Environ. Sci. Technol. 2005, 39, 5999-6005
Arsenic Occurrence and Species in Near-Shore Macroalgae-Feeding Marine Animals J . K I R B Y , * ,† W . M A H E R , A N D D. SPOONER‡ EcoChemistry Laboratory, Division of Science and Design, University of Canberra, Bruce, ACT 2616, Australia
This study was undertaken to improve the understanding of arsenic species and their pathways of formation in marine animals: fish (Odax cyanomelas), abalone (Haliotis rubra), and sea urchins (Heliocidaris erythrogramma and Centrostephanus rodgersii) that are directly exposed through their diets to dimethyl arsenoriboses in macroalgae (Phyllospora comosa and Halopteris platycena). The identification of dimethyl arsenoriboses (phosphate, sulfonate, and glycerol) in both dominant macroalgae species, and especially digestive tissues of marine animals that consume them, suggests these arsenic species, are to some degree accumulated directly from their diets without degradation or conversion. An unknown arsenic species in H. rubra intestinal tissue was identified using tandem mass spectrometry as 2′,3′-dihydroxypropyl 5-deoxy-5-trimethyl arsonioriboside (trimethyl glycerol arsenoribose). The concentration of trimethyl glycerol arsenoribose in H. rubra intestinal tissue was estimated to account for 28% (5.0 µg g-1 dry mass) of the methanol-water-soluble arsenic fraction. The presence of a trimethyl glycerol arsenoribose in marine animal tissues may be due to microbialmediated processes that promote the reduction and methylation of dimethyl arsenoriboses released during the breakdown of macroalgae in their diets. Arsenobetaine formation may then occur in the lumen of the digestive tract (i.e., mediated by microorganisms) or in the liver catalyzed by enzymes. The identification of a large amount of trimethyl glycerol arsenoribose in H. rubra intestinal tissue suggests this species is a main constituent in the pathway for arsenic in this marine animal.
Introduction In general, arsenic in marine macroalgae is present as dimethyl arsenoriboses, while in animals the most abundant arsenic species is arsenobetaine (AsB) (1, 2). Since the discovery of AsB by Edmonds et al. (3) in the western rock lobster (Panulirus cygnus), there has been extensive research into determining the formation pathway (s) for this species in marine animals. Two main pathways have been proposed to explain the formation of AsB in marine animals, based on bioconversion from arsenoriboses (1, 2, 4, 5) and a process * Corresponding author phone: +61-0883038478; fax: +610883038565; e-mail:
[email protected]. Current position: CSIRO Land and Water, Glen Osmund, Adelaide, SA, Australia, 5867. † Current address: CSIRO Land and Water, Glen Osmond, SA 5867, Australia. ‡ Current address: NIWA Australia, Bowen Hills, QLD 4051, Australia. 10.1021/es050546r CCC: $30.25 Published on Web 07/12/2005
2005 American Chemical Society
of “arsenylation” using dimethylarsenic acid that parallels “amination” in amino acid synthesis (6). The main pathway for AsB formation in marine animals is believed to occur through the degradation of dimethyl arsenoriboses to dimethylarsinylethanol (DMAE) followed by conversion to dimethylarsinylacetic acid (DMAA) and/or arsenocholine (AsC) (1, 2, 4). However, the conversion of dimethyl arsenoriboses to AsB by this pathway in marine animals still has to be established (7, 8) and the proposed intermediate arsenic species (i.e., DMAE, DMAA, and AsC) have only been identified at trace concentrations (9-11). The presence of dimethyl arsenoriboses together with AsB in marine animals provides some evidence for this proposed pathway (1, 2). A recent study by McSheedy et al. (9) using liquid chromatography-tandem mass spectrometry has provided additional support for this pathway. These authors identified AsB, DMAA, dimethyl arsenoriboses, and successive degradation products of arsenoriboses together in the kidney tissue of the giant clam, Tridacna derasa. The results suggest arsenoriboses present in the kidney tissue of the giant clam have the potential to undergo successive oxidation and decarboxylation reactions to produce DMAA, which on further methylation can be converted to AsB (1, 9). The discovery of trimethyl arsenoriboses in marine macroalgae (12, 13) and animals (5) allows an alternative pathway for the synthesis of AsB from dimethyl arsenoriboses in marine animals to be proposed. Trimethyl arsenoriboses have been shown to degrade in anaerobic conditions with the aid of associated microorganisms to produce almost quantitative amounts of AsC (15). The formation of AsB in marine animals from trimethyl arsenoriboses may then occur through a two-stage conversion pathway: trimethyl arsenoribose degradation to AsC followed by oxidation to AsB (1, 2). However, trimethyl arsenoriboses have only been identified at low concentrations in aquatic organisms and are currently considered unlikely to explain the high amount of AsB found in marine animals. This study was undertaken to determine arsenic species present in marine animals: fish (Odax cyanomelas), abalone (Haliotis rubra), and sea urchins (Heliocidaris erythrogramma and Centrostephanus rodgersii) that are directly exposed through their diets to dimethyl arsenoriboses occurring in macroalgae (Phyllospora comosa and Halopteris platycena). The information gained will be used to assess the potential role dimethyl arsenoriboses have in the formation of AsB.
