Biovolatilization of Metal(loid)s by Intestinal Microorganisms in the

Jun 9, 2009 - In this study, an in vitro gastrointestinal model, the Simulator of the Human ... MS).SignificantvolatilizationwasfoundforSe,As,andTe(ma...
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Environ. Sci. Technol. 2009, 43, 5249–5256

Biovolatilization of Metal(loid)s by Intestinal Microorganisms in the Simulator of the Human Intestinal Microbial Ecosystem† R O L A N D A . D I A Z - B O N E * ,‡ A N D TOM R. VAN DE WIELE§ Institute of Environmental Analytical Chemistry, University of Duisburg-Essen, Universita¨tstrasse 3-5, 45141 Essen, Germany, and Laboratory of Microbial Ecology and Technology, University of Gent, Belgium

Received February 19, 2009. Revised manuscript received March 30, 2009. Accepted May 8, 2009.

Methylation and hydrogenation of metal(loid)s by microorganisms are widespread and well-known processes in the environment by which mobility and in most cases toxicity are significantly enhanced in comparison to inorganic species. The human gut contains highly diverse and active microbiocenosis, yet little is known about the occurrence and importance of microbial metal(loid) methylation and hydrogenation. In this study, an in vitro gastrointestinal model, the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), was used for investigating volatilization of metal(loid)s by intestinal microbiota. Suspensions from different compartments of the SHIME system analogous to different parts of the human intestinal tract were incubated with different concentrations of inorganic Ge, As, Se, Sn, Sb, Te, Hg, Pb, and Bi and analyzed by gas chromatography and inductively coupled plasma mass spectrometry (GC-ICPMS). Significant volatilization was found for Se, As, and Te (maximal hourly production rates relative to the amount spiked; 0.6, 2, and 9 ng/mg/h, respectively). In addition, volatile species of Sb and Bi were detected. The occurrence of AsH3 and (CH3)2Te was toxicologically important. Furthermore, mixed Se/S and mixed As/S metabolites were detected in significant amounts in the gas phase of the incubation experiments of which two metabolites, (CH3)2AsSSCH3 and CH3As(SCH3)2, are described for the first time in environmental matrices. The toxicology of these species is unknown. These data show that the intestinal microbiota may increase the mobility of metal(loid)s, suggesting a significant modulation of their toxicity. Our research warrants further studies to investigate the extent of this process as well as the availability of metal(loid)s from different sources for microbial transformations.

Introduction Biomethylation and Hydrogenation in the Environment. Conversion of metal(loid)s to their methyl and hydride derivatives by organisms is a well-known phenomenon in the environment and has been shown for a wide range of * Corresponding author phone: +49 201 183 3963; fax: +49 201 183 3951; e-mail: [email protected]. † For comparability of data, all amounts and concentrations indicated in this manuscript refer to the mass of the metal(loid) and not to the mass of the species. ‡ University of Duisburg-Essen. § University of Gent. 10.1021/es900544c CCC: $40.75

