Improvement scheme for the determination of arsenic species in mussel and fish tissues

Six interlaboratory studies were organised by the Standard, Measurement and Testing Programme of the European Commission on the determination of arsenic species (arsenobetaine, arsenocholine, monomethylarsonic acid, dimethylarsinic acid, As(III) and As(V)) in marine matrices and soil. A step-by-step approach was used and a meeting was held at the end of each study to help the participants to discover errors and to improve their analytical methods. The successive steps investigated the calibration procedures on various solutions, the separation and derivatisation techniques on solutions and extracts and the extraction on mussel and fish tissues. All materials used for the study were monitored for their stability. Verified calibration solutions and compounds were distributed to the participants in each exercise in order to trace calibration problems. The agreement between the results improved regularly and at the end of the six campaigns allowed the certification of a reference material of tuna-fish tissue (BCR-CRM 627) for its total arsenic, arsenobetaine and dimethylarsinic acid contents. Received: 31 March 1998 / Revised: 20 July 1998 / Accepted: 25 July 1998     

Certification of total arsenic, dimethylarsinic acid and arsenobetaine contents in a tuna fish powder (BCR-CRM 627)

Tuna fishes were collected in the Straits of Messina (Italy), were dissected and dorsal muscles minced, freeze-dried, ground and sieved. The obtained powder was stabilised by γ-irradiation and filled into brown glass bottles. The homogeneity and stability at +20 °C and +40 °C were verified with regards to the total arsenic, dimethylarsinic acid and arsenobetaine contents. Ten laboratories participated in the certification study. All participants had demonstrated beforehand their ability to produce accurate results for the As speciation in fish tissue. The certified values are: total arsenic (4.8 ± 0.3) mg/kg, arsenobetaine (52 ± 3) μmol/kg, dimethylarsinic acid (2.0 ± 0.3) μmol/kg. The material is available from the BCR since early 1998. Received: 31 March 1998 / Revised: 20 July 1998 / Accepted: 25 July 1998     

Certification of total arsenic, dimethylarsinic acid and arsenobetaine contents in a tuna fish powder (BCR-CRM 627)

Tuna fishes were collected in the Straits of Messina (Italy), were dissected and dorsal muscles minced, freeze-dried, ground and sieved. The obtained powder was stabilised by γ-irradiation and filled into brown glass bottles. The homogeneity and stability at +20?°C and +40?°C were verified with regards to the total arsenic, dimethylarsinic acid and arsenobetaine contents. Ten laboratories participated in the certification study. All participants had demonstrated beforehand their ability to produce accurate results for the As speciation in fish tissue. The certified values are: total arsenic (4.8 ± 0.3) mg/kg, arsenobetaine (52 ± 3) μmol/kg, dimethylarsinic acid (2.0 ± 0.3) μmol/kg. The material is available from the BCR since early 1998.     

The fate of organoarsenic compounds in marine ecosystems

Microbial degradation experiments were performed with each standard arsenical [arsenobetaine, trimethylarsine oxide, dimethylarsinic acid, methanearsonic acid, inorganic arsenic(V) and inorganic arsenic(III)]. As typical origins for marine micro-organisms, sediments, macro-algae, mollusc intestine and suspended substances were used. The results were from these experiments led us to the following conclusions: (1) there is an arsenic cycle which begins with the methylation of inorganic arsenic on the route to arsenobetaine and terminates with the complete degradation of arsenobetaine to inorganic arsenic; (2) all the organoarsenic compounds which are derived from inorganic arsenic in seawater, through the food chains, have the fate that they, at least in part, finally return to the original inorganic arsenic.     