Methods Study Locations and Sample Collection. The marine fish (O. cyanomelas), abalone (H. rubra), red sea urchin (H. erythrogramma), and black sea urchin (C. rodgersii) along with the two dominant macroalgae species, P. comosa and H. platycena, were collected at locations (Mosquito Bay, Wimbie, and Lilli Pilli) on the south coast of New South Wales, Australia. The four marine animal species were selected for collection due to their reported diets that consist mainly of macroalgae (15-17). At each location, 15 individuals of each animal species and 10 samples of each macroalgae species were randomly collected from macroalgae beds situated approximately 10-50 m from the shoreline. The absence of H. rubra from Mosquito Bay prevented the collection of this species at all locations. The macroalgae samples were washed with deionized water (Millipore) to remove epiphytic algae, sediment particles, and seawater. The animals were dissected into identifiable tissues: muscle, liver, intestine, and gonad for O. cyanolelas; muscle and intestine for H. rubra; gonad and visceral for H. erythrogramma and C. rodgersii. Individual VOL. 39, NO. 16, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Total arsenic concentrations in near-shore marine macroalgae and macroalgae-feeding animals. tissues were washed in deionized water (Millipore), freezedried for 48 h to a constant mass, homogenized, and stored in a desiccator until analysis. Total Arsenic Analysis. Total arsenic concentrations in macroalgae and marine animal tissues were determined using a microwave nitric acid digestion procedure developed by Baldwin et al. (18). The 10% (v/v) nitric acid digests were analyzed for total arsenic using inductively coupled plasmamass spectrometry (19). Arsenic Species Analysis. High-Performance Liquid Chromatography-Inductively Coupled Plasma-Mass Spectrometry. The 50% (v/v) methanol-water-soluble arsenic species in macroalgae and marine animal tissues were extracted and measured using a microwave-assisted extraction procedure followed by high-performance liquid chromatographyinductively coupled plasma-mass spectrometry developed by Kirby and Maher (20). This procedure has successfully been applied to the identification and quantification of arsenic species in biological tissues from marine and estuarine environments (11, 21). Stock standard solutions (1000 mg L-1) of arsenous acid (As(III)), arsenic acid (As(V)), methylarsonic acid (MA), and dimethylarsenic acid (DMA) were prepared by dissolving sodium arsenite (AJAX Laboratory Chemicals), sodium arsenate heptahydrate (AJAX Laboratory Chemicals, Australia), disodium methyl arsenate (Alltech-Specialists), and dimethylarsinic acid sodium salt (Alltech-Specialists), respectively, in 0.01 M hydrochloric acid (Trace Pur, Merck)deionized water (Millipore). Arsenobetaine (AsB), arsenocholine (AsC), trimethylarsine oxide (TMAO), and tetramethylarsonium ion (TETRA) were kindly supplied by Prof. Erik Larsen (Danish Institute for Food and Veterinary Research, Denmark) and Dr. Walter Goessler (Institute of Chemistry, Karl-Franzens-University, Graz, Austria). The glycerol, sulfonate, and sulfate arseno6000
