Biomethylation of arsenic and its excretion by the alga Chlorella vulgaris

Experimental results in this paper lead to the following conclusions. (1) Cell homogenates of Chlorella vulgaris adsorbed the inorganic arsenic compound Na₂HAsO₄ but no methylation of the arsenic occurred in vitro. (2) A small part of the arsenic bioaccumulated by C. vulgaris was methylated in vivo. The quantity of arsenic methylated in the cell increased with an increase of arsenic concentration in the medium. (3) When the arsenic-accumulating cells were transferred into arsenic-free media, the arsenic was excreted and the relative quantity of the methylated arsenic in the excrement was larger than that in the cell. (4) In the growth phase of C. vulgaris, a small fraction of the arsenic accumulated in the cell was first transformed to monomethyl and dimethyl-arsenic compounds during the early exponential phase, and after a short time a fraction was transformed to trimethylarsenic species.     

Biomethylation and biotransformation of arsenic in a freshwater food chain: Green alga (chlorella vulgaris)→shrimp (neocaridina denticulata)→killifish (oryzias iatipes)

Tolerance, bioaccumulation, biotransformation and excretion of arsenic compounds by the fresh–water shrimp (Neocaridina denticulata) and the killifish (Oryzias latipes) (collected from the natural environment) were investigated. Tolerances (LC₅₀) of the shrimp against disodium arsenate [abbreviated as As(V)], methylarsonic acid (MAA), dimethylarsinic acid (DMAA), and arsenobetaine (AB) were 1.5, 10, 40, and 150μg As ml^?1, respectively. N. denticulata accumulated arsenic from an aqueous phase containing 1 μg As ml^?1 of As(V), 10 μg As ml^?1 of MAA, 30 μg As ml^?1 of DMAA or 150 μg As ml^?1 of AB, and biotransformed and excreted part of these species. Both methylation and demethylation of the arsenicals were observed in vivo. When living N. denticulata accumulating arsenic was transferred into an arsenic–free medium, a part of the accumulated arsenic was excreted. The concentration of methylated arsenicals relative to total arsenic was higher in the excrement than in the organism. Total arsenic accumulation in each species via food in the food chain Green algae (Chlorella vulgaris) → shrimp (N. denticulata) → killifish (O. latipes) decreased by one order of magnitude or more, and the concentration of methylated arsenic relative to total arsenic accumulated increased successively with elevation in the trophic level. Only trace amounts of monomethylarsenic species were detected in the shrimp and fish tested. Dimethylarsenic species in alga and shrimp, and trimethylarsenic species in killifish, were the predominant methylated arsenic species, respectively.     

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.     

Lipophilic arsenic compound(s) in the liver of a tiger shark (Galeocerdo cuvier)

Homogenized aliquots (100 g) of the liver (8.4 kg, 5 m?g As g^?1) of a tiger shark (Galeocerdo cuvier) were extracted with chloroform/methanol, and the extracts purified by countercurrent extraction (hexane/87% ethanol), silica gel column chromatography (chloroform/methanol mixtures as mobile phases), and silica gel thin-layer chromatography (chloroform/methanol/acetic acid). The purified samples (24 mg arsenic g^?1) gave no ³¹P NMR signal, but gave ¹H and ¹³C NMR signals with similarities to those of dipalmitoylphosphatidic acid and salad on and also signals indicative of the presence of methylated arsenic compounds. The sample could contain a diacyl glyceride with a methylated arsenic group.     

Effect of cadmium on the accumulation of arsenic in a marine green alga,Dunaliellasp.

To investigate the effect of cadmium on the accumulation of arsenic by Dunaliella sp., the arsenic accumulated in the alga was determined as a function of time for coexistence of the algae with arsenic and cadmium, with batch methodology. Growth of Dunaliella sp. was affected by addition of arsenic (Na₂HAsO₄.7H₂O) and cadmium (CdCl.2.5H₂O). Growth inhibition of Dunaliella sp. was accelerated by coexistence of arsenic and cadmium. The content of arsenic in Dunaliella sp. became a maximum at 15 h after exposure. The arsenic content in the cells was influenced by addition of cadmium to the solution; the arsenic content in the alga derived from growth in a 10 mg As dm ^?3 solution decreased from 2.7 mg g^?1 in the absence of cadmium to 0.35 mg g^?1 for the addition of 100 mg Cd dm^?3. Dunaliella sp. accumulated cadmium in large quantities but, in conditions of coexistence with arsenic and cadmium, the cadmium content in cells decreased with an increase in the concentration of arsenic in the growth medium Cadmium accumulation by Dunaliella sp. was observed in dead cells although arsenic accumulation was not observed. About 85% of arsenic in the cells was in the water-soluble fraction. On the other hand, about 42% of cadmium in the cells was in the water-soluble fraction, and about 55% was in a fraction soluble in cold trichloroacetic acid.     

