Arsenic Speciation in Blue Mussels (Mytilus edulis) Along a Highly

Feb 14, 2012 - Arsenic is naturally present in marine ecosystems, and these can become contaminated from mining activities, which may be of toxicologi...
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Arsenic Speciation in Blue Mussels (Mytilus edulis) Along a Highly Contaminated Arsenic Gradient K. J. Whaley-Martin,† I. Koch,† M. Moriarty,† and K. J. Reimer*,† †

Environmental Sciences Group, Royal Military College of Canada, P.O. Box 17000 Station Forces, Kingston, Ontario K7K 7B4, Canada S Supporting Information *

ABSTRACT: Arsenic is naturally present in marine ecosystems, and these can become contaminated from mining activities, which may be of toxicological concern to organisms that bioaccumulate the metalloid into their tissues. The toxic properties of arsenic are dependent on the chemical form in which it is found (e.g., toxic inorganic arsenicals vs nontoxic arsenobetaine), and two analytical techniques, high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and X-ray absorption spectroscopy (XAS), were used in the present study to examine the arsenic species distribution in blue mussels (Mytilus edulis) obtained from an area where there is a strong arsenic concentration gradient as a consequence of mining impacted sediments. A strong positive correlation was observed between the concentration of inorganic arsenic species (arsenic compounds with no As−C bonds) and total arsenic concentrations present in M. edulis tissues (R2 = 0.983), which could result in significant toxicological consequences to the mussels and higher trophic consumers. However, concentrations of organoarsenicals, dominated by arsenobetaine, remained relatively constant regardless of the increasing As concentration in M. edulis tissue (R2 = 0.307). XANES bulk analysis and XAS two-dimensional mapping of wet M. edulis tissue revealed the presence of predominantly arsenic−sulfur compounds. The XAS mapping revealed that the As(III)-S and/or As(III) compounds were concentrated in the digestive gland. However, arsenobetaine was found in small and similar concentrations in the digestive gland as well as the surrounding tissue suggesting arsenobetaine may being used in all of the mussel’s cells in a physiological function such as an intracellular osmolyte.

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concentration of arsenic but also the arsenic species.2,3 Only one of the studies involved a known contaminated site, at which unusually high proportions of inorganic arsenic (>40%) were found in clams that contained the highest concentrations of arsenic reported to date in their tissue, up to 230 mg·kg−1 (wet weight).2 Mussels with concentrations of arsenic higher than 3 mg·kg−1 (wet weight) also contained high proportions of inorganic arsenic ranging from 30 to 42%,3 but contamination at collection sites was not reported.3 Therefore the conditions under which inorganic arsenic is retained in an organism are unclear, and a study examining arsenic speciation changes in marine organisms along a sediment concentration gradient would be valuable. Blue mussels (M. edulis) were chosen as the study specimen because of mussels’ common use in biomonitoring programs,4,5 their widespread distribution, sedentary life habits, and intimate relationship with the local environmental conditions. These

INTRODUCTION Arsenic’s ubiquitous presence in marine environments is a result of natural phenomena, but mining practices have amplified the input of this metalloid in various coastal ecosystems around the world. This is of concern since arsenic is capable of posing serious toxicological threats to the organisms in these environments. Low trophic organisms that inhabit marine environments (i.e., plankton, mussels, clams, etc.) and their consumers (i.e., large fish, seals, humans) are all potential receptors. The toxicological properties of arsenic are widely recognized to be dependent on the concentration as well as the chemical form of arsenic to which an organism is exposed. Thus, characterizing the arsenic species distribution within organisms can provide insight into how arsenic is being cycled and transformed throughout an ecosystem and whether a resulting health risk is present. Usually in marine animals, the nontoxic organoarsenical arsenobetaine (AB) is the dominant form of arsenic of the overall composition of arsenic species.1 Only two studies have reported the arsenic characterization in marine organisms with higher than normal arsenic concentrations, and they show substantial differences when compared with those assumed to be at natural concentrations, not only with respect to the Published 2012 by the American Chemical Society