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riboses (OH-arsenoribose, SO3-arsenoribose, OSO3-arsenoribose, respectively) were isolated in-house from marine macroalgae certified reference material Fucus 140 (IAEA, Monaco) (20). The PO4-arsenoribose was isolated in-house from the marine animal certified reference material, oyster 1566a (NIST, USA) (20). Trimethylarsoniopropionate (TMAP) was isolated in-house from the marine animal certified reference material, lobster hepatopancreas (TORT-2) (NRCCNRC, Canada) (20). Quality Control Procedures. The accuracy of arsenic species extraction followed by HPLC-ICP-MS was determined using the certified reference material, dogfish muscle (DORM2) (NRC-CNRC, Canada) (n ) 3). The concentrations found for AsB (16.1 ( 0.62 µg g-1) and TETRA (0.263 ( 0.026 µg g-1) were in close agreement to certified values (AsB, 16.4 ( 1.1 µg g-1; TETRA, 0.248 ( 0.054 µg g-1). Tandem Mass Spectrometry. A large quantity of an unknown arsenic species in H. rubra was identified using tandem mass spectrometry following purification by cation and anion exchange chromatography. The unknown arsenic species was collected after multiple injections (n ) 10, 100 µL) onto a Supelco LC-SCX cation exchange column (20 mM pyridine-formic acid buffer, pH 2.6, flow rate 1.5 mL min-1, and column temperature 40 °C). The collected eluent (∼2 mL) that contained the unknown arsenic species was evaporated to dryness using a stream of ultrapure nitrogen (BOC Gases). The dried residue was re-suspended in 0.5 mL of deionized water (Millipore) and further purified on a Supelco LC-SAX column (250 mm × 4.6 mm, 5 µm) (Supelco) using a 20 mM ammonium carbonate (Suprapur, Merck)1% methanol (HiPerSolv, BDH, Australia) mobile phase at pH 5.1 (1 mL min-1; column temperature 40 °C). The eluent (∼2 mL) was again evaporated to dryness using a stream of ultrapure nitrogen (BOC Gases). The structure of the unknown arsenic species was identified by tandem mass
TABLE 1. Total and Methanol-Water Extracted Arsenic from Near-Shore Marine Macroalgae and Macroalgae-Feeding Animals
type
species
tissues
total As (µg g-1)
methanol-water extracted As (µg g-1)
methanol-water extracted As (%)
fish
O. cyanomelas
muscle liver intestine gonad
5.4 16.3 20.0 10.5
4.6 13.1 16.8 8.2
85.2 80.4 84.0 78.1
mollusks
H. rubra
muscle intestine
76.1 29.3
39.4 18.2
51.8 62.1
echinoderms
H. erythrogramma
gonad intestine
15.5 95.4
11.5 57.7
74.2 60.5
C. rodgersii
gonad intestine
15.1 106.6
12.6 77.8
83.4 73.0
P. comosa H. platycena
whole whole
49.3 71.1
37.8 57
76.7 80.2
macroalgae
TABLE 2. Anionic Arsenic Compounds in Near-Shore Marine Macroalgae and Macroalgae-Feeding Animalsa,b type
species
tissue
DMA (µg g-1)
MA (µg g-1)
PO4-arsenoribose (µg g-1)
As(V) (µg g-1)
0.03 (0.65) 0.39 (2.9) 0.67 (4.0) 0.06 (0.70)
n/d 0.03 (0.23) 0.05 (0.29) n/d
1.2 (25) 2.5 (19) 1.2 (7.3) 0.56 (6.8)
0.03 (0.68) 0.10 (0.45) 1.9 (11.4) 0.05 (0.56)
0.03 (0.65) 0.21 (1.6) 0.51 (3.0) n/d
n/d n/d n/d n/d
0.1 (0.22) 1.90 (10.5)
n/d 0.09 (0.5)
n/d 1.1 (6.2)
n/d n/d
0.12 (1.1) 1.5 (2.5)
0.01 (0.07) 0.33 (0.6)
n/d 0.30 (0.5)
n/d n/d
n/d n/d
n/d 0.25 (0.32)
n/d n/d
fish
O. cyanomelas
muscle liver intestine gonad
mollusks
H. rubra
muscle 0.03 (0.07) n/d intestine 0.15 (0.82) 0.03 (0.16)
echinoderms H. erythrogramma gonad 0.14 (1.2) 0.03 (0.25) intestine 0.46 (0.79) n/d
macroalgae
C. rodgersii
gonad visceral
0.01 (0.08) n/d n/d 0.3 (0.4)
n/d n/d
P. comosa H. platycena
whole whole
0.03 (0.08) n/d 0.03 (0.05) n/d
3.8 (10) 4.9 (8.6)
14 (37) 35 (61)
SO3-arsenoribose OSO3-arsenoribose (µg g-1) (µg g-1)
17 (46) 15 (27)
n/d n/d
a Numbers in brackets show percentage determined by total arsenic concentration of the 50% (v/v) methanol-water fraction. b n/d ) not detected.
