Distribution of arsenic species in the freshwater crustacean Procambarus clarkii

The concentrations of total arsenic and arsenic species in the complete organism of the crayfish Procambarus clarkii and its various parts (hepatopancreas, tail, and remaining parts) were analyzed in order to discover the distribution of arsenic and its species. With this information it will be possible to establish where the chemical forms of this metalloid tend to accumulate and what risks may derive from the contents and species present in the edible parts of this crustacean. The total arsenic content in the complete organism and in the various parts analyzed ranged from 2.5 to 12 µg g^?1 dry mass (DM), with inorganic arsenic representing 18 to 34% of total arsenic. The arsenical composition varied according to the part of the crayfish considered. The hepatopancreas had the highest levels of total arsenic (9.2–12 µg g^?1 DM) and inorganic arsenic (2.7–3.2 µg g^?1 DM). The tail (edible part) had the lowest levels of both total arsenic (2.5–2.6 µg g^?1 DM) and inorganic arsenic (0.46–0.64 µg g^?1 DM). The predominant organoarsenical species were the dimethylarsinoylribosides: glycerol riboside in the hepatopancreas, sulfate riboside in the tail, and sulfonate and phosphate ribosides in the remaining parts. Copyright © 2002 John Wiley & Sons, Ltd.     

Arsenobetaine as the major arsenic compound in the muscle of two species of freshwater fish

The chemical form of arsenic contained in the muscle of certain freshwater fish was examined using cultured specimens of rainbow trout (Salmo gairdneri) and wild specimens of Japanese smelt (Hypomesus nipponensis). More than 95% of the total arsenic of both species was extracted with methanol and recovered in the water-soluble fraction. The major arsenic compound of both species was purified by cation-exchange chromatography on Dowex 50, gel filtration on Bio-Gel P-2 and HPLC on Asahipak GS-220H. Behavior in the above purification procedure and analyses of the purified compounds by HPLC–ICP and TLC confirmed that the major arsenic compound of both species was arsenobetaine. Arsenobetaine found in cultured rainbow trout seems to be derived from the commercial assorted feed containing arsenobetaine as the major arsenical. On the other hand, the result with wild Japanese smelt suggested that arsenobetaine is a naturally occurring compound in the freshwater environment.     

Arsenobetaine and other arsenic species in mushrooms

Arsenic species in arsenic accumulating mush- rooms (Sarcosphaera coronaria, Laccaria amethystina, Sarcodon imbricatum, Entoloma lividum, Agaricus haemorrhoidaius, Agaricus placomyces, Lycoperdon perlatum) were determined. HPLC/ICP MS and ion-exchange chromatogra- phy–instrumental neutron activation analysis (NAA) combinations were used. The remarkable accumulator Sarcosphaera coronaria (up to 2000 mg As kg^?1 dry wt) contained only methylarsonic acid, Entoloma lividum only arsenite and arsenate. In Laccaria amethystina dimethylarsinic acid was the major arsenic compound. Sarcodon imbricatum and the two Agaricus sp. were found to contain arsenobetaine as the major arsenic species, a form which had previously been found only in marine biota. Its identification was confirmed by electron impact MS.     

Intake of different chemical species of dietary arsenic by the japanese,and their blood and urinary arsenic levels

We calculated the intake of each chemical species of dietary arsenic by typical Japanese, and determined urinary and blood levels of each chemical species of arsenic. The mean total arsenic intake by 35 volunteers was 195±235 (15.8-1039) μg As day^?1, composed of 76% trimethylated arsenic (TMA), 17.3% inorganic arsenic (As_i), 5.8% dimethylated arsenic (DMA), and 0.8% monomethylated arsenic (MA): the intake of TMA was the largest of all the measured species. Intake of As_i characteristically and invariably occurred in each meal. Of the intake of As_i, 45-75% was methylated in vivo to form MA and DMA, and excreted in these forms into urine. The mean measured urinary total arsenic level in 56 healthy volunteers was 129±92.0 μg As dm^?3, composed of 64.6% TMA, 26.7% DMA, 6.7% As_i and 2.2% MA. The mean blood total arsenic level in the 56 volunteers was 0.73±0.57 μg dl^?1, composed of 73% TMA, 14% DMA and 9.6% As_i. The urinary TMA levels proved to be significantly correlated with the whole-blood TMA levels (r = 0.376; P<0.01).     