Received: Revised: Accepted: Published: 3110

October 26, 2011 February 9, 2012 February 14, 2012 February 14, 2012 dx.doi.org/10.1021/es203812u | Environ. Sci. Technol. 2012, 46, 3110−3118

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intermediate tide level. Sediment, surface water, and porewater samples were immediately frozen after sampling and shipped with dry ice to the Royal Military College of Canada and stored at −18 °C until analysis. All water samples were filtered though 0.45 μm filters (Millipore polypropylene 25 mm diameter hydrophilic PVDF durapore membrane) prior to analysis. Composite samples from each site were made from the whole body tissues of ten to fifteen mussels, after removal from shells and washing (2×) with DDW. Dry biological tissue samples were prepared by freeze-drying wet tissue for at least 24 h immediately after deshelling. Dry tissue samples were then ground to a fine powder with a ceramic mortar and pestle. Arsenic Species Extraction from Mytilus edulis. Five replicate extractions were carried out for mussels from each site. Freeze-dried samples (0.5 g) were weighed into 50 mL Fisherbrand disposable polypropylene centrifuge tubes. Extraction was carried out using a two-step sequential extraction as described in detail elsewhere11 with 1:1 methanol/water used in the first step and 2% HNO3 (at 70 °C for two hours) in the second step. Methanol was removed by evaporation at 60 °C, and all extracts were filtered through 0.45 μm filters and kept frozen until analysis. Total Arsenic Digestion. Coddles Harbour (CH) sediment samples were digested with aqua regia (8 mL of HNO3 and 2 mL of HCl), and samples were heated to 120 °C until the sample was reduced to 1−2 mL. Total arsenic within mussel tissues was determined by adding total arsenic in extracts with total arsenic in residues from the two-step extraction. Residues were digested with 10 mL of 70% HNO3 at 120 °C until the sample was reduced to 1−2 mL following by heating for an additional hour with 2 mL of H2O2 and final dilution with DDW to 10 mL. All samples were filtered though 0.45 μm filters (Millipore polypropylene 25 mm diameter hydrophilic PVDF durapore membrane) prior to analysis. Total Arsenic Analysis. Total arsenic in dried Seal Harbour sediment samples was determined with a hand-held XRF. CH sediment was found to be below the detection limit of the XRF ( SH Site 4 > Coddles Harbour. Surface water had a similar trend except that SH Site 2 had slightly higher arsenic concentrations than SH Site 1 (Figure 1). The results of the sediment, surface water, and porewater analysis show that M. edulis inhabiting Seal Harbour and especially at SH Site 1 and 2 are potentially being exposed to arsenic through dermal contact with the sediment and water column and ingestion of sediment at concentrations that are higher than those expected under natural conditions. Similar 3113

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Seal Harbour Site 4 mussels additionally contained low concentrations of Sugar 4 (0.110 ± 0.002 mg/kg). bCoddles Harbour mussels additionally contained low concentrations of four unknown arsenic species visible using anion exchange: UK1: 6.3 min (0.104 ± 0.003 mg/kg); UK2: 9.1 min (0.24 ± 0.01 mg/kg); UK5: 10.3 min (0.58 ± 0.02 mg/kg); UK6: 11.5 min (0.24 ± 0.01 mg/kg). cAverages represent the arithmetic mean ± standard error of five replicates (SH Site 3 only had 4 replicates). Inorganic arsenic is the sum of As(III) and As(V). (Percent moisture and total As in sample residues are reported in Supporting Information, Table S5.) a

62 ± 2 0.10 ± 0.02 0.22 ± 0.02 6.223±0.003 0.19±0.01 1.5±0.005 1.3 ± 0.1 0.2 ± 0.1 16 ± 1 grocery store mussels

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46 ± 1 0.75 ± 0.02 0.39 ± 0.01 0.71 ± 0.02 7.2 ± 0.2 0.25±0.01 7.92± 0.02 2.23 ±0.03 34 ± 1 CH musselsb