Formation of arsenobetaine from arsenocholine by micro-organisms occurring in sediments

As one of the experiments to pursue marine circulation of arsenic, we studied microbiological conversion of arsenocholine to arsenobetaine, because arsenocholine may be a precursor of arsenobetaine in these ecosystems. Two culture media, 1/5 ZoBell 2216E and an aqueous solution of inorganic salts, were used in this in vitro study. To each medium (25 cm³) were added synthetic arsenocholine (0.2%) and about 1 g of the sediment, and they were aerobically incubated at 25°C in the dark. These conversion experiments were performed in May and July 1990. In both seasons, two or three metabolites were derived in each mixture. These metabolites were purified using cation-exchange chromatography. Their structures were confirmed as arsenobetaine, trimethylarsine oxide and dimethylarsinic acid by high-performance liquid chromatography, thin-layer chromatography, FAB mass spectrometry and a combination of gas-chromatographic separation with hydride generation followed by a cold-trap technique and selected-ion monitoring mass spectrometric analysis. From this and other evidence it is concluded that, in the arsenic cycle in these marine ecosystems, as recently postulated by us, the pathway arsenocholine → arsenobetaine → trimethylarsine oxide → dimethylarsinic acid → methanearsonic acid → inorganic arsenic can be carried out by micro-organisms alone.     

Metabolism of arsenic compounds by the blue mussel mytilus edulis after accumulation from seawater spiked with arsenic compounds

Blue mussels (Mytilus edulis) were exposed to 100 μg As dm^?3 in the form of arsenite, arsenate, methylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide, tetramethylarsonium iodide or dimethyl-(2-hydroxyethyl)arsine oxide in seawater for 10 days. The seawater was renewed and spiked with the arsenic compounds daily. Analyses of water samples taken 24 h after spiking showed that arsenobetaine and arsenocholine had been converted to trimethylarsine oxide, whereas trimethylarsine oxide and tetramethylarsonium iodide were unchanged. Arsenobetaine was accumulated by mussels most efficienty, followed in efficiency by arsenocholine and tetramethylarsonium iodide. None of the other arsenic compounds was significantly accumulated by the mussels. Extraction of mussel tissues with methanol revealed that control mussels contained arsenobetaine, a dimethyl-(5-ribosyl)arsine oxide and an additional arsenic compound, possibly dimethylarsinic acid. Mussels exposed to arsenobetaine contained almost all their experimentally accumulated arsenic as arsenobetaine, and mussels exposed to tetramethylarsonium iodide contained it as the tetramethylarsonium compound. Mussels exposed to arsenocholine had arsenobetaine as the major arsenic compound and glycerylphosphorylarsenocholine as a minor arsenic compound in their tissues. The results show that arsenobetaine and arsenocholine are efficiently accumulated from seawater by blue mussels and that in both cases the accumulated arsenic is present in the tissues as arsenobetaine. Consequently arsenobetaine and/or arsenocholine present at very low concentrations in seawater may be responsible for the presence of arsenobetaine in M. edulis and probably also among other marine animals. The quantity of arsenobetaine accumulated by the mussels decreases with increasing concentrations of betaine. HPLC-ICP-MS was found to be very powerful for the investigation of the metabolism of arsenic compounds in biological systems.     

Preparation of pure calibrants (arsenobetaine and arsenocholine) for arsenic speciation studies and certification of an arsenobetaine solution – CRM 626

Interlaboratory studies have been organised by the Standards, Measurement and Testing Programme in order to certify the content of some chemical forms of arsenic in fish and mussel tissue, soil and sediment. Among the six compounds of interest (arsenite, arsenate, dimethylarsinic and monomethylarsonic acids, arsenobetaine and arsenocholine) only four are commercially available. Arsenobetaine and arsenocholine of well defined purity had therefore to be prepared by the pilot laboratories in charge of the management of the project. The present work deals with the synthesis and characterisation of these two compounds and the certification of the content of arsenobetaine in a standard solution (BCR-CRM 626). Received: 31 March 1998 / Revised: 20 July 1998 / Accepted: 25 July 1998     

Arsenic Compounds in Terrestrial Organisms I: Collybia maculata,Collybia butyracea and Amanita muscaria from Arsenic Smelter Sites in Austria