Published on Web 06/09/2009

 2009 American Chemical Society

elements (1). Though the prerequisites for methylation can vary significantly depending on the element, volatile species of As, Se, Sn, Sb, Te, Hg, Pb, and Bi can be detected simultaneously in the gas phase of environmental compartments with high microbiological activity like waste deposits, fermentation reactors, and soils (2–5). Although methylation by microorganisms has been shown for all of these elements, only As, Se, and Te have been shown to be methylated by mammalian cells (1). Furthermore, biohydrogenation has only been proven for microorganisms. For geochemical and toxicological reasons, biomethylation and hydrogenation are highly relevant processes as mobility and in most cases toxicity are significantly enhanced in comparison to inorganic species (6). While inorganic hydride species exhibit a high acute toxicity resulting in very low air quality standards (e.g., threshold recommendation for arsine is 16 µg/m3), (7) permethylated Sn, Pb, and Hg species are well-known for their severe neurotoxic effects (6), which to a lesser extent have also been shown for dimethyltellurium (8). The toxicity of dimethylselenium is lower in comparison to inorganic selenium or selenium dihydrogen (9), but toxicity of methylated compounds is markedly enhanced by parallel administration of arsenite (10). For methylated arsenic species, the situation is most complex; as pentavalent oxoforms of methylated arsenic species have a low toxicity, biomethylation has been considered as a detoxification process. Yet, recent studies have shown that other arsenic metabolites (trivalent methylated and pentavalent thioforms of methylated arsenic) have a high toxicity, hence detoxification of arsenic by biomethylation seems questionable (11). Finally, (CH3)3As and (CH3)2AsH as well as (CH3)3Sb have been reported to be genotoxic (12, 13). Intestinal Biomethylation. Though the human gut contains a highly diverse and active microbial community, little is known about the occurrence and importance of biomethylation in the human gut. For arsenic, the first indication of volatilization of arsenic by intestinal microorganisms dates back to 1917 (14). Putoni attributed the garlic odor after oral treatment of dimethylarsenic acid to intestinal biovolatilization as he detected the same garlic odor (presumably (CH3)3As) in bacterial cultures isolated from feces amended with dimethylarsenic acid. Challenger and co-workers (15) were unable to reproduce these results with cultures of these organisms obtained from a culture collection. Rowland et al. (16) and Hall et al. (17) demonstrated that arsenic was methylated by rat and mouse cecum material to mono- and dimethylarsenic acid, (volatile species were not analyzed). Upon oral administration of colloidal bismuth subcitrate (CBS), volatile (CH3)3Bi was detected in the breath as well as the blood of voluntary probands (18). While maximal conversion ranged to 0.03% of the ingested bismuth, a large interindividual variation was observed. By incubating stool samples of these probands, the ability of the fecal microorganisms to produce volatile methyl and hydride species of Bi was demonstrated; in addition, volatile methyl and hydride species of As, Sn, Sb, Te, and Pb were detected (19). In Vitro Simulation of Human Gut Microbiota. For detailed investigation of biovolatilization of metal(loid)s, in vivo studies are limited in respect to reproducibility and repeatability as well as the choice of metal(loid)s that can be administered. Additionally, studying microbial biovolatilization requires the exclusion of eukaryotic metabolism from the experimental setup. Hence, the use of an in vitro model of the human intestinal microbiota is necessary. The SHIME VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Inorganic Metal(loid) Standards and Composition of Multielement Spike Solutions A and B compound

chemical formula

oxidation number

supplier

purity [%]

primary solvent

spike A spike B

GeO2

4

ABCR

99.999

water

+

AsHNa2O4 * 7H2O

5

Fluka

98.5+

water

+

AsNaO2 H2SeO4 H2SeO3 SnCl2 anhydride SnCl4

3 6 4 2 4

Fluka Aldrich Fluka Lancaster Acros

99 99.9 97+ 98 98+

water water water water water

K[Sb(OH)6]

5

Merck

p.a.

water

SbCl3 H6TeO6 HgCl2 C4H6HgO4 Pb(NO3)2 Pb(CH3COO)4

3 6 2 2 2 2

BDH Fluka Acros Merck Alfa-Aesar Lancaster

p.a. 99+ reagent ACS p.a. 99 95

bismuth nitrate

Bi(NO3)3

3

Fluka

99

bismuth ammonium citrate

C6H10BiNo8

3

Alfa Aesar 98

ethanol water water water water water 100 mM EDTA, 20 mM Tris/HCl water

germanium(IV) oxide arsenate (Na-hydrogen arsenate) arsenite [Na-(meta) arsenite] selenic acid selenous acid tin(II) chloride tin(IV) chloride potassium hexahydroxy-antimonate (V) antimony trichloride telluric acid mercury(II) chloride mercury(II) acetate lead(II) nitrate lead acetate