0.49±0.01 1.28±0.04 -

56 ± 4 2.04 ± 0.03 0.266 ± 0.004 0.225 ± 0.004 5.45 ± 0.09 4.66 ±0.07 16.4 ± 0.2 60 ± 2 Seal Harbour Site 1 mussels

2.71±0.05 2.33±0.04 1.31±0.02

58 ± 1 0.94 ± 0.01 1.10 ± 0.02 2.82 ± 0.04 2.35 ± 0.04 0.45 ± 0.01 4.27 ± 0.07 0.58±0.01 0.34±0.01 0.85±0.01 7.4 ± 0.1 1.83 ±0.03 15.1 ± 0.2

0.65±0.01 6.35±0.08 0.43±0.01 23 ± 1 75 ± 3 Seal Harbour Site 3 mussels

2.08 ±0.03

33 ± 1 109 ± 3

Seal Harbour 65 ± 2 Site 4 musselsa

57 ± 2 1.12 ± 0.01 0.70 ± 0.01 2.68 ± 0.04 1.95 ± 0.03 0.298 ± 0.001 3.19 ± 0.04

0.469 ± 0.004 0.208 ± 0.002

EE (%) 3114

Seal Harbour Site 2 mussels

Table 2. Total Arsenic, Arsenic Species Concentration (mg/kg dry weight), and Extraction Efficiency (EE) of Mytilus edulisc

Tetra

arsenic and its nearest neighbors; in the present study the data support the identification of the species as being trivalent in arsenic and bonded to three sulfurs (i.e., As(III)-S). Although thioarsenosugars (which contain an As−C bond) have been observed in mussels previously (e.g.,18), HPLC-ICP-MS analysis did not indicate the presence of these compounds (although they may have been unstable in the method used, which had not been optimized for the detection of these compounds). The XAS behavior of such compounds is unknown, but we hypothesize that As(V) thiolated species would demonstrate absorptions at higher energies.19 Sulfur is a common component of molecular species in biological tissue (e.g., cysteine residues of amino-acids in proteins), and therefore the interaction between these biomolecules and arsenic or its metabolites may be expected. While the binding of inorganic arsenic with the thiol moieties has been proposed as a likely candidate for a mechanism that explains arsenic’s toxicity,20,21 this type of interaction could also be involved in detoxification processes. Specifically, the As(III)-S identified in the present study could be arsenic bound to metallothioneins. The formation of this ubiquitous class of cysteine-rich proteins (30% of the amino acids)22 is induced when the intracellular concentration of metals and/or metalloids becomes too high or too toxic and have been observed to be produced by M. edulis.23 Metallothionein-bound arsenic could be transported to the intracellular lysosomal system in M. edulis, which hypothetically might allow the organism to avoid acute toxicity of free arsenic and to bioccumulate the metalloid without adverse affects. The As(III)-S compounds identified by XAS were not detectable with the HPLC-ICP-MS method employed in this study because the arsenic−sulfur bonds in these compounds were likely unstable during freeze-drying, a procedure that in another study led to similar molecules presenting themselves as As(V) and As(III).24 XANES-identified As(III)-S compounds are therefore referred to as “inorganic arsenic” for the purposes of comparing HPLC and XANES data in the present study. The results of the arsenic speciation analysis conducted by HPLC-ICP-MS of M. edulis are listed in Table 2. Six unknown (UK) species were found at low concentrations in extracts on the anion exchange chromatography system (UK1: 6.3 min; UK2: 9.1 min; UK3: 9.8 min; UK4; 10.1 min; UK5: 10.3 min; UK6: 11.5 min). Four of these unknown anionic arsenic species (UK1, UK2, UK5, and UK6) were only found in extracts from Coddles Harbour mussels. UK3 and UK4 were found in extracts of three of the six groups of mussels (Table 2). Large concentrations of inorganic arsenic were found in mussels from contaminated sites, and a decrease in extraction efficiency was observed as total arsenic and inorganic arsenic increased. Extraction efficiency in the most contaminated mussels was 42 ± 2%, while extraction efficiencies in mussels

0.075 ± 0.001

Data were fit with frozen AB (white line energy 11872.6 eV); frozen As(V) (white line energy 11875.3 eV); frozen As(III) (white line energy 11871.7 eV); and As(glutathione)3 (white line energy 11870.0 eV).

