Fresenius J Anal Chem (1997) 359 : 434437 Springer-Verlag 1997

W. Goessler W. Maher K. J. Irgolic D. Kuehnelt C. Schlagenhaufen T. Kaise

Arsenic compounds in a marine food chain

Received: 18 November 1996 / Revised: 17 February 1997 / Accepted: 18 February 1997 Abstract A three-organism food chain within a rock pool at Rosedale, NSW, Australia, was investigated with respect to arsenic compounds by high performance liquid chromatography hydraulic high pressure nebulization inductively coupled plasma mass spectrometry (HPLC-HHPN-ICP-MS). Total arsenic concentration was determined in the seaweed Hormosira banksii (27.2 g/g dry mass), in the gastropod Austrocochlea constricta (74.4 g/g dry mass), which consumes the seaweed, and in the gastropod Morula marginalba (233 g/g dry mass), which eats Austrocochlea constricta. The major arsenic compounds in the seaweed were (2R)-dimethyl[1-O-(2,3-dihydroxypropyl)-5-deoxy--D-ribofuranos-5-yl]arsine oxide and an unidentified compound. The herbivorous gastropod Austrocochlea constricta transformed most of the arsenic taken up with the seaweed to arsenobetaine. Traces of arsenite, arsenate, dimethylarsinic acid, arsenocholine, the tetramethylarsonium cation, and several unknown arsenic compounds were detected. Arsenobetaine accounted for 95% of the arsenic in the carnivorous gastropod Morula marginalba. In Morula marginalba the concentration of arsenocholine was higher, and the concentrations of the minor arsenic compounds lower than in the herbivorous gastropod Austrocochlea constricta.

For the determination of arsenic compounds an efficient separation step followed by an adequate detection step is necessary. These conditions are met by liquid chromatography coupled to detection systems such as hydride generation atomic absorption spectrometry (HG-AAS) [5, 6], hydride generation inductively coupled plasma mass spectrometry (HG-ICP-MS) [7, 8], inductively coupled plasma atomic emission spectrometry (ICP-AES) [9], and inductively coupled plasma mass spectrometry (ICP-MS) [10, 11]. The hydride generation technique provides excellent detection limits for arsenic compounds reducible to volatile hydrides but cannot detect arsenobetaine, arsenocholine, and the tetramethylarsonium cation without prior chemical conversion. The detection limits for ICP-AES are too high for minor arsenic compounds in biological samples. HPLC combined with ICP-MS provides the required low detection limits and is capable of detecting hidden arsenic species. High-efficiency nebulizers such as the hydraulic high pressure nebulizer can lower the detection limits by an order of magnitude [12]. A three-organism food chain in a rock pool at Rosedale, NSW, Australia, was investigated by HPLC-HHPN-ICP-MS. Concentrations of total arsenic and of arsenic compounds were determined in the seaweed Hormosira banksii, in the gastropod Austrocochlea constricta (which consumes the seaweed), and in the gastropod Morula marginalba (which eats A. constricta) to obtain information about the conversion and accumulation of arsenic compounds and their biologically mediated transformations within this food chain.

Materials and methods

The organisms were dissected. The soft tissues were removed with stainless steel forceps. For the determination of total arsenic about 200 mg of the freeze-dried tissues (weighed to 0.1 mg) were digested with 3 mL HNO3 and 0.5 mL H2O2 in the microwave digestion system MLS 1200 Mega (MLS, Leutkirch, Germany). The clear, colorless digests were quantitatively transferred into 50-mL volumetric flasks. As internal standard for the ICP-MS measurements a solution of 10 g/mL In was added to each flask to achieve a final concentration of 50 ng/mL. Total arsenic was determined with a VG PlasmaQuad 2 Turbo Plus ICP-MS (VG Elemental, Winsford, UK). All concentrations are based on dry mass. For the determination of arsenite, arsenate, methylarsonic acid (MA), dimethylarsinic acid (DMA), arsenobetaine (AB), arsenocholine (AC), trimethylarsine oxide (TMAO), tetramethylarsonium ion (TETRA), and (2R)-dimethyl[1-O-(2,3dihydroxypropyl)-5-deoxy--D-ribofuranos-5-yl]arsine oxide (Ribose 1, Fig. 1) by HPLC-HHPN-ICP-MS about 200 mg of the freeze-dried tissues were weighed to 0.1 mg into 50-mL polyethylene vials. Methanol/water (9 :1, v/v, 20 mL) was added. The samples were shaken for 14 h and then centrifuged. The supernatants were transferred into round-bottom flasks. The residues were washed three times with 10 mL methanol/water (9 :1), the mixtures centrifuged, and the supernatants transferred into the round-bottom flasks. The extracts were evaporated to dryness on a rotavapor. The residues were resuspended in 20 mL of water, centrifuged, and filtered through 0.2-m cellulose nitrate filters. Aliquots (100 L) of the filtrates were analyzed by HPLC-HHPN-ICP-MS. The experimental parameters for the hydraulic high pressure nebulizer, for the ICP-MS and the chromatographic conditions are summarized in Table 1 [12, 13].