Three mushroom species from two old arsenic smelter sites in Austria were analyzed for arsenic compounds. The total arsenic concentrations were determined by ICP–MS. Collybia maculata contained 30.0 mg, Collybia butyracea 10.9 mg and Amanita muscaria 21.9 mg As kg⁻¹ dry mass. The arsenic compounds extracted with methanol/water (9:1) from the dried mushroom powders were separated by HPLC on anion-exchange and reversed-phase columns and detected by ICP-MS using a hydraulic high-pressure nebulizer. In Collybia maculata almost all arsenic is present as arsenobetaine. Collybia butyracea contained mainly arsenobetaine (8.8 mg As kg⁻¹ dry mass) and dimethylarsinic acid (1.9 mg As kg⁻¹). Amanita muscaria contained arsenobetaine (15.1 mg As kg⁻¹), traces of arsenite, dimethylarsinic acid and arsenate, and surprisingly arsenocholine (2.6 mg As kg⁻¹) and a tetramethylarsonium salt (0.8 mg As kg⁻¹). © 1997 by John Wiley & Sons, Ltd.     

Determination of arsenic species in human urine using HPLC with on-line photooxidation or microwave-assisted oxidation combined with flow-injection HG-AAS

An improved analytical procedure is presented for the separation and simultaneous determination of hydride-forming (toxic) and not hydride-forming (non-toxic) arsenic species in human urine. Separation was performed by cation-exchange chromatography using a new solid phase type based on the continuous bed chromatography (CBC) technology. This column permits by a factor of 4 higher flow rates than conventional columns resulting in a drastical reduction of retention times without any loss of resolution. Using this type of column, arsenobetaine (AsBet), arsenocholine (AsChol), and dimethylarsinic acid (DMA) were separated from the more toxic arsenic species arsenous acid (As(III)), arsenic acid (As(V)), and methylarsonic acid (MA) within only 4 min. The HPLC system was coupled via a flow injection system and either a UV or a microwave (MW) reactor to the HG-AAS instrument. UV photolysis and MW digestion were used to transform AsBet and AsChol to hydride-forming species and to make them accessible to HG-AAS. UV photolysis turned out to be more suitable for this application than MW digestion, because the latter technique led to peak broadening and poorer performance. The described procedure was applied to the determination of arsenic species in urine samples of non-occupationally exposed persons before and 12 h after seafood consumption. Detection limits were about 1 μg/L for each arsenic species. After consumption, the AsBet and DMA excretion increased by at least a factor of 150 for AsBet and by a factor of 6 for DMA, respectively, while the excretion of the other species did not increase significantly. This invalidates the use of total urinary arsenic as well as total hydride-forming arsenic as an indicator for exposure to inorganic arsenic. Received: 12 August 1998 / Revised: 30 October 1998 / Accepted: 24 November 1998     

Speciation and health risk considerations of arsenic in the edible mushroom Laccaria amethystina collected from contaminated and uncontaminated locations

Samples of the edible mushroom Laccaria amethystina, which is known to accumulate arsenic, were collected from two uncontaminated beech forests and an arsenic-contaminated one in Denmark. The total arsenic concentration was 23 and 77 μg As g⁻¹ (dry weight) in the two uncontaminated samples and 1420 μg As g⁻¹ in the contaminated sample. The arsenic species were liberated from the samples using focused microwave-assisted extraction, and were separated and detected by anion- and cation-exchange high-performance liquid chromatography with an inductively coupled plasma mass spectrometer as arsenic-selective detector. Dimethylarsinic acid accounted for 68–74%, methylarsonic acid for 0.3–2.9%, trimethylarsine oxide for 0.6–2.0% and arsenic acid for 0.1–6.1% of the total arsenic. The unextractable fraction of arsenic ranged between 15 and 32%. The results also showed that when growing in the highly arsenate-contaminated soil (500–800 μg As g⁻¹) the mushrooms or their associated bacteria were able to biosynthesize dimethylarsinic acid from arsinic acid in the soil. Furthermore, arsenobetaine and trimethylarsine oxide were detected for the first time in Laccaria amethystina. Additionally, unidentified arsenic species were detected in the mushroom. The finding of arsenobetaine and trimethylarsine oxide in low amounts in the mushrooms showed that synthesis of this arsenical in nature is not restricted to marine biota. In order to minimize the toxicological risk of arsenic to humans it is recommended not to consume Laccaria amethystina mushrooms collected from the highly contaminated soil, because of a genotoxic effect of dimethylarsinic acid observed at high doses in animal experiments. © 1998 John Wiley & Sons, Ltd. No Abstract.     