1.49±0.01 2.54±0.02 1.26±0.01

a

5.0 ± 0.5

UK4 (anion, 10.1 min)

0.0008 0.0007 0.0006 0.002

UK3 (anion, 9.8 min)

28 24 11 5

AC

14 11 14 0

TMAO

16 18 19 36

AB

42 47 56 59

Sugar 3

Seal Harbour Site 1 Seal Harbour Site 3 Seal Harbour Site 4 Coddles Harbour

Sugar 2

reduced X2

Sugar 1

% As(V)

DMA

% As(III)

MMA

% As(III)sulfur

inorganic As

% AB

total As (dry wt)

site

2.46 ±0.02

Table 1. X-ray Absorption Near-Edge Structure Fitting Results for Blue Mussels (Mytilus edulis) along a Concentration Gradienta

42 ± 2

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Figure 2. a) HPLC-ICP-MS results of arsenic species concentration (mg/kg dry weight) of inorganic arsenic, unextracted arsenic, and complex organoarsenicals (AB, AC, MMA, DMA, TMAO, Tetra, and Arsenosugars) in Mytilus edulis across a total arsenic concentration gradient in whole mussel soft tissue. b) XANES analysis results of arsenic species proportions (%) of inorganic arsenic (As(III)+As(V)+As(III)-sulfur) and complex organoarsenicals in wet M. edulis tissue across a total arsenic concentration gradient in whole mussel soft tissue.

from a local grocery store were considerably higher (62 ± 2%). The data were further examined for patterns in the inorganic arsenic with respect to total tissue arsenic concentrations. Organoarsenicals (AB, AC, MMA, DMA, TMAO, Tetra, and arsenosugars) were summed together, representing metabolized arsenic species. Organoarsenicals can be present in mussels as a result of two pathways, either alone or in combination: metabolism of ingested simpler arsenicals by the mussels or ingestion and retention of organoarsenicals. In the former scenario, high proportions of inorganic arsenic might result from saturation of biochemical pathways (within the mussel or associated organisms) responsible for the transformation of inorganic arsenicals (from food, water, and/or sediment) into arsenobetaine and other complex organoaresenicals, The results of this study show that the concentration of the summed organoarsenic species (AB, AC, MMA, DMA, TMAO, Tetra, and Arsenosugars) remains relatively constant regardless of an increase in total arsenic concentration in the tissues (R2 = 0.307, p > 0.05) (Figure 2). These results support the hypothesis of an arsenic biotransformation threshold in which the pathways responsible for organoarsenical creation

from inorganic arsenic become saturated within the marine ecosystem, leaving more unmetabolized inorganic arsenic. If organoarsenicals are being ingested and retained, their constant concentration may suggest the amount in the mussels’ food sources remains constant, assuming inconsequential concentrations of such compounds in water or sediment. The relatively constant concentration of arsenobetaine found along the concentration gradient also supports the theory that it serves as an osmolyte in these organisms.25−28 That is, marine organisms are thought to passively take up or create and subsequently retain arsenobetaine in their cells because it can act as a surrogate compound for a common intracellular organic osmolyte, glycine betaine.27,28 Glycine betaine is one of the most abundant osmolytes in molluscs and crustaceans in shallow depths,29 and arsenobetaine possesses an analogous chemical configuration to this osmolyte. Concentrations of inorganic arsenicals (As(III) and As(V)) and the unextracted arsenicals present in the mussel tissue both increase as the total concentration in the tissue increases (R2 = 0.9825 and R2 = 0.9381 respectively, p < 0.05) (Figure 2a). The proportion of inorganic arsenic identified by XANES (As(III)-S, 3115