system (Simulator of the Human Intestinal Microbial Ecosystem) is an in vitro gastrointestinal model developed to mimic the microbial community present in the human colon (20). The SHIME system mimics several physicochemical, enzymatic, and especially microbiological processes that occur in the human gut. It allows the control of stomach pH, small intestine bile salt, and enzyme concentrations as well as the use of human colon microbiota at different pH, Eh, and residence time values. This provides access to research that studies the dissolution of ingested contaminants in the gut and also the changes in speciation that are taking place during digestion. The SHIME system has previously been used to investigate the influence of intestinal microorganisms on the bioaccessibility of arsenic from ingested mine tailings (21) as well as the dissolution and microbial bioactivation of polycyclic aromatic hydrocarbons (22). Aim of This Study. The aim of this study is to explore the metabolic potency of the gut microbiome toward different elements and compare the microbial conversion between different regions of the colon. Therefore, different concentration levels as well as different inorganic redox forms of these elements were added to aliquots from the SHIME colon and rectum compartments. This study focuses on the volatilization of these elements as volatile species of all investigated elements can be simultaneously detected by the use of gas chromatography and inductively coupled plasma mass spectrometry (GC-ICP-MS). For identification of unknown volatile species, parallel detection by ICP-MS and electron ionization mass spectrometry (GC-EI-MS/ICPMS) was applied.

Experimental Section Preparation of Spike Solutions. The metal(loid) compounds listed in Table 1 were used for preparation of the different spike solutions used in this work. For all compounds, stock solutions were prepared with a final concentration of 500 mg (element/L) in pure laboratory water, if not indicated as following. Antimony trichloride stock solution was prepared in ethanol. Twenty millimolar bismuth nitrate was solubilized using 100 mM 1,3-propanediol, 100 mM EDTA, and 20 mM tris/HCl prior to dilution to 500 mg/L in water. Bismuth ammonium citrate was not sufficiently soluble. Upon sedimentation of the precipitate, the supernatant was used for further dilutions. Concentration of the supernatant was determined by ICP-MS after 1:100 dilution in 1.3% HNO3 as 300 mg Bi/L. Multielement stock solutions containing 50 mg element/L (Hg(II) and Sb(III), both 5 mg/L), were prepared by combining one compound per element according to Table 5250

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+ +

+ + + + + + +

+ + +

+ + + +

1 in water. All spike solutions were prepared in pure laboratory water directly before the experiment and dilutions were controlled by weighing. Dynamic SHIME. The SHIME (Simulator of the Human Intestinal Microbial Ecosystem) is a continuous reactor model of the human gut. It consists of five reactor compartments mimicking physicochemical, enzymatic, and microbiological conditions in the stomach, small intestine, and different colon regions [ascending (C3), transverse (C4), and descending colon (C5)]. The colon compartments harbor an anaerobically cultured microbial community with a composition and metabolic activity resembling that of the human in vivo situation. Colon effluent was collected in a separate rectum compartment (C6), from which larger sample volumes could be collected for conducting a larger number of batch experiments. The microbial community in the colon compartments received a nutritional medium that passed the stomach and small intestine compartments. Composition of the medium can be consulted in Van de Wiele et al. (23). The suspension in the stomach compartment was maintained at pH 2 and mixed constantly at 37 °C for 2 h. Following stomach incubation, a pancreatic solution containing 12.5 g L-1 NaHCO3, 6 g L-1 Oxgall (Difco), and 0.9 g L-1 porcine pancreatin powder (Sigma, Belgium) was pumped into the small intestine vessel in a 2:1 (stomach:pancreatic solution) ratio, thereby creating a mixture of pH 6.5. Following a 5 h retention time, the small intestine suspension was pumped into the ascending colon compartment and subsequently into the transverse and descending colon compartments. These colon compartments were inoculated with fecal microbiota from a healthy adult male individual. Freshly excreted feces (10 g) were diluted and homogenized with sterile phosphate buffer (0.1 M, pH 7.0, 100 mL) to which 100 mg of sodium thioglycolate was added. Particulate matter was removed by centrifugation (500 g, 1 min), and 40 mL of the microbiota-containing supernatant was inoculated into the different colon compartments. The pH in the ascending, transverse, and descending colon vessels was maintained at 5.7, 6.3, and 6.7, respectively. Other reactor specifications can be found in Table 2. Aliquots of the suspension from the colon compartments and rectum compartment of the SHIME provided the suspensions used in static batch experiments to monitor metal(loid) speciation changes. Samples from the SHIME colon compartments were only taken after a stabilization period of 3 weeks. This way, the residing microbial community had reached equilibrium in terms of community composition as metabolic activity (24). This

TABLE 2. Setup of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) with Reactor Volumes and Retention Times compartment

volume (mL)

residence time (h)

C1, stomach C2, small intestine C3, ascending colon C4, transverse colon C5, descending colon C6, rectum

