Georg Raber

Environmental context: The marine ecosystem is highly elevated in arsenic in comparison to the terrestrial environment. More than 100 different naturally occurring organoarsenic compounds have been found in different marine organisms. In order to understand how highly toxic AsHCs are formed and bioaccumulate in different marine organisms, large scale monitoring of AsHCs from a range of different laboratories is necessary. Despite their toxicity, AsHCs have not been extensively studied due in part to the lack of low-cost analytical methods and the lack of commercially available standards. Rational: This study aims to develop a new analytical method for the quantitative determination of arsenic-containing hydrocarbons (AsHCs) with gas chromatography–mass spectrometry (GC-MS). The new method is an alternative approach to determine these compounds in environmental samples. Methodology: AsHCs were extracted and purified from fish oil and seaweed samples. Due to the thermal conversion of oxo-AsHCs to their trivalent forms in the injection port of the GC, the conversion is incomplete resulting in low signal intensities and large carry overs. We therefore chemically reduced AsHCs to their trivalent forms and analysed them with GC-MS, gas chromatography–tandem mass spectrometry (GC-MS/MS) and high performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICPMS). The possibility for compound independent quantification with commercially available standards was investigated. Results: After optimisation of conditions for reduction of AsHCs, these compounds could be determined in marine samples using authentic standards with the new method. Results showed good agreement with results from HPLC-ICPMS. The novel GC-MS method showed compound independent quantification using triphenylarsine (TPA) as a commercially available standard. Discussion: With the developed method we could demonstrate the applicability of GC-MS for the quantitative determination of AsHCs in marine samples for the first time. The compound independent quantification with TPA opens up a quantification for AsHCs species for which no standards are commercially available. The broad applicability might enhance screening for these toxic compounds in the environment.

Although the natural occurrence of arsenic-containing lipids (arsenolipids) in marine organisms is now well established, the possible role of these unusual compounds in organisms and in the cycling of arsenic in marine systems remains largely unexplored. We report the finding of arsenolipids in 61 plankton samples collected from surface marine waters of high- and low-nutrient content along a transect spanning the Gulf Stream in the North Atlantic Ocean. Using high-performance liquid chromatography (HPLC) coupled to both elemental and molecular mass spectrometry, we show that all 61 plankton samples contained six identifiable arsenolipids, namely, three arsenosugar phospholipids (AsPL958, 10–13%; AsPL978, 13–25%; and AsPL1006, 7–10% of total arsenolipids), two arsenic-containing hydrocarbons (AsHC332, 4–10% and AsHC360, 1–2%), and a methoxy-sugar arsenolipid that contained phytol (AsSugPhytol, 1–3%). The relative amounts of the six arsenolipids showed clear dependence on the nutrient status of the ambient water with plankton collected from high-nutrient waters having less of the arsenosugar phospholipids and more of the three non-P containing arsenolipids compared to low-nutrient waters. By combining these first field data of arsenolipids in plankton with reported global phytoplankton productivity, we estimate that the oceans’ phytoplankton transform per year 50 000–100 000 tons of arsenic into arsenolipids.

Arsenolipids include a wide range of organic arsenic species that occur naturally in seafood and thereby contribute to human arsenic exposure. Recently arsenic-containing phosphatidylcholines (AsPCs) were identified in caviar, fish, and algae. In this first toxicological assessment of AsPCs, we investigated the stability of both the oxo- and thioxo-form of an AsPC under experimental conditions, and analyzed cell viability, indicators of genotoxicity and biotransformation in human liver cancer cells (HepG2). Precise toxicity data could not be obtained owing to the low solubility in the cell culture medium of the thioxo-form, and the ease of hydrolysis of the oxo-form, and to a lesser degree the thioxo-form. Hydrolysis resulted amongst others in the respective constituent arsenic-containing fatty acid (AsFA). Incubation of the cells with oxo-AsPC resulted in a toxicity similar to that determined for the hydrolysis product oxo-AsFA alone, and there were no indices for genotoxicity. Furthermore, the oxo-AsPC was readily taken up by the cells resulting in high cellular arsenic concentrations (50 μM incubation: 1112 ± 146 μM As cellular), whereas the thioxo-AsPC was substantially less bioavailable (50 μM incubation: 293 ± 115 μM As cellular). Speciation analysis revealed biotransformation of the AsPCs to a series of AsFAs in the culture medium, and, in the case of the oxo-AsPC, to as yet unidentified arsenic species in cell pellets. The results reveal the difficulty of toxicity studies of AsPCs in vitro, indicate that their toxicity might be largely governed by their arsenic fatty acid content and suggest a multifaceted human metabolism of food derived complex arsenolipids.