Introduction
Concentrations of total arsenic and of arsenic compounds have been determined in many marine biota [1, 2]. Marine animals are known to contain arsenic mostly in the form of arsenobetaine. Arsenocholine and the tetramethylarsonium ion were identified as minor constituents. High concentrations of arsenobetaine (up to 100 mg As/kg) were found in gastropods [2]. Marine algae and higher plants do not contain arsenobetaine but rather arsenosugars. In marine algae dimethylarsinylribosides account for most of the arsenic [3, 4]. Pathways that lead from arsenic-containing ribosides to arsenobetaine were proposed [2].

Poster award winner W. Goessler () K. J. Irgolic D. Kuehnelt C. Schlagenhaufen Institute for Analytical Chemistry, Karl-Franzens-Universitaet Graz, Universitaetsplatz 1, A-8010 Graz, Austria W. Maher CRC for Freshwater Ecology, University of Canberra, PO Box 1, Belconnen ACT. 2616, Australia T. Kaise Laboratory of Environmental Chemistry, School of Life Science, University of Pharmacy and Life Science, 14321 Horinouchi, Hachijoji, Tokyo 192-03, Japan

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Fig. 1 Formulae for arsenic compounds available as synthetic standards

Results and discussion

Total arsenic concentrations The total arsenic concentrations increased within the food chain from 27.2 g/g in the seaweed Hormosira banksii to 74.4 g/g in the gastropod Austrocochlea constricta and 233 g/g in the gastropod Morula marginalba. Within each step of this food chain the concentration of total arsenic increased approximately threefold. From Hormosira banksii only 30% of the arsenic was extracted by methanol/water (9 :1), from Austrocochlea constricta 74%, and from Morula marginalba 95%. Arsenic compounds The extracted arsenic compounds were separated on anion- and cation-exchange columns. The arsenic compounds shown in Fig. 1 served as standards. From the seaweed Hormosira banksii two major arsenic compounds were extracted that produced chromatographic signals at retention times of ~ 180 s and ~ 330 s (anion-exchange) and ~ 145 s and ~ 250 s (cationexchange) (Fig. 2). The compound responsible for the signals at 180 s (anion-exchange) and 250 s (cation-exchange) was identified by spiking experiments as (2R)-dimethyl[1-O-(2,3dihydroxypropyl)-5-deoxy--D-ribofuranos-5-yl]arsine oxide (Ribose 1, Fig. 1). The second major peaks at ~ 330 s and 145 s

were enhanced in intensity after the solutions had been spiked with methylarsonic acid. Methylarsonic acid was hardly ever found to be a major arsenic compound in biological samples. The two major arsenic compounds from H. banksii when chromatographed on a Hamilton PRP-X100 column produced two signals with retention times clearly different from the retention time of methylarsonic acid. Consequently, methylarsonic acid cannot be the cause for the second major signals obtained with the Supelcosil columns. Riboses 2, 3, and 4 (Fig. 1) had retention times different from the retention time of the unknown peak in the seaweed extract. Arsenite was found to be a minor constituent (Fig. 2). Most of the arsenic in the extracts of the gastropod Austrocochlea constricta was arsenobetaine (Fig. 3A, 3B). Arsenate, arsenite, DMA, AC, TETRA, and the Ribose 1 were present at low concentrations. Additionally, the anion-chromatogram had two unassignable signals and the cation-chromatogram three. The herbivorous A. constricta converted most of the Ribose 1 and the unknown arsenic compound from the seaweed into arsenobetaine. Arsenocholine, a proposed intermediate in the biogenesis of arsenobetaine, was not observed in the seaweed extract (Fig. 2) but was identified in the extract of A. constricta (Fig. 3). The tetramethylarsonium ion in A. constricta could have as precursor a trimethyl(ribosyl)arsonium compound that could have experienced an exchange of the ribosyl group for a methyl group. In the carnivorous gastropod Morula marginalba arsenobetaine accounted for 96% of the arsenic (Fig. 4A, 4B). The concentration of arsenocholine was much higher in M. marginalba than in A. constricta (Fig. 3B, 4B). The two unknown arsenic compounds in A. constricta (retention times of ~ 290 s and ~ 365 s on the anion-exchange column, Fig. 3A) were present