200 200 500 800 600 variable

2 5 20 32 24 variable

pH

redox potential (mV)

net short fatty acid production (mM)

5.6-5.9 6.1-6.4 6.6-6.9

-170 -200 -230 -220

46 8 5

TABLE 3. Experimental Designa compartment concentration dependence

interelement cross influence

rectum

0 µg

rectum rectum rectum rectum rectum rectum rectum

spike A 0.1 µg spike A 0.5 µg spike A 1 µg spike A 5 µg spike A 10 µg spike A 50 µg spike A 1 µg + AsV 5 µg spike A 1 µg + AsV 50 µg spike A 1 µg + AsV 500 µg spike A 1 µg + SeVI 5 µg spike A 1 µg + SeVI 50 µg spike A 1 µg

rectum rectum rectum rectum colon compartment dependence

oxidation state dependence matrix dependence

spike amount (per element)

ascending colon transverse colon descending colon rectum transverse colon rectum

spike A 1 µg spike A 1 µg spike B 1 µg spike B 1 µg BGS soil 100 mg

a

The metal(loid)s in spike solutions A and B are listed in TABLE 1. Hg(II) and Sb(III) (solubilized in ethanol) were used at one-tenth of the concentration indicated in order to reduce cytotoxic effects.

approach increases the reproducibility of our batch incubations and lowers the variability in the experimental data set. SHIME Batch Incubations. Suspensions (50, 70, 50, and 800 mL, respectively) were sampled anaerobically from the ascending (C3), transverse (C4), descending (C5), and colon and rectum (C6) compartments. Subsequently, 10 mL aliquots were transferred to 20 mL glass vials with butyl rubber stoppers. Anaerobic conditions were obtained in these serum bottles by consecutive cycles of overpressure (1 bar N2, 2 min) and underpressure (1 bar vacuum, 2 min) for 20 min beforehand. Then, different amounts of elements were added to the batches according to the experimental design in Table 3. All incubation experiments were conducted as triplicates. First, in order to investigate the concentration dependence, each 1 mL of different levels of multielement solution A diluted in pure laboratory water (Table 1) was added to aliquots of rectum suspension. Second, batches containing rectum suspension and 1 µg per element of solution A were additionally amended with different amounts of arsenic or selenium in order to determine interelement effects of biovolatilization. Third, volatilization of 1 µg per element of solution A by suspension from different SHIME colon compartments was compared. Fourth, the effect of different initial redox forms of the inorganic element spike was tested