Determination of inorganic arsenic and organic arsenic compounds in marine organisms by hydride generation/cold trap/gas chromatography— mass spectrometry

The behavior of arsenite, methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, dimethyl-R-arsine oxides, and trimethyl-R-arsonium compounds (R = carboxymethyl, 2-carboxyethyl, 2-hydroxyethyl) toward sodium borohydride and hot aqueous sodium hydroxide was investigated. The arsines obtained by sodium borohydride reduction of the undigested and digested solutions were collected in a liquid-nitrogen cooled trap, separated with a gas chromatograph, and detected with a mass spectrometer in the selected-ion-monitoring mode. The investigated arsenic compounds were stable in hot 2 mol dm^?3 sodium hydroxide except arsenobetaine [trimethyl(carboxymethyl)arsonium zwitterion] that was converted to trimethylarsine oxide, and dimethyl(ribosyl)arsine oxides that were decomposed to dimethylarsinic acid. Hydride generation before and after digestion of extracts from marine organisms allowed inorganic arsenic, methylated arsenic, arsenobetaine, and ribosyl arsenic compounds to be identified and quantified. This method was applied to extracts from shellfish, fish, crustaceans, and seaweeds.     

Speciation of arsenic in natural waters by HPLC-NAA

Neutron activation analysis (NAA) in combination with mainly high-performance liquid chromatography (HPLC) has been developed for the determination of low levels of five arsenic species, namely As(III), As(V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) in water samples. Organically bound arsenic (OBAs) and total arsenic have also been determined. In addition to anion-exchange HPLC, solid phase extraction and open-column cation-exchange chromatographic methods have also been used. The detection limits of the method have been found to be 0.005 ng·cm⁻³ for OBAs, 0.02 ng·cm⁻³ for AsB, DMA, MMA, As(III), and As(V) and 0.12 ng·cm⁻³ for total arsenic. This revised version was published online in July 2006 with corrections to the Cover Date.     

Arsenic Speciation in Sargassum fusiforme by Microwave-Assisted Extraction and LC-ICP-MS

Sargassum fusiforme, the common Chinese edible seaweeds, was investigated for total arsenic concentration by ICP-MS and for individual arsenic species by LC-ICP-MS. For this purpose, a microwave-assisted procedure was used for the extraction of arsenic species in freeze-dried seaweed and an analytical procedure for the sensitive and efficient speciation of the arsenic species As(III), dimethylarsinic acid, monomethyl arsonic acid, As(V), arsenobetaine and arsenocholine was optimized. Arsenic compounds were extracted from the seaweed with a methanol/water mixture; the extracts were evaporated to dryness, redissolved in water, and chromatographed on an anion exchange column. The arsenic species in Sargassum fusiforme are abundant. In some sample, the majority of arsenic compounds detected in the extracts were inorganic species, with a predominance of As (V). In addition, some significant amounts of unidentified arsenic compounds were also observed in the extracts.