The discovery of hidden arsenic species in coastal waters

The analysis of ultraviolet (UV)-irradiated and untreated seawater samples has shown that the dissolved arsenic content of marine waters cannot be completely determined by hydride generation–atomic absorption spectrophotometry without sample pretreatment. Irradiation of water samples obtained during a survey of arsenic species in coastal waters during the summer of 1988 gave large increases in the measured speciation. Average increases in the measured speciation. Average increases in total arsenic, monomethylarsenic and dimethylarsenic were 0.29 μg As dm^?3 (25%), 0.03 μg As dm^?3 (47%) and 0.12 μg As dm^?3 (79%), respectively. Overall, an average 25% increase in the concentration of dissolved arsenic was observed following irradiation. This additional arsenic may be derived from compounds related to algal arsenosugars or to their breakdown products. These do not readily yield volatile hydrides when treated with borohydride and are not therefore detected by the normal hydride generation technique. This has important repercussions as for many years this procedure, and other analytical procedures which are equally unlikely to respond to such compounds, have been accepted as giving a true representation of the dissolved arsenic speciation in estuarine and coastal waters. A gross underestimate may therefore have been made of biological involvement in arsenic cycling in the aquatic environment.     

Species differences in the metabolism of arsenic compounds

Humans are exposed via air, water and food to a number of different arsenic compounds, the physical, chemical, and toxicological properties of which may vary considerably. In people eating much fish and shellfish the intake of organic arsenic compounds, mainly arsenobetaine, may exceed 1000 μg As per day, while the average daily intake of inorganic arsenic is in the order of 10–20 μg in most countries. Arsenobetaine, and most other arsenic compounds in food of marine origin, e.g. arsenocholine, trimethylarsine oxide and methylarsenic acids, are rapidly excreted in the urine and there seem to be only minor differences in metabolism between animal species. Trivalent inorganic arsenic (AsIII) is the main form of arsenic interacting with tissue constituents, due to its strong affinity for sulfhydryl groups. However, a substantial part of the absorbed AsIII is methylated in the body to less reactive metabolities, methylarsonic acid (MMA) and dimethylarsinic acid (DMA), which are rapidly excreted in the urine. All the different steps in the arsenic biotransformation in mammals have not yet been elucidated, but it seems likely that the methylation takes place mainly in the liver by transfer of methyl groups from S-adenosylmethionine to arsenic in its trivalent oxidation state. A substantial part of absorbed arsenate (AsV) is reduced to AsIII before being methylated in the liver. There are marked species differences in the methylation of inorganic arsenic. In most animal species DMA is the main metabolite. Compared with human subjects, very little MMA is produced. The marmoset monkey is the only species which has been shown unable to methylate inorganic arsenic. In contrast to other species, the rat shows a marked binding of DMA to the hemoglobin, which results in a low rate of urinary excretion of arsenic.     

Determination of lipid‐soluble arsenic species in seaweed‐eating sheep from Orkney

This work is part of an ongoing research study towards an understanding of the complete metabolism of arsenosugars in mammalian organisms when ingesting seaweed, using the North Ronaldsay (NR) sheep as a model organism. We focus on the analysis of only those arsenic species bound to the lipids of the feed (Laminaria digitata), faeces and the tissues of the NR sheep using a novel enzymatic hydrolytic method that is simple and reliable. This rare breed of sheep, found in the remote Orkney Islands in the north of Scotland, live the entire year on the beaches and eat seaweed that is washed ashore (up to 3 kg daily). Previous studies on arsenic fractionation in muscle, kidney and liver tissues revealed that most of the arsenic is concentrated in the fat fractions of these tissues (muscle fat: 61%; liver fat: 66%; kidney fat: 25%) rather than in the non‐lipid fractions. Hence, this study was undertaken in order to determine the arsenic species bound to lipids in the muscle, kidney and faeces of NR sheep and to compare these with the arsenic species bound to the lipids of the L. digitata consumed. The enzymatic hydrolytic procedure has been successfully employed for the first time to cleave the arsenic species cleanly from the rest of the lipid structure. This makes the arsenic species water soluble and enables their direct determination by high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry. Dimethylarsinic acid (DMA(V)) and monomethylarsonic acid (MA(V)) were found to be the major hydrolysed arsenic species bound to the kidney and muscle lipids, whereas arsenosugar‐1 was found to be the major hydrolysed arsenic species in L. digitata lipids. On the other hand, DMA(V) was found to be the major arsenical obtained after the enzymatic hydrolysis of the faeces lipids. These results seem to suggest that both direct absorption and biotransformation of the absorbed organoarsenicals are the likely reasons for their occurrence and accumulation in the NR sheep tissues. Copyright © 2003 John Wiley & Sons, Ltd.     