by amending 1 µg per element of solution B (Table 1) to batches with either rectum or transverse suspension. Finally, to investigate whether volatilization of elements bound to a soil matrix takes place, we placed 100 mg of a reference soil (BGS 102, British Geological Service) in the 20 mL glass vials prior to flushing with nitrogen as indicated above. Then, 10 mL of rectum suspension was added. Masses of aliquots and spike solutions added were controlled by weighing. After spiking, all batches were incubated in the dark at 37 °C without shaking, with exception of the soil incubation experiments, which were incubated under moderate shaking (180 rpm) to prevent settlement of the soil. The gas phase was completely removed and replaced with He after 40 h and analyzed for volatile heteroelement compounds (VHEC) after 90 h with the exception of the soil incubation experiments, which were analyzed after 90 h incubation without intermediate headspace removal. Sterile Control Experiments. Ten milliliter aliquots of suspension from rectum reactors were transferred to 12 100 mL glass serum bottles. Then, six batches were autoclaved at 120 °C for 20 min twice. After autoclaving, 5 µg per element of multielement solution A was added via sterile 0.2 µm filters to each three autoclaved and nonautoclaved batches. All batches were incubated in the dark at 37 °C and analyzed for VHEC after 48 h. Analysis of VHEC by GC-ICP-MS. VHEC were analyzed by gas chromatography and inductively coupled plasma mass spectrometry (GC-ICP-MS) using a self-developed semiautomated GC system with packed columns, which is a further development of the system described by Diaz-Bone and Hitzke (25). In contrast to this system, self-developed software allowed to reproducibly control the time-programmed heating of the resistance wire, gas flows, and valve positions, and to trigger the data acquisition of the ICP-MS. Losses of VHEC in the GC system were below 5% for this system (25). The GC efflux was mixed to the efflux from the spray chamber of a 7500a ICP-MS (Agilent, Yokohama, Japan) by using a T-piece inserted between the spray chamber and torch. Wet aerosol from the liquid sample introduction system is used for introduction of a continuous internal standard solution (10 µg/L Ga, Tl, and 100 µg/L In) and for postcolumn quantification using interaggregate calibration (IAC) (25). Polyatomic interferences were identified by controlling the isotope ratio of polyisotopic analytes. In order to minimize the effect of eluting organic matrix compounds on plasma stability and ionization efficiency, 5% n-propanol was added to the aqueous standard. Operating parameters for the GCICP-MS are presented in Table S1 of the Supporting Information. For analysis of VHEC, headspace of the incubation vials was purged with 100 mL He/min for 3 min. The volatile analytes were focused on-column by immersing the column in liquid nitrogen. At the beginning of the GC run, the liquid nitrogen was removed, and the column was immersed for 60 s in an n-pentane cold bath adjusted with liquid nitrogen to -80 ( 5 °C in order to achieve baseline separation of VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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arsine from CO2. Then, the column was heated by a computercontrolled electric current time profile to a final temperature of ∼200 °C. Standard addition was performed with hydride and methylated species of Ge, As, Sn, and Sb. The sample matrix had no significant effect on the analytical separation and quantification of the investigated VHEC. Recoveries were satisfactory in the range of 100 ( 10% for the species tested with the exception of (CH3)3Sb (88.5%) (Figure S1 of the Supporting Information). Identification of VHEC. VHEC were identified by retention time comparison with volatile standards listed in Tables S2 and S3 of the Supporting Information or by GC-EI-MS/ICPMS. The system has been described in Ko¨sters et al. (26); instrumental parameters are listed in Table S4 of the Supporting Information.

Results Detection of Volatile Heteroelement Compounds (VHEC). The use of purge and trap GC-ICP-MS allowed the direct cryotrapping of the entire headspace (without the necessity of matrix removal) and analysis of analytes with boiling points between -62 and 200 °C, with a total analysis time of 10 min per analysis (25). Sample chromatograms can be consulted in panels a-d of Figure S2 of the Supporting Information. Simple hydride and methylated species were identified by retention time comparison with standards listed in Tables S2 and S3 of the Supporting Information. For other compounds, identification was achieved by GC-EI-MS/ICP-MS analysis of selected samples. The ICP-MS mass trace was used for determination of the retention time of the compounds when the EI-MS fragment pattern was extracted. With these spectra, it was possible to identify (CH3)2Te, CH3SeSCH3 (methyl-methylthio-selenide), CH3SeSSCH3 (methyl-methyldithio-selenide), (CH3S)2Se [di(methylthio)-selenide], and (CH3)2AsSCH3 (dimethyl-methylthio-arsine) by comparison with literature spectra (Figure S3 of the Supporting Information). In addition to these compounds, (CH3)2AsSSCH3 (dimethyl-methyldithio-arsine) and CH3As(SCH3)2 [methyl-di(methylthio)-arsine] were identified by their characteristic EI-MS mass fragments 184 and 169 and by retention time comparison with synthesized standards (Table S5 of the Supporting Information). A detailed description of these new arsenic species, including full mass spectra and synthesis as well as discussion of the uniqueness of the identification, can be consulted elsewhere (27). In addition to these compounds, several quantitatively less important high-boiling species of As, Se, and Te were detected, which could not be identified so far. For Ge, Sn, Pb, and Hg, no volatile species were detected in this study. Factors Modulating Biovolatilization. Element Concentration. Incubation of a concentration series of a mixed element solution with SHIME rectum suspension showed a dose-dependent relationship toward the amount volatilized. Most surprisingly, the dominant As species detected in batches with rectum suspension was AsH3, showing a clear increase in relation to the amount spiked and a leveling off for the 10 and 50 µg spiked samples (Figure 1). In contrast, only trace amounts of (CH3)3As were detected, and production of (CH3)3As decreased with an increasing amount of element spiked. (CH3)2Te production showed a concentration dependence similar to AsH3. While in nonamended batches these species were only present in minute amounts close to the detection limit, significant quantities of volatile Se species were found in these batches, which stem from the high background concentration of the SHIME suspension (60 µg Se/L or 0.6 µg Se absolute per batch). With the exception of the highest level amended (50 µg per element), the amount of volatile sulfur compounds was not affected by the concentration of elements added, which implies that the 5252