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Figure 3. XAS mapping of the digestive gland and surrounding tissue interface in a highly contaminated Blue mussel (Mytilus edulis). A) Microscope photograph of M. edulis tissue prior to mapping showing the mapped region outline in black. B) Total copper map of tissue showing the presence and location of digestive tubules in the digestive gland (photon energy = 13696 eV). C) Total arsenic map (photon energy = 13696 eV). D) Arsenobetaine map (photon energy = 11872.6 eV). E) As(III) + As(III)-sulfur map (photon energy = 11871.7 eV). F) As(V) map (photon energy = 11875.3 eV).

specimen (from Seal Harbour Site 1) was carried out to elucidate pathways possibly connected to arsenic metabolism and detoxification in the organism, and the results are shown in Figure 3. A higher concentration of arsenic was present in the digestive gland of the mussel than in the surrounding tissue. The arsenic likely entered the digestive gland in food or accompanying sediment particles via the gills to the labial palps, into the mouth, down the esophagus, and into the stomach. From the stomach the arsenic-containing material, which as mentioned has been selected based primarily on size, enters the digestive gland through tubules, from which absorption into the mussel’s tissues takes place. Digestive tubules were identified in the digestive gland of M. edulis by comparing histopathological images (Supporting Information, Figure S6) and XAS mapping of the copper distribution in digestive gland tissues (Figure 3b). This is based on the similarity of the copper results shown in Figure 3b with those in Zorita et al.42 where the digestive tubules of the lumen were characterized by higher copper concentrations (identified with autometallography imaging). The digestive tubules are locations from which nutrients can be absorbed, or where waste products (expelled via lysosomes) from mussel tissue can be excreted. Arsenic is notably not found in the digestive tubules (Figure 3c), suggesting that it has been absorbed into the mussel’s tissue. Mapping carried out at the K-edge photon energies characteristic of three groups of arsenic species (As(III)-O and As(III)-S together at 11871.7 eV, AB at 11872.6 eV, and As(V) at 11875.3 eV; where As(V) and AB signals were corrected for the contributions from lower-energy compounds as described in the methods) helped to further elucidate the bioaccumulation pattern of arsenic. The majority of the total arsenic present was in the digestive gland as As(III) and/or As(III)-S species and were probably absorbed from the tubules into the digestive gland but then sequestered there. These mapping results should be considered together with the linear combination fitting results

As(III)-O, and As(V)) also increased with increasing mussel tissue arsenic (Figure 2b). The latter results reflect the nature of arsenic speciation prior to extraction, since wet samples subjected to minimal sample preparation were analyzed. They are also a better reflection of the “total” amount of inorganic arsenic in the sample (within the constraints of XANES linear combination fitting techniques), since extraction for HPLC-ICP-MS analysis was incomplete. Previous studies that have examined the arsenic species distribution in blue mussels and the closely related species of mussels, Mytilus galloprovincialis, from uncontaminated environments, found very low levels of toxic inorganic arsenic species and high proportions of arsenobetaine in their tissues.30−32 The presence of high proportions of inorganic arsenic in contaminated mussels in the present study (also found in clams from the same site2) may have significant toxicological implications, although the toxic effects of chronic exposure to inorganic arsenic are not well understood for low trophic marine organisms. Studies that have examined toxicity of inorganic arsenic to low trophic marine organisms have only examined acute exposure and high variability has been observed, dependent on the study organism.33−38 To date, no study has examined the effects of chronic exposure on inorganic arsenic to adult M. edulis. No visible stress was observed in the contaminated mussels in the present study, but the possibility that arsenic exposure may be affecting the mussels on a population, community, and/or genotoxic level cannot be discounted. Inorganic arsenic accumulation within the mussel’s tissues also poses a hazard to higher trophic organisms who may be consuming these organisms as part of their diet. This includes risk to humans who commonly consume M. edulis as seafood. XAS Mapping of M. edulis. XAS mapping has been used in previous studies to investigate the in situ distribution of arsenic compounds within unaltered biological samples.39−41 Using similar methods, XAS two-dimensional mapping of the digestive gland and adjacent tissue of a highly contaminated mussel 3116