Arsenic Compounds in Zoo- and Phyto-plankton of Marine Origin

Major water-soluble arsenic compounds accumulated in some zoo- and phyto-plankton were identified. Zooplankton were collected at sampling stations in the Sea of Japan by a Norpac net towed from 600 m depth to the surface. Phytoplankton were cultivated under axenic conditions. Water-soluble arsenic compounds were extracted repeatedly from plankton tissues by aqueous methanol. The arsenic compounds in the extracts were analyzed by HPLC–ICP/MS. Among zooplankton analyzed in the present study, two carnivorous species, i.e. Amphipoda ( Themisto sp.) and Sagittoidea ( Sagitta sp.), contained arsenobetaine as the dominant arsenic species. Arsenobetaine was the major species in Euphausiacea ( Euphausia sp.), also. The most abundant arsenic compound in the herbivorous Copepoda species ( Calanus sp.), on the other hand, was an arsenic-containing ribofuranoside with a sulfate ester group, and arsenobetaine was only a minor component. Phytoplankton contained arsenic-containing ribofuranosides apparently in a species-speific manner. The arsenic compounds in zooplankton seem to reflect their feeding habit; i.e. carnivorous species eating zooplankton or other small animals accumulate arsenobetaine, while herbivorous ones eating phytoplankton accumulate arsenic-containing ribofuranosides as major arsenic compounds.     

Enzyme-assisted extraction of arsenic species from plant material

Investigations regarding the transfer and metabolism of arsenic species in plants require mild extraction conditions to conserve the original composition of arsenic species. Beside the use of water or water/methanol for extraction of arsenic species from plant samples, enzymes can assist this procedure by digestion of cellulose and other constituents of cell walls, resulting in a faster, more efficient extraction technique which preserves the arsenic species. The investigations presented here were focused on the stability of certain arsenic species in enzymatic solutions, optimal conditions for their chromatographic separation and detection namely by means of ion chromatography–inductively coupled plasma mass spectrometry and improvements with respect to extraction efficiency. With commercially available enzymes and enzyme mixtures, the digestion rate of soluble starch as model cellulose was determined using high-performance anion exchange chromatography–pulsed amperometric detection analysis of glucose as the major digestion product. The most effective digestion rate (80% within 4?h) was obtained with Viscozyme®. This enzyme mixture was applied to extracted arsenic species from algae and terrestrial plant materials. Qualitative and quantitative differences in the results between enzyme-assisted and water extractions were obtained and discussed. The results show that the application of enzymes in mild extraction protocols should be evaluated as an additional step for the identification of As-metabolics in organisms. Careful selection of suitable enzyme mixtures can overcome the disadvantage that extraction efficiency is very organism-specific.     

利用氢化物发生-冷阱捕集-原子吸收光谱联用技术测定雄黄染毒大鼠血中砷的含量

采用氢化物发生-冷阱捕集-原子吸收光谱法(HG-CT-AAS)测定了雄黄染毒大鼠血中不同形态砷的含量.结果表明,雄黄在大鼠体内的代谢产物为无机砷、一甲基胂酸和二甲基胂酸,其中二甲基胂酸是主要代谢产物;3种形态砷的精密度、准确度、回收率及线性关系良好.     

The association mode of arsenic accumulated in the freshwater alga Chlorella vulgaris

Arsenic accumulated in living Chlorella vulgaris cells was solvent-fractionated with chloroform/methanol (2:1), and the fractions were analyzed for arsenic. A large part of the accumulated arsenic was localized in the extract residues. The extract residue from the same extraction of C. vulgaris, which had been, however, cultured in any arsenic-free Detmer medium (MD), adsorbed arsenic physico-chemically at a concentration of 1.1 mg As g^?1 dry weight. Arsenic was found to be combined with protein with molecular weight around 3000 in the arsenicaccumulated living cells. The arsenic-bound protein was analyzed for amino acids. The experimental results showed that no metallothionein-like protein was inductively biosynthesized in C. vulgaris on the exposure to arsenic.     

Biomethylation of Arsenic in an Arsenic-rich Freshwater Environment

Arsenic circulation in an arsenic-rich freshwater ecosystem was elucidated to detect arsenic species in the river water and in biological samples living in the freshwater environment. Water-soluble arsenic compounds in biological samples were extracted with 70% methanol. Samples containing arsenic compounds in the extracts were treated with 2 mol dm³ of sodium hydroxide and reduced with sodium borohydride. The detection of arsenic species was accomplished using a hydride generation/cold trap/cryofocus/gas chromatography-mass spectrometry (HG/CT/CF/GC-MS) system. The major arsenic species in the river water, freshwater algae and fish are inorganic arsenic, dimethylarsenic and trimethylarsenic compounds, respectively. Trimethylarsenic compounds are also detected in aquatic macro-invertebrates. The freshwater unicellular alga Chlorella vulgaris, in a growth medium containing arsenate, accumulated arsenic and converted it to a dimethylarsenic compound. The water flea Daphnia magna, which was fed on arsenic-containing algae, converted it to a trimethylarsenic species. © 1997 by John Wiley & Sons, Ltd.     