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element concentrations up to 1 µg/mL were below acute toxicity. Finally, only trace amounts of volatile Sb and Bi species were detected in all experiments (Table S6 of the Supporting Information). As the amount of species detected in the headspace was independent from the amount added, it must be assumed that the inorganic antimony and bismuth added was not volatilized and that the volatile species detected were produced from the metal(loid) background concentration of the SHIME suspension (both 40 µg/L). Interelement Cross Influence. For the batches with the highest spikes, 10 µg and in particular 50 µg per element, a decrease in volatilization efficiency was observed for As and Se. In order to investigate if this lower volatilization was attributed to higher concentrations of the element itself or if it was due to interelemental cross influence, we conducted parallel experiments with different relative element spike ratios (Figure 2). Incubations containing 1 µg multielement spike with an additional 5 µg As or Se (Figure 1), resulted in a higher volatilization of As (five times more) and Se (three times more) compared to the 5 µg multielement batches (Figure 2a,d). For the 50 µg level, volatilization of As was seven times higher and volatilization of Se three times higher in comparison to the 50 µg multielement spike. This indicates an inhibition of the volatilization of As and Se by the high concentration of other metal(loid)s. Furthermore, As spiked at the highest level of 500 µg resulted in a decrease of As volatilization (Figure 2a), indicating self-inhibition of this element. Comparison of interelement cross influence of As and Se showed that volatilization of 1 µg As was decreased by a factor of 5 and 10 when adding 5 µg and 50 µg Se, respectively (Figure 2b). In contrast, volatilization of 1 µg Se was not affected with increasing As concentration (Figure 2c). With respect to volatilization of the other compounds in the 1 µg multielement spike, there was little effect from the spiked amounts of As and Se (Table S6 of the Supporting Information). Dependence on Colon Region and Volatilization Rates. To investigate whether volatilization is colon region dependent, we sampled suspension from the different SHIME colon compartments and supplemented with 1 µg per element. Upon incubation, volatilization rates from colon compartments were up to 2 orders of magnitude higher compared to the rectum compartment (Figure 3). In addition to absolute volatilization, the As species pattern differed fundamentally between different colon compartments. While hydrogenation of As dominated in the rectum compartment, high-boilingmixed As/S species were the most important compounds detected in the headspace of the colon compartments. In terms of volatilization rates, the highest rates were measured for As and Te in batches with suspension from the ascending and transverse colon compartments. Maximal hourly production rates relative to the amount spiked were 2 and 9 ng/(mg h) for As and Te, respectively. For Se, differences between the different compartments were less pronounced. Highest rates for Se [0.6 ng/(mg h)] were found in batches additionally amended with selenium. Sterile Control Experiments. To investigate whether the observed VHEC were formed by microbiological activity, we conducted sterile control experiments, in which the metal(loid) mixture was incubated in a heat sterilized suspension of the SHIME rectum compartment, thereby inactivating the intestinal microbiota (Table S7 of the Supporting Information). In comparison to the nonsterile batches, only trace amounts of VHEC were detected in the headspace of sterilized batches. The trace amounts of volatile selenium, tellurium, and antimony species detected in amended and nonamended sterile batches likely stem from methylated species formed prior to sterilization as amended and nonamended batches