     

Tissue accumulation and distribution of arsenic compounds in three marine fish species: relationship to trophic position

In this study the accumulation and distribution of arsenic compounds in marine fish species in relation to their trophic position was investigated. Arsenic compounds were measured in eight tissues of mullet Mugil cephalus (detritivore), luderick Girella tricuspidata (herbivore) and tailor Pomatomus saltatrix (carnivore) by high performance liquid chromatography–inductively coupled plasma‐mass spectrometry. The majority of arsenic in tailor tissues, the pelagic carnivore, was present as arsenobetaine (86–94%). Mullet and luderick also contained high amounts of arsenobetaine in all tissues (62–98% and 59–100% respectively) except the intestines (20% and 24% respectively). Appreciable amounts of dimethylarsinic acid (1–39%), arsenate (2–38%), arsenite (1–9%) and trimethylarsine oxide (2–8%) were identified in mullet and luderick tissues. Small amounts of arsenocholine (1–3%), methylarsonic acid (1–3%) and tetramethylarsonium ion (1–2%) were found in some tissues of all three species. A phosphate arsenoriboside was identified in mullet intestine (4%) and from all tissues of luderick (1–6%) except muscle. Pelagic carnivore fish species are exposed mainly to arsenobetaine through their diet and accumulate the majority of arsenic in tissues as this compound. Detritivore and herbivore fish species also accumulate arsenobetaine from their diet, with quantities of other inorganic and organic arsenic compounds. These compounds may result from ingestion of food and sediment, degradation products (e.g. arsenobetaine to trimethylarsine oxide; arsenoribosides to dimethylarsinic acid), conversion (e.g. arsenate to dimethylarsinic acid and trimethylarsine oxide by bacterial action in digestive tissues) and/or in situ enzymatic activity in liver tissue. Copyright © 2001 John Wiley & Sons, Ltd.     

Separation of organic and inorganic arsenic species by HPLC-ICP-MS

The HPLC separation of eight anionic, cationic or neutral arsenic species (arsenite, arsenate, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine, arsenocholine, trimethylarsine oxide and tetramethylarsonium ion) on a high-capacity, anion-exchange column (Ion Pac AS 7, Dionex) was studied. The separation was performed during one run with a nitric acid gradient ranging from pH 4–1.3. The influence of sodium dodecyl sulfate (SDS), sodium octyl sulfate (SOS) and 1,2-benzenedisulfonic acid (BDSA) as ion pairing eluent modifiers was investigated. In addition the effect of elevated temperatures (30 to 40 °C) was studied. The best results were obtained at room temperature of 20 °C with 0.05 mM benzenedisulfonic acid as the eluent modifier. The chromatograph was connected to an ICP-MS via a cross-flow nebulizer. Detection limits obtained with the optimized chromatographic separation were 0.16–0.60 μg As L^–1 for different species. The proposed speciation method was applied to the determination of arsenic species in the DORM-2 reference material (Dogfish Muscle) and in aqueous extracts of mushrooms collected on arsenic contaminated ground. Received: 3 August 1998 / Revised: 17 September 1998 / Accepted: 21 September 1998     

The retention behavior of arsenic compounds on PRP-X100 column under alkaline condition.

系统地研究了碱性条件下（ｐＨ８～１０．８）Ａｓ３＋,Ａｓ５＋,ＭＡ,ＤＭＡ和ＡＢ等砷化合物在ＰＲＰ－Ｘ１００阴离子交换柱上的保留行为。用火焰原子吸收光谱（ＦＡＡＳ）测定从ＨＰＬＣ分离的砷化合物,即通过一根１ｍ×０．２３ｍｍｉ．ｄ．不锈钢毛细管,将ＨＰＬＣ柱出口与ＦＡＡＳ的雾化器连接起来,采用乙炔／空气火焰,在１９３．７ｎｍ处测定。具体研究了两个流动相（２０ｍｍｏｌ／ＬＮＨ４ＨＣＯ３和２．５ｍｍｏｌ／Ｌ对－羟基苯甲酸－１．０ｍｍｏｌ／Ｌ苯甲酸水溶液）。     