Refractory methylated arsenic compounds in estuarine waters: Tracing back elusive species

Water from the Tagus estuary, Portugal, was concentrated and purified through evaporation, solvent extraction, ion exchange and HPLC, and peaks of refractory arsenicals were detected by difference between total arsenic (GF AA) and hydride-forming arsenic species (HG QF AA). DCI mass spectra of these fractions presented peaks at m/z 139, 157 and 159; the proportion of m/z 157 and 159 peaks, approx. 3:1, suggested a chlorinated moiety. DCI MS/MS daughter-ion fragmentation of these peaks seems compatible with dimethylarsenic (cacodylic) acid and structures of the type Me₂As(O)Cl or Me₃As(OH)F. The refractory character of these fractions, however, cannot be explained by these structures. Further work with mixtures of halogen and arsenic species injected in the HPLC system showed that fluoride and iodide can shift DMA (dimethylarsenic) and TMAO (trimethylarsine oxide) to shorter retention times but not to R_f values similar to refractory arsenicals. These latter are attained by mixtures of sodium chloride + arsenobetaine, and sodium fluoride and chloride + arsenocholine. We suggest that peaks at m/z 139 and 157 correspond to fragments of a heavier refractory molecule mainly formed by halogenated betaines including chloroarsenobetaine and chloro- and fluoro-arsenocholine.     

Evidence of the Presence of Dimethylated,Trimethylated and "Refractory' Arsenic Compounds in Estuarine Salt-marsh Halophytes

Five species of halophytes were sampled in the salt marshes of the Tagus estuary, dried, ground and digested. They were further extracted with ethanol and the extracts passed through weak and strong cationic ion-exchange resins, purified through TLC and submitted to pyrolysis mass spectrometry and HPLC–ICP/MS. Arsenic content and hydride-forming arsenic species were verified, in each step, by GF–AA and HG–QFAA. A high content of arsenic was found in the samples of halophytes studied, both di- and tri-methylated arsenic compounds being present. A considerable fraction of this arsenic content seems to be refractory to hydride generation. Moreover, the arsenic fraction found seems to have the same ion-exchange behaviour as the refractory fractions formerly studied in estuarine water. A partial characterization of these structures by pyrolysis–GC–MS suggests the presence of arsenobetaine and arsenocholine compounds. Furthermore, HPLC–ICP/MS data seem to confirm the presence of these compounds. In addition, the latter hyphenated technique strongly suggests the presence of a number of other organoarsenicals including tetramethylarsonium (TMAs), trimethylarsine oxide (TMAO), cacodylate (DMA) and possibly an arsenosugar-type compound. © 1997 by John Wiley & Sons, Ltd.     

Tolerance,bioaccumulation and biotransformation of arsenic in freshwater prawn (Macrobrachium rosenbergii)

Tolerance bioaccumulation and biotransformation of arsenic compounds by a freshwater prawn (Macrobrachium rosenbergii) were investigated. M. rosenbergii was exposed to 10, 20, 30 and 35 μg As cm⁻³ of disodium arsenate [abbreviated as As(V)], 25, 50, 100 and 120 μg As cm⁻³ of methylarsonic acid (MMAA), or 100,200, 300 and 350 μg As cm⁻³ of dimethylarsinic acid (DMAA). Tolerances (50% lethal concentration: LC₅₀) of the prawn against As(V), MMAA, and DMAA were 30, 100, and 300 μg As cm⁻³, respectively. The prawn accumulated arsenic compounds directly from aqueous phase and biotransformed them in part. Both methylation and demethylation of the arsenicals were observed in vivo. Highly methylated and less toxic arsenicals were less accumulated in M. rosenbergii.     