A GC-MS/MS method with low solvent consumption and easy operation was developed to simultaneously determine ten new-generation pesticides, namely fenobucarb, acetochlor, pretilachlor, fipronil, trifloxystrobin, fluazifop-p-butyl, isoprothiolane, tebuconazole, cypermethrin and difenoconazole in leafy vegetables. Influences from ultrasonic sample extraction and the clean-up steps to reduce matrix effects were investigated. Under suitable conditions, good linearity (deviation of back calculated concentration from true concentration lower than 20%) was achieved for all studied pesticides; the method detection limits (MDLs) ranged from 1.4 to 3.6 ng g⁻¹ wet weight. For mustard green and green onion the method yielded good recoveries at two spiking levels (20¹ and 100 ng g⁻¹) ranging from 80% to 111% (n = 5). The repeatability, expressed as relative standard deviation (RSD), was lower than 11% (n = 5). The method was successfully used to quantify pesticide residues in 207 vegetable samples (green onions, mustard greens, and lettuce) collected in Thua Thien Hue and Quang Binh Provinces, Central Vietnam. The insecticide cypermethrin was found at critical levels in 98% of the vegetable samples. Green onions had high pesticide residues with a significant number of samples containing pesticides at concentrations exceeding the maximum residue levels (MRLs).

Lipid-soluble arsenicals, so called arsenolipids, occur in appreciable quantities in various marine organisms including fish. In this study, arsenolipids as well water-soluble arsenic species were investigated in brain (9.1–17.4 mg As per kg; dry mass) and muscle (4.0–5.8 mg As per kg; dry mass) tissues in five specimens of the marine fish skipjack tuna (Katsuwonus pelamis). For this purpose, we developed a sequential extraction method whereby the freeze-dried tissue was first treated with pyridine (organic extract) followed by aqueous ammonium bicarbonate (water extract) to extract arsenolipids and water-soluble species, respectively. When the method was applied to the tuna tissues, the arsenic distribution for brain was 55% (organic extract), 30% (water extract) and 15% (pellet), whereas for muscle tissue the corresponding values were 20%, 55%, and 25%. Arsenic species in water and organic extracts of muscle and brain tissues were investigated by HPLC/mass spectrometry. For both tissues, the water extracts contained arsenobetaine as the major arsenic species together with small amounts of dimethylarsinate and trimethylarsine oxide; trace amounts of dimethylarsinoyl propionic acid were found only in brain tissues. Arsenic-containing hydrocarbons (AsHCs), were the major arsenolipids in both tuna brain and muscle. The arsenolipid content in brain ranged from 3.8–5.9 mg As per kg, whereas it was substantially lower in muscle (0.3–0.8 mg As per kg) reflecting arsenolipids' potential to cross the blood brain barrier and accumulate in the fish brain.

Arsenosugars are arsenic-containing ribosides that play a substantial role in arsenic biogeochemical cycles. Arsenosugars were identified more than thirty years ago, and yet their mechanism of biosynthesis remains unknown. In this study we report identification of the arsS gene from the cyanobacterium Synechocystis sp. PCC 6803 and show that it is involved in arsenosugar biosynthesis. In the Synechocystis sp. PCC 6803 ars operon, arsS is adjacent to the arsM gene that encodes an As(III) S-adenosylmethionine (SAM) methyltransferase. The gene product, ArsS, contains a characteristic CX3CX2C motif which is typical for the radical SAM superfamily. The function of ArsS was identified from a combination of arsS disruption in Synechocystis sp. PCC 6803 and heterologous expression of arsM and arsS in Escherichia coli. Both genes are necessary, indicating a multi-step pathway of arsenosugar biosynthesis. In addition, we demonstrate that ArsS orthologs from three other freshwater cyanobacteria and one picocyanobacterium are involved in arsenosugar biosynthesis in those microbes. This study represents the identification of the first two steps in the pathway of arsenosugar biosynthesis. Our discovery expands the catalytic repertoire of the diverse radical SAM enzyme superfamily and provides a basis for studying the biogeochemistry of complex organoarsenicals.

The release of a toxicant from a food matrix during the gastrointestinal digestion is a crucial determinant of the toxicant’s oral bioavailability. We present a modified setup of the human simulator of the gut microbial ecosystem (SHIME), with four sequential gastrointestinal reactors (oral, stomach, small intestine, and colon), including the salivary and colonic microbiomes. Naturally arsenic-containing rice, mussels and nori seaweed were digested in the presence of microorganisms, and in vitro oral bioaccessibility, bioavailability and metabolism of arsenic species were evaluated. When food matrices were digested with salivary bacteria, the soluble arsenic in the gastric digestion stage increased for mussel and nori samples, but no coincidence impact was found in the small intestinal and colonic digestion stages. However, the simulated small intestinal absorption of arsenic was increased in all food matrices (1.2-2.7 fold higher) following digestion with salivary microorganisms. No significant transformation of the arsenic species occurred except for the arsenosugars present in mussels and nori. In those samples, conversions between the oxo arsenosugars were observed in the small intestinal digestion stage whereupon the thioxo analogs became major metabolites. These results expand our knowledge on the likely metabolism and oral bioavailability of arsenic during the human digestion and provide information valuable for future risk assessments of dietary arsenic.