液相色谱-电感耦合等离子体质谱法测定稻米中的5种砷形态

建立了稻米中砷酸根[As（Ⅴ）]、亚砷酸根[As（Ⅲ）]、砷甜菜碱（AsB）、一甲基砷（MMA）和二甲基砷（DMA）的液相色谱-电感耦合等离子体质谱（LC-ICP-MS）检测方法。以0.3 mol/L硝酸水溶液为提取试剂,样品在石墨消解仪中于95 ℃消解1.5 h,上清液供LC-ICP-MS分析。5种砷形态采用Dionex IonPac AS19阴离子交换柱（250 mm×4 mm）分离,经ICP-MS检测。比较了4种提取液对稻米中5种砷形态的提取效率,并对提取溶剂的浓度、提取温度和提取时间等条件进行了优化。通过加标回收试验结合测定标准物质考察了方法准确度及精密度,在2个加标水平上各形态的回收率为89.6%~99.5%,RSD（n=5）不大于3.6%,大米标准物质中各形态之和的测定结果与其标准值吻合,5种砷形态的线性范围AsB和DMA为0.05~200 μg/L,As（Ⅲ）和MMA为0.10~400 μg/L,As（V）为0.15~600 μg/L,方法检出限为0.15~0.45 μg/kg。结果表明,本方法简单、灵敏、耐用,可用于稻米中5种砷形态的准确定量和风险评估。     

Adsorption of Inorganic and Organic Arsenic Compounds by Aluminium-loaded Coral Limestone

Coral limestones were treated with an aqueous solution of aluminium sulfate and thereby aluminium-loaded coral limestones (Al-CL) were prepared. By use of Al-CL as an adsorbent, the adsorption of inorganic arsenic compounds (arsenate [As(V)] and arsenite [As(III)] and of organic arsenic compounds (methylarsonic acid, dimethylarsinic acid, and arsenobetaine) was examined. The adsorption ability of Al-CL is superior to that of iron(III)-loaded coral limestone (Fe-CL) for As(V), As(III), methylarsonic acid and dimethylarsinic acid. The adsorption of As(V) and As(III) is almost independent of the initial pH over a wide range (2 or 3 to 11). The addition of other anions, such as chloride, nitrate, sulfate and acetate, in the solution does not affect the adsorption of As(V) and As(III), whereas the addition of phosphate greatly interferes with the adsorption. Arsenic adsorption is effectively applied to a column-type operation and the adsorption capability for As(V) is 150 μg/g coral limestone.     

Validated determination of total arsenic species of toxicological interest (arsenite, arsenate and their metabolites) by atomic absorption spectrometry after separation from dietary arsenic by liquid extraction: toxicological applications

A validated method for the selective extraction of total As species of toxicological interest (arsenite, arsenate and mono- and dimethylated arsenic species) from urine, followed by atomic absorption spectrometric determination, is described. The mechanisms involved in extraction were studied and the extraction method was optimized. The urine sample was acidified with concentrated HCl and KI and sodium hypophosphite were added. Under these conditions, As species were reduced to their corresponding iodide arsines, extracted with toluene and back-extracted with 1 mmol l-1 NaOH solution. Only inorganic arsenic and its metabolites in humans (monomethylarsonic and dimethylarsinic acid) were extracted. Arsenobetaine of dietary origin was not extracted. This method can detect if any As increase in urine originates from inorganic As intoxication or only from dietary non-toxic As species such as arsenobetaine.     

Coupled high performance liquid chromatography-microwave digestion-hydride generation-atomic absorption spectrometry for inorganic and organic arsenic speciation in fish tissue

A high performance liquid chromatography-microwave digestion-hydride generation-atomic absorption spectrometry (HPLC-MW-HG-AAS) coupled method is described for As(III), As(V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC) determination. A Hamilton PRP-X100 anion-exchange column is used for carrying out the arsenic species separation. As mobile phase 17 mM phosphate buffer (pH 6.0) is used for As(III), As(V), MMA and DMA separation, and ultrapure water (pH 6.0) for AsB and AsC separation. Prior to injection into the HPLC system AsB and AsC are isolated from the other arsenic species using a Waters Accell Plus QMA cartridge. A microwave digestion with K(2)S(2)O(8) as oxidizing agent is used for enhancing the efficiency of conversion of AsB and AsC into arsenate. Detection limits achieved were between 0.3 and 1.1 ng for all species. The method was applied to arsenic speciation in fish samples.