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coast of the middle Adriatic sea using Mytilus galloprovincialis. Nucl. Instrum. Methods Phys. Res., Sect. B 2006, 245 (2), 495−500. (5) Taleb, Z. M.; Benali, I.; Gherras, H.; Ykhlef-Allal, A.; BachirBouiadjra, B.; Amiard, J.; et al. Biomonitoring of environmental pollution on the Algerian west coast using caged mussels Mytilus galloprovincialis. Oceanologia 2009, 51 (1), 63−84. (6) Cowden, C.; Young, C. M.; Chia, F. S. Differential predation on marine invertebrate larvae by two benthic predators. Mar. Ecol.: Prog. Ser. 1984, 14, 145−149. (7) Lehane, C.; Davenport, J. Ingestion of mesozooplankton by three species of bivalves: Mytilus edulis, Cerastoderma edule and Aequipecten opercularis. J. Mar. Biol. Assoc. U.K. 2002, 82, 615−619. (8) Parsons, J. G.; Aldrich, M. V.; Gardea-Torresdey, J. Environmental and biological applications of extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES) spectroscopies. Appl. Spectrosc. Rev. 2002, 37 (2), 187−222. (9) Parsons, M. B.; Yeats, P. A.; Milligan, T. G.; Parrott, D. R.; Smith, J. N.; Little, M. E. Marine environmental impacts of historical gold mining activities, Isaacs, Seal, and Wine harbours, Nova Scotia. Geochem.: Explor., Environ., Anal., Submitted for publication. (10) Bates, J. Gold in Nova Scotia; Nova Scotia Department of Mines and Energy, Information Series, 1987, 13. (11) Whaley-Martin, K. J.; Koch, I.; Reimer, K. J. Arsenic Species Extraction of Biological Marine Samples (Periwinkles, Littorina littorea) from a Highly Contaminated Site. Talanta 2012, 88, 187− 192. (12) Smith, P. G.; Koch, I.; Gordon, R. A.; Mandoli, D. F.; Chapman, B. D.; Reimer, K. J. X-ray absorption near-edge structure analysis of arsenic species for application to biological environmental samples. Environ. Sci. Technol. 2005, 39 (1), 248−254. (13) Smith, P. G.; Koch, I.; Reimer, K. J. Arsenic speciation analysis of cultivated white button mushrooms (Agaricus bisporus) using highperformance liquid chromatography-inductively coupled plasma mass spectrometry, and X-ray absorption spectroscopy. Environ. Sci. Technol. 2007, 41 (20), 6947−6954. (14) Ravel, B.; Newville, M. ATHENA, ARTEMIS, HEPHAESTUS: Data analysis for X-ray absorption spectroscopy using IFEFFIT. J. Synchrotron Radiat. 2005, 12 (4), 537−541. (15) Francesconi, K.; Edmonds, J. S. Arsenic species in marine samples. Croat. Chem. Acta 1998, 71 (2), 343−359. (16) Ebdon, L.; Walton, A. P.; Millward, G. E.; Whitfield, M. Methylated arsenic species in estuarine porewaters. Appl. Organomet. Chem. 1987, 1 (5), 427−433. (17) Duester, L.; Vink, J.; Hirner, A. V. Methylantimony and -arsenic species in sediment pore water tested with the sediment or fauna incubation experiment. Environ. Sci. Technol. 2008, 42 (16), 5866− 5871. (18) Schmeisser, E.; Raml, R.; Francesconi, K. A.; Kuehnelt, D.; Lindberg, A. L.; Soros, C.; Goessler, W. Thio arsenosugars identified as natural constituents of mussels by liquid chromatography-mass spectrometry. Chem. Commun. 2004, 1824−1825. (19) Suess, E.; Scheinost, A. C.; Bostick, B. C.; Merkel, B. J.; Wallschlaeger, D.; Planer-Friedrich, B.; et al. Discrimination of thioarsenites and thioarsenates by X-ray absorption spectroscopy. Anal. Chem. 2009, 81, 8318−8326. (20) Kitchin, K. T.; Wallace, K. Arsenite binding to synthetic peptides based on the Zn finger region and the estrogen binding region of the human estrogen receptor. Toxicol. Appl. Pharmacol. 2005, 206, 66−72. (21) Kitchin, K. T.; Wallace, K. The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity. J. Inorg. Biochem. 2008, 102 (3), 532−539. (22) Ngu, T.; Lee, J. A.; Rushton, M. K.; Stillman, M. J. Arsenic metalation of seaweed Fucus vesiculosus metallothionein: The importance of the interdomain linker in metallothionein. Biochemistry 2009, 48, 8806−8816. (23) Leignel, V.; Hardivillier, Y.; Laulier, M. Small metallothionein MT-10 genes in coastal and hydrothermal mussels. Mar. Biotechnol. 2005, 7, 236−244.

