spe­ ciation of As in the colon digests, all samples were flash frozen with liquid nitrogen upon incubation and subsequently stored at –80°C. Before analysis with highp­erformance liquid chromatography (HPLC) coupled with induc­ tively coupled plasma (ICP) mass spectrom­ etry (MS), samples were thawed and diluted appropriately with 20 mmol/L (NH4)2CO3 at pH 9.0 to minimize sulfur–oxygen exchange while awaiting analysis (Conklin et al. 2008). Upon complete thawing, the sample was vor­ texed and centrifuged for 10 min at 10,400 rel­ ative centrifugal force with an Eppendorf 5810R centrifuge (Brinkman Instruments, Westburg, NY, USA) to separate soluble As species from soil­bound As. The supernatant was filtered through a MillexL­CR 0.45 µm fil ­ ter (Millipore) with a LuerL­ok 10m­L syringe (BD, Franklin Lakes, NJ, USA). Finally, filtrates were diluted with the mobile phase and injected into the HPLC. The sum of the As species in the filtrate observed chromato­ graphically was considered the bioaccessible fraction. We measured total As concentration in the digest filtrates using ICP optical emis­ sion spectroscopy (ICPO­ES). This allowed us to calculate chromatographic recovery, which quantifies the extent to which the sum of the chromatographic As species comprises the total amount of As in the digest filtrates. As speciation analysis by HPLC/ICP-MS. Sample supernatants were analyzed with HPLC (Agilent 1100) and ICPM­S (Agilent 7500ce ; Agilent, Palo Alto, CA, USA) for As specific detection at m/z 75. Separation of As oxides was performed on a PRPX­100 HPLC column (250 mm × 4.1 mm, 5 µm). The mobile phase was a solution of NH4NO3 (10 mmol/L), NH4H2PO4 (10 mmol/L), and EDTA (500 mg/L) at pH 4.57 in distilled water (sepa­ ration 1). The flow rate was 1.0 mL/min, and the sample injection volume was 100 µL. The retention times of the separated compounds were 3.6 min for AsIII, 4.2 min for DMAV, 5.5 min for MMAIII, 7.1 min for MMAV, and 8.9 min for AsV, similar to those previ­ ously reported by Yathavakilla et al. (2008). We used this separation for quantification of the As species of interest. Arsenic sulfides were identified by retentiont­ime matching between samples and fortified samples. Using chromato­ graphic separation 1, monothioarsonic acid eluted at 15 min, whereas MMMTAV eluted at 18.6 min. See the Supplemental Material (doi:10.1289/ehp.0901794) for details on syn­ thesis, chromatographic confirmation of these As sulfides, and sample analysis. A second chromatography [separation 2; see Supplemental Material (doi:10.1289/ ehp.0901794)] was used for ICP­MS and electrospray ionization (ESI)­MS detection, because the mobile phase of separation 1 was not compatible with ESIM­S detection. Results In the first experiment, we assessed the meta­ bolic potency of the human fecal microbial Table 2. Influence of cofactors toward microbial metabolism during in vitro fecal incubation of iAsV at 90 mg/L. MMAV MMAIII Recovery (%) 113 93 118 104 86 104 86 102 94 97 inoculum toward high levels of iAsV (90 mg/L). iAsV was efficiently (> 94%) reduced to iAsIII after the 48­ hr incubation with both active and sterilized fecal microbiota (Table 2), prob­ ably because of the highly reducing conditions (redox potential was –180 mV). Incubation with sterilized fecal microbiota did not lead to thiolated or methylated arsenicals. In contrast, incubation of iAsV with active fecal microbiota resulted in the production of monothioarsonic acid (mTA; mean ± SD) in nonamended (2.2 ± 3.1 mg/L) and methionine­amended (0.8 ± 1.1 mg/L) samples. Interestingly, we observed methylation only in the presence of methylcobalamin. Addition of the methyl­ cobalamin displayed a significant methylation of iAs (18%), with MMAV (13.0 ± 1.4 mg/L) b e i n g m o r e d o m i n a n t t h a n M M A III (2.6 ± 1.4 mg/L). The addition of both methyl­ cobalamin and glutathione as a reducing agent increased the methylation to 28%, with MMAIII (10.5 ± 5.4 mg/L) becoming equally as important as MMAV (11.3 ± 5.6 mg/L). These preliminary data convinced us that the selected microbial community had the potency to actively metabolize iAsV. We therefore inoculated the SHIME reactor with this fecal microbial inoculum; after 3 weeks of adaptation, a stable microbial community was obtained in the proximal and distal colon compartments. We regularly sampled the distal colon compartment to perform colon incubations on iAsV and Asc­ontaminated soil samples that had already gone through a gas­ tric and intestinal digestion. Characterization of the colon digests consisted of determin­ ing As bioaccessibility and As speciation. The bioaccessibility determination was based on the sum of all chromatographically detected (HPLC/ICP­MS) As species in the filtrates (0.45 µm) of the colon digests; therefore, the chromatographic recovery was calculated first. The sum of the concentrations of chromato­ graphically detected As species in the colon Table 3. Percentage bioaccessibility of As and biomass-specific production rate of methyl- ated arsenicals by colon microorganisms after in vitro colon digestion of iAsV (225 μg/L) and As-contaminated soils. Sample Na2HAsO4·7H2O Soil 1 Soil 2 Soil 3 Soil 4 Percent Methylation rate bioaccessibilitya (μg/hr/g biomass)b 76 ± 14.3 10.0 ± 4.0 24 ± 4.3 10.0 ± 1.8 44 ± 3.9 4.2 ± 1.6 36 ± 1.5 28.9 ± 3.4 0.3 ± 0.1 ND ND, not detected. Values are mean ± SD of three experiments. aCalculated by dividing the sum of detected As species (iAsV, iAsIII, mTA, MMAV, MMAIII, and MMMTAV) in the colon digest filtrate by the total amount of As that was incubated under colon conditions. bCalculated by divid- ing the sum of methylated arsenicals (MMAV, MMAIII, and MMMTAV) in the colon digest filtrate by the initial biomass concentration and the incubation time. ovlume 118 | number 7 | July 2010 • Environmental Health Perspectives Microbiota Active microbiota No cofactor Me-B12 Meth GSH Me-B12/Meth/GSH Sterilized microbiotaa No cofactor Me-B12 Meth GSH Me-B12/Meth/GSH 1006 1.7 ± 2.3 1.7 ± 2.3 2.0 ± 1.2 AsV 2.6 ND 5.6 4.6 4.6 ND 3.5 AsIII 97.6 ± 2.5 65.3 103.4 ± 3.1 93.2 ± 18.8 53.6 ± 9.7 87.6 72.7 86.8 84.6 83.7 Abbreviations: GSH, glutathione; Me-B12, methylcobalamin; Meth, methionine; ND, not detected. Values are mean ± SD (mg/L) from duplicate incubation experiments aIncubation tests with sterilized microbiota were performed once, so no SD is available. 13.0 ± 1.4 2.6 ± 1.4 11.3 ± 5.6 10.5 ± 5.4 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND mTA 2.2 ± 3.1 0.8 ± 1.1 ND ND ND ND ND ND ND ND