Uptake and excretion of total inorganic arsenic by the freshwater alga Chlorella vulgaris

Arsenic-tolerant freshwater alga Chlorella vulgaris which had been collected from an arsenicpolluted environment were tested for uptake and excretion of inorganic arsenic. Approximately half the quantity of arsenic taken up by C. vulgaris was estimated to be adhered to the extraneous coat (10 wt %) of the cell. The remainder was bioaccumulated by the cell. Both adhered and accumulated arsenic concentrations increased with an increase in arsenic(V) concentration of the aqueous phase. Arsenic(V) accumulation was affected by the growth phse: arsenic was most actively accumulated when the cell was exposed to arsenic during the early exponential phase and then accumulation decreased with an increase in culture time exposed to arsenic. The alga grew well in the modified Detmer (MD) medium containing 1 mg As(III) dm^?3 and the growth curve was approximated by a ‘logistic equation’. Arsenic(III) was accumulated up to the second day of the culture time and arsenic(III) accumulation decreased with an increase in the culture time after that. Arsenic accumulation was also largely affected by various nutrients, especially by managanese, iron and phosphorus compounds. A modified MD medium with the three nutrients was proposed for the purpose of effective removal of arsenic from the aqueous phase. Using radioactive arsenate (Na₂H⁷⁴AsO₄), the arsenic accumulated was found to be readily excreted under conditions which were unfavourable for the multiplication of C. vulgaris.     

Preservation of arsenic species in water samples using phosphoric acid--limitations and long-term stability

The preservation of arsenic species in water samples is an indispensable method to avoid their changes during storage, if it is not possible to analyse them immediately. The aim of this investigation was to demonstrate the limitations of the suggested method by using phosphoric acid as a preservation agent. The samples remain stable for 3 months, even if they show evidence of high concentrations of iron or manganese. Critical is an increasing pH > 3. Theoretically, a precipitation of strengite (Fe₃(PO₄)₂) could occur, which should be avoided. Phosphoric acid with a final concentration of 10 mM is recommended as a preservation agent, combined with keeping the samples cool (6 °C) and dark. Filtration of samples before preservation may be carried out with respect to the analytical aim to distinguish between the total and soluble fraction (without colloids). It was shown that filtered and non-filtered samples can be preserved by utilising the above mentioned scheme.     

Bioaccumulation and excretion of arsenic compounds by a marine unicellular alga,polyphysa peniculus

Polyphysa peniculus was grown in artificial seawater in the presence of arsenate, arsenite, monomethylarsonate and dimethylarsinic acid. The separation and identification of some of the arsenic species produced in the cells as well as in the growth medium were achieved by using hydride generation–gas chromatography–atomic absorption spectrometry methodology. Arsenite and dimethylarsinate were detected following incubation with arsenate. When the alga was treated with arsenite, dimethylarsinate was the major metabolite in the cells and in the growth medium; trace amounts of monomethylarsonate were also detected in the cells. With monomethylarsonate as a substrate, the metabolite is dimethylarsinate. Polyphysa peniculus did not metabolize dimethylarsinic acid when it was used as a substrate. Significant amounts of more complex arsenic species, such as arsenosungars, were not observed in the cells or medium on the evidence of flow injection–microwave digestion–hydride generation–atomic absorption spectrometry methodology. Transfer of the exposed cells to fresh medium caused release of most cell–associated arsenicals to the surrounding environment.     

离子色谱-等离子体质谱联用测定热泉水样的砷形态

利用阴离子色谱与六极碰撞等离子体质谱联用的方法,在线同时测定水样的4种砷形态(As(Ⅴ),As(Ⅲ),MMA,DMA),并用于实际样品-热泉水中砷形态的测定.使用K2HPO4-KH2PO4为淋洗液等度淋洗,用Hamilton PRP-X100阴离子色谱柱分离,4种砷形态在7 min之内完全分离.调节淋洗液中K2HPO4与KH2PO4的比例可以优化峰的分离.地下水(含热泉水)基质、样品及淋洗液中的Cl-对砷形态的分离测定没有影响,淋洗液中的盐份在样品锥和截取锥上的积累对测定的影响很小.检出限分别为As(Ⅴ) 0.23 μg/L,As(Ⅲ) 0.30 μg/L,MMA 0.26 μg/L,DMA 0.54 μg/L.     

Refractory arsenic species in estuarine waters

Total digestion of estuarine water samples by dry ashing shows that a significant fraction of dissolved arsenic does not form hydrides with sodium tetrahydroborane (NaBH₄) and is therefore not detected by the normal hydride generation-atomic absorption analytical technique. It is also refractory to alkaline digestion with sodium hydroxide. Sequential ultrafiltration suggests a molecular weight below 210 for this new arsenic fraction. The magnitude and molecular weight of this fraction may open new perspectives on the biogeochemical cycling of arsenic. Ecological reasons can explain the discrepancy between these findings and previous research.