of bulk tissue, which suggest that at least half of the observed signal at 11871.7 eV was contributed by As(III)-S species in the contaminated mussel tissue (Table 1). The potentially high proportions of arsenic−sulfur compounds found to be concentrated within the digestive gland of the mussel may be due to the thiol moieties of proteins binding to arsenic to sequester the metalloid and limit toxicological effects to the mussel. On the other hand, arsenobetaine was found in small and similar concentrations in the digestive gland as well as the surrounding tissue. This suggests that arsenobetaine is digested and probably passes through the digestive tubules to become absorbed into the mussel’s tissue and distributed via the circulatory system. As previously mentioned, mussels may have evolved this capability in response to arsenobetaine being used in a physiological function such as an osmolyte.

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ASSOCIATED CONTENT

S Supporting Information *

Chemicals and reagents, an illustration of the study site, anion and cation arsenic standards chromatographs, QAQC results, raw data from the arsenic in water samples not included in the paper, XANES results of sediment (fittings and raw data) and mussels (fittings) and histopathological images of M. edulis. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 613-541-6000 x 6161. Fax: (613) 541-6596. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Robert Gordon for assistance on this work. PNC/XSD facilities at the Advanced Photon Source, and research at these facilities, are supported by the US Department of Energy - Basic Energy Sciences, a Major Resources Support grant from NSERC, the University of Washington, Simon Fraser University, and the Advanced Photon Source. Use of the Advanced Photon Source, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357. The award of a Discovery Grant to K.J.R. from the Natural Sciences and Engineering Research Council and the financial support of the Academic Research Program (Royal Military College of Canada) is gratefully acknowledged.

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REFERENCES

(1) Reimer, K. J.; Koch, I.; Cullen, W. R. Organoarsenicals. distribution and transformation in the environment. Metal ions in life sciences; RSC Publishing: Cambridge, 2010; Vol. 7, pp 165−229. (2) Koch, I.; McPherson, K.; Smith, P.; Easton, L.; Doe, K. G.; Reimer, K. J. Arsenic bioaccesibility and speciation in clams and seaweed from a contaminated marine environment. Mar. Pollut. Bull. 2007, 54, 586−594. (3) Sloth, J. J.; Julshamn, K. Survey of total and inorganic arsenic content in blue mussels (Mytilus edulis L.) from Norwegian fiords: Revelation of unusual high levels of inorganic arsenic. Agric. Food Chem. 2008, 58, 1269−1273. (4) Orescanin, V.; Lovrencic, I.; Mikelic, L.; Barisic, D.; Matasin, Z.; Lulic, S.; et al. Biomonitoring of heavy metals and arsenic on the east 3117

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dx.doi.org/10.1021/es203812u | Environ. Sci. Technol. 2012, 46, 3110−3118