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4
Fig. 2A, B HPLC-HHPN-ICP-MS chromatograms of an extract of the seaweed Hormosira banksii obtained with a Supelcosil LC-SAX1 anion-exchange column (mobile phase 15 mmol/L aqueous NH4H2PO4, pH 5.1; flow 1.5 mL/min; 100 L injected, A) and with a Supelcosil LC-SCX cation-exchange column (mobile phase 20 mmol/L aqueous pyridine, pH 2.9; flow 1.5 mL/min; 100 L injected, B) Fig. 3A, B HPLC-HHPN-ICP-MS chromatograms of an extract of the herbivorous gastropod Austrocochlea constricta obtained with a Supelcosil LC-SAX1 anion-exchange column (mobile phase 15 mmol/L aqueous NH4H2PO4, pH 5.1; flow 1.5 mL/min; 100 L injected, A) and with a Supelcosil LCSCX cation-exchange column (mobile phase 20 mmol/L aqueous pyridine, pH 2.9; flow 1.5 mL/min; 100 L injected, B) Fig. 4A, B HPLC-HHPN-ICP-MS chromatograms of an extract of the carnivorous gastropod Morula marginalba obtained with a Supelcosil LC-SAX1 anion-exchange column (mobile phase 15 mmol/L aqueous NH4H2PO4, pH 5.1; flow 1.5 mL/min; 100 L injected, A) and with a Supelcosil LC-SCX cation-exchange column (mobile phase 20 mmol/L aqueous pyridine, pH 2.9; flow 1.5 mL/min; 100 L injected, B)

in M. marginalba as traces if at all (Fig. 4A). The two unknown compounds in A. constricta (retention times of ~ 295 s and ~ 325 s on the cation-exchange column, Fig. 3B) are also present in M. marginalba albeit at much lower concentration (Fig. 4B), whereas the unknown compound with a retention time of ~ 440 s (cation-exchange column) was not detected in M. marginalba. The concentration of the tetramethylarsonium cation was lower in M. marginalba than in A. constricta.

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Table 1 Operating conditions for the HPLC-HHPN-ICP-MS Hydraulic high pressure nebulizer heating 150 C cooling 2 C nebulizer gas (argon) 1.00 L/min back pressure ~ 200 bar Inductively coupled plasma mass spectrometer Plasma rf power Argon gas flows cooling gas auxiliary gas Vacuum expansion intermediate analyzer Ion sampling sample cone skimmer cone Measuring parameters monitored signal time/slice total analysis time forward reflected 13.5 L/min 1.1 L/min 1.8 mbar < 1.0 104 mbar 4.8 106 mbar Nickel orifice, 1.00 mm diameter Nickel orifice, 0.75 mm diameter
75As, 40Ar37Cl,

Conclusions
Arsenic is not only accumulated within this three-organism marine food chain but also metabolized. The Ribose 1 and the unknown arsenic compound, the major arsenic compounds in the seaweed Hormosira banksii, were converted mainly to arsenobetaine by the gastropods. Several signals in the chromatograms could not be assigned to arsenic compounds for which synthetic standards were available (Fig. 1). More research is needed to identify the unknown arsenic compounds. A conversion of dimethyl(ribosyl)arsine oxides to dimethyl (2-hydroxyethyl)arsine oxide, which in turn is transformed via arsenocholine or dimethyl(carboxymethyl)arsine oxide to arsenobetaine was proposed but remains unproved [2]. The results obtained with the food chain Hormosira banksii Austrocochlea constricta Morula marginalba agree with the proposed pathway.

desolvation

1.4 kW <1 W

References
1. Cullen WR, Reimer KJ (1989) Chem Rev 89:713764 2. Francesconi KA, Edmonds JS (1993) Oceanogr Mar Biol Annu Rev 31:111151 3. Jin K, Hayashi T, Shibata Y, Morita M (1988) Appl Organomet Chem 2:365369 4. Shibata Y, Morita M, Edmonds JS (1987) Agr Biol Chem 51:391398 5. Stummeyer J, Harazim B, Wippermann T (1996) Fresenius J Anal Chem 354:344351 6. Zhang X, Cornelis R, De Kiempe J, Mees L (1996) Anal Chim Acta 319:177185 7. Magnuson ML, Creed JT, Brockhoff CA (1996) J Anal At Spectrom 11:893898 8. Le X-C, Cullen WR, Reimer KJ (1994) Talanta 41:495502 9. Mrer AJL, Abildtrup A, Poulsen OM, Christensen JM (1992) Analyst 117:677680 10. Sheppard BS, Caruso JA, Heitkemper DT, Wolnick KA (1992) Analyst 117:971975 11. Larsen EH, Pritzl G, Hansen SH (1993) J Anal At Spectrom 8:557563 12. Kuehnelt D, Goessler W, Irgolic KJ (1997) Appl Organomet Chem (in press) 13. Gailer J, Irgolic KJ (1994) Appl Organomet Chem 8:129 140

or 77Se 0.51 s column dependent (400500 s)

Chromatographic conditions Column Supelcosil LC-SAX1 Supelcosil LC-SCX Hamilton PRP-X100 Mobile phase 15 mmol/L aqueous NH4H2PO4 20 mmol/L aqueous pyridine 30 mmol/L aqueous NH4H2PO4 pH 5.1 2.9 6.0