FIGURE 1. Volatilization of As, Se, Te, and S dependent on the amount of element spiked. Each 10 mL amount of reactor C6 (rectum) of the SHIME system was amended with multielement spike solution A containing Ge, As, Se, Sn, Sb, Te, Hg, Pb, and Bi according to Table 3 and incubated anaerobically for 90 h. Note that the amount of Hg was one-tenth of the amount indicated, and that the spike solution did not contain sulfur. n ) 3. show similar concentrations. Thus, we infer, that the As) was added to batches with 10 mL of rectum suspension. volatilization of the elements investigated is predominantly Significant volatilization was detected in the batches amended caused by biological processes. with soil (Table S8 of the Supporting Information). Surprisingly, Dependence on Valence. Different inorganic redox forms the element pattern as well as conversion rates differed of As, Se, Sn, Sb, and Pb were spiked to suspension from significantly from experiments with inorganic metal(loid) salts. rectum and transverse colon compartments (Table S6 of the While the relative As volatilization was low, significant amounts Supporting Information). Only a little difference between of permethylated Sb, Bi, Se, and Te species were detected. The these batches was observed. specific conversion rates for Sb, Bi, Se, and Te were 5, 6, 12, and Dependence on Matrix of Element Amendment. Ingested 64 ng/(mg h), respectively. soils are important metal(loid) sources, particularly for children Discussion (21). In order to investigate if metal(loid)s bound to a soil matrix This study is the first to explore the metabolic potency of are also available for intestinal biovolatilization, 100 mg of a human intestinal bacteria toward a mixture of different reference soil (BGS 102, British Geological Service, 104 mg/kg VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Volatilization of As (graphs a,d) and Se (graphs b,c) dependent on the amount of As or Se added. In addition to the As and Se spike indicated, 1 µg of Ge, Sn, Sb, Te, Pb, and Bi and 0.1 µg Hg were added to all batches according to Table 3. Each 10 mL amount of suspension from reactor C6 (rectum) was incubated anaerobically for 90 h. n ) 3. metal(loid)s and nonmetal Se starting from inorganic salts. Intestinal bacteria sampled from the in vitro Simulator of the Human Intestinal Microbial Ecosystem (SHIME) and subsequently incubated with a metal(loid) mixture were capable of volatilizing As, Se, Te, Sb, and Bi into methylated species. These findings together with the occurrence of arsine and methylthio species of As and Se warrant further research into this topic as it may have toxicological consequences that go beyond our current knowledge of the capabilities of the intestinal microbial community. Highest volatilization rates were observed for Te, followed by As and Se, whereas Bi and Sb volatilization was minimal. No volatile Ge, Sn, Pb, and Hg species were detected in this study. While permethylated species represent the most abundant volatile species in different environmental matrices (waste deposits (2), sewage sludge fermentation (3), and soils (4)), hydrogenation and addition of methylthio groups seem to play important roles in volatilization by intestinal microorganisms. Much to our surprise, the highly toxic arsine (AsH3) was the most dominant As volatile produced by intestinal bacteria from the rectum compartment. Arsine has a hemolytic potential by releasing the heme group when interacting with hemoglobin (28). Because of the high amounts of volatile sulfur compounds in the intestinal headspace sample, the detection of Se/S and As/S species is not surprising. Various mixed Se/S species have been detected in the headspace of bacterial cultures, soils, and plants as well as human breath after ingestion of garlic (5, 29, 30). In contrast, little evidence on methylthio species of arsenic has been gathered in environmental samples so far. (CH3)2AsSCH3 has been detected in compost samples and hydrothermal gas from Yellowstone National Park by Ko¨sters et al. (31) and Planar-Friedrich et al. (32). To the best of our knowledge, neither (CH3)2AsSSCH3 nor (CH3)As(SCH3)2 have been described in environmental matrices previously. As demonstrated by our own synthesis experiments with (CH3)2AsI and CH3SH, (CH3)2AsSCH3, (CH3)2AsSSCH3, and CH3As(SCH3)2 can be formed from these educts (27). The toxicology of methylthio species of arsenic and selenium species is unknown. The finding of these new metabolites may thus lead to new research initiatives to assess the actual toxicity and risks posed toward human health by these compounds. While significant methylation was found for Te and Se as well as formation of arsine and mixed Se/S and As/S species, surprisingly only trace amounts of volatile Sb and Bi species 5254