spectrometry using a procedure previously outlined by McSheedy et al. (22). The dried residue was re-suspended in 30% (v/v) methanol (ChromAR HPLC, Mallinckrodt, SelbyBiolab)-acidified with hydrochloric acid (Trace Pur, Merck) and infused directly into the mass spectrum of a PerkinElmer SCIEX API 300 (Perkin-Elmer) using a syringe pump. The instrument was operated in positive ion acquisition mode in a scanning m/z range of 60-500.
Results and Discussion Total Arsenic Concentrations. Arsenic concentrations in brown macroalage (P. comosa and H. platycena) (28-57 µg g-1 dry mass) were similar to those previously reported for macroalgae collected along the New South Wales coast, Australia (23). Arsenic concentrations in fish (O. cyanomelas) (4-27 µg g-1 dry mass), mollusc (H. rubra) (20-96 µg g-1 dry mass), and echinoderm (H. erythrogramma and C. rodgersii) (5-135 µg g-1 dry mass) tissues are higher than those normally found in Australian fish and mollusc species from uncontaminated marine environments (24). Arsenic concentrations in tissues were found to be similar between sample locations (Figure 1). The higher arsenic concentrations found in marine animal tissues from this study is attributed to the consumption of arsenic-rich macroalgae. 50% (v/v) Methanol-Water Extracted Arsenic. Arsenic extraction efficiency from P. comosa (77%) and H. platycena (80%) (Table 1) using 50% (v/v) methanol-water is consistent with previous findings for brown macroalgae species collected from Australian waters (23). The more variable arsenic
extraction efficiencies for marine animal tissues in this study (52-86%) is also consistent with those found in previous studies (11, 20, 21). Arsenic extraction efficiency from marine animals has been found to be dependent on the extractant (e.g., water, methanol-water, phosphoric acid, or nitric acid), technique (e.g., mechanical agitation, sonication, accelerated solvent extraction, and microwave), animal species, and tissue analyzed (20, 25-28). The high arsenic extraction efficiencies found in this study from macroalgae and marine animals are the result of efficient sample preparation (e.g., small sample particle size and acetone extraction) and the use of microwave-assisted extraction. Arsenic Species. Macroalgae. The majority of arsenic in P. comosa was present as sulfonate arsenoribose (46%) and As(V) (37%) (Tables 2 and 3). A typical HPLC-ICP-MS chromatogram of arsenic compounds in P. comosa tissue is presented in Figure 2. Small amounts of arsenic in P. comosa were also present as phosphate (10%) and glycerol arsenoriboses (7.6%) (Tables 2 and 3). In H. platycena, the majority of arsenic was present as As(V) (61%) and sulfonate arsenoribose (27%) (Tables 2 and 3). A small amount of arsenic in H. platycena was also present as the phosphate (8.6%) and glycerol arsenoriboses (5.2%) (Tables 2 and 3). Trace amounts of DMA (