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were detected upon amendment of the metal(loid) salts, although biomethylation is known to be very effective for these elements (2–4). A possible explanation for this phenomenon is that cross influence between metal(loid)s suppressed biomethylation of these elements (33), thus single element experiments will have to be conducted. This study illustrates such an inhibitory effect from Se toward As volatilization. Further research with binary metal(loid) mixtures would be needed to further elucidate the cross influence of metal(loid)s with respect to their volatilization by intestinal microbiota. Incubation of the metal(oid) mixtures in microbial communities sampled from different colon compartments resulted in much higher volatilization rates for the colon suspensions compared to the rectum suspensions. The higher volatilization efficiency may be attributed to higher microbial fermentation activity as nondigested dietary constituents from the small intestine compartment become available to the resident colon microbiota. This, for instance, is reflected by the higher net short-chain fatty acid concentration in the ascending colon compartment (46 mM) versus the transverse colon (8 mM) and descending colon (5 mM) (Table 2). Yet, a difference in microbial community composition may also form the basis for the difference in volatilization efficiency. Characterization and identification of the responsible microorganisms triggering the volatilization is therefore an important future research area. The difference in oxidationreduction potential between the different compartments did not affect the volatilization process. Redox potentials were highest in the ascending colon (-170 mV) and lower in the transverse colon (-200 mV), descending colon (-230 mV), and rectum (-220 mV) (Table 2). Apart from the AsH3 formation, which was lower in the ascending colon, metal(loid) volatilization was influenced more by the degree of biological activity than redox potential. This conclusion is fairly similar to the observations from Eckley and Hentelmann (34), who described lake sediment anoxic conditions to be a prerequisite for mercury methylation, but the degree of methylation depends on microbial activity. Finally, we show that the initial oxidation of the elements seem to play little role on the volatilization rate. This is probably due to the strongly reducing conditions that prevail in every compartment of the colon. With these experiments we have clearly demonstrated metal(loid) volatilization in the human intestine. Because of the complexity of this process, further research will be necessary to estimate the toxicological relevance of this process. While some of the compounds formed are highly toxic [AsH3 and (CH3)2Te], the overall conversion rates found in these experiments were relatively low. Yet higher conversion rates can be expected under optimized incubation conditions (e.g., single element incubations), sampling times, and metal(loid) concentration. Furthermore, the matrix in which the metal(loid)s are embedded can enhance volatilization as in the case of soil incubation. Investigating metal(loid) volatilization under optimized conditions will allow a more in-depth study of this process and makes it possible to calculate reliable biomass-specific conversion rates. This further demonstrates the need for research on the fundamental parameters that influence this process. While acute toxicity of the volatile compounds might be low due to the low absolute amounts formed, the process of intestinal biovolatilization has to be taken into account when assessing the risk from orally exposed metal(loid)s. Because of their ability to pass cell membranes, volatile metal(loid) compounds can penetrate the intestinal mucosa as well as the blood-brain barrier and thus contribute to metal(loid) toxicity. Hence, to what extent microbial arsine formation contributes to chronic toxicity of arsenic exposure is not known.

FIGURE 3. Volatilization of As, Se, Te, and S by the microbiocenosis of different compartments of the SHIME system. Each 10 mL suspension from the different compartments were spiked with multielement spike solution A (containing 1 µg per element Ge, As, Se, Sn, Sb, Te, Pb, and Bi and 0.1 µg Hg) according to Table 3 and incubated anerobically for 90 h. n ) 3. Clearly, interindividual variation of the composition and activity of the intestinal microbiome will strongly affect the biotransformation of metal(loid)s as demonstrated for bismuth by Boertz et al. (18). Hence, interindividual differences in susceptibility to metal(loid)s need to be investigated in future studies. Finally, oral exposure to metal(loid)s is highly diverse and can stem from soils, dust, drinking water, and metal(loid) contaminated foods as well as metal(loid) containing pharmaceuticals or personal care products. We therefore conclude that the role of the intestinal microbial community in metal(loid) biotransformation needs to be further addressed to assess to what extent this metabolic potency may pose health hazards to the human body.

Acknowledgments R.A.D.-B. acknowledges the University of Duisburg-Essen for funding in the “Sponsorship for Young Scientists” program. T.V.d.W. is a postdoctoral research fellow of the FWO (Fonds Wetenschappelijk Onderzoek Vlaanderen). We thank Prof. A.V. Hirner for fruitful discussions and Joerg Hippler for technical assistance.

Supporting Information Available GC-ICP-MS operational parameters, sample chromatograms, and standard addition recovery as well as the amounts of volatile species detected in tabulate form.This material is available free of charge via the Internet at http://pubs.acs.org. VOL. 43, NO. 14, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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