Methylated arsenic species in estuarine porewaters

Inorganic arsenic, monomethylarsenic and dimethylarsenic species have been observed in samples of sediment porewater collected from the Tamar Estuary in South-West England. Porewater samples were collected using in situ dialysis. The arsenic species were separated by hydride generation and concentrated by liquid nitrogen trapping, prior to analysis by directly coupled gas chromatography-atomic absorption spectroscopy. The predominant dissolved arsenic species present was inorganic arsenic (5-62 m?g dm^?3). However, this is the first time significant concentrations of methylated arsenic species have been quantified in estuarine porewaters (0.04–0.70 m?g dm^?3), accounting for between 1 and 4% of the total dissolved arsenic. The presence of methylated arsenic compounds in porewaters is attributed to in situ environmental methylation, although the possibility of methylated arsenic species being derived from biological debris cannot be excluded.     

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.     

Arsenic metabolism in a freshwater food chain: Blue–green alga (Nostoc sp.)→ shrimp (Neocaridina denticulata)→ carp (Cyprinus carpio)

Bioaccumulation and biomethylation of inorganic arsenic were investigated in a three-step fresh-water food chain consisting of an autotroph (blue- green alga: Nostoc sp.), a herbivore (shrimp: Neocaridina denticulata) and a carnivore (carp: Cyprinus carpio). The autotroph, herbivore and carnivore survived in arsenic-containing water below 1000, 2 and 60 mg As(V) dm^?3, respectively. Bioaccumulation of arsenate by Nostoc sp. was decreased with an increase in the nitrogen concentration of the medium. Arsenic(V) was accumulated from the water phase and part-methylated by the carp, as well as by the algae and shrimp. Arsenic was mostly accumulated in the gut of the carp. The predominant arsenical in the guts was the monomethylarsenic species. Arsenic accumulation via food in the above three-step food chain decreased by one order of magnitude and the relative concentration of methylated arsenic to the total arsenic accumulated increased successively with an elevation in the trophic level. When arsenicals were transferred via the food chain, no monomethylarsenic, or only a trace amount, was detected in the three organisms. Dimethylarsenic in the alga, both dimethyl- and trimethyl-arsenic in shrimp, and trimethyl-arsenic in carp, were the predominant methylated arsenic species, respectively.     

Metabolism of methylated arsenic compounds by arsenic-resistant bacteria (Klebsiella oxytoca and Xanthomonas sp.)

Two bacteria exhibiting resistance to toxic arsenic were isolated. These had been contaminated with arsenic in a Chlorella sp. culture medium containing arsenic. The two bacteria were identified as Klebsiella oxytoca and Xanthomonas sp., and grew well in a peptone medium at neutral pH at 30°C, reaching the stationary phase in ca 100h and 70h, respectively. The growth of the bacteria was not affected by arsenic(V) concentrations in the medium as high as 1000mg dm^?3. The bacteria bioaccumulated arsenic, a part of the arsenic being methylated. The bioaccumulation exhibited its peak around the turing point from the log phase to the stationary phase. The relative content of methylated arsenic in the excrement was greater than that in the bacterial cells. Adaptation treatment of inorganic arsenic caused an increase in the bioaccumulation of inorganic arsenic by K. oxytoca. Such a situation was not observed in the case of Xanthomonas sp. The bacteria also bioaccumulated methylated arsenic compounds, and demethylation of these species was observed. When the bacteria were killed by ethanol, arsenic was not taken up by the cells.     

Effects of arsenic on the organic component of the alga Dunaliella salina

The unicellular marine alga, Dunaliella salina 19/30 was grown in seawater containing an inorganic arsenic concentration (Na₂HAsO₄) up to 2000 mg dm^?3. The cells survived even at 5000 mg dm^?3. The arsenic concentration of the cells increased with an increase of the surrounding arsenic concentration. Arsenic in D. salina was also greatly affected by addition of phosphorus. The arsenic-tolerance behavior of D. salina seemed to suggest that the algae have a function to prevent accumulation of inorganic arsenic by increasing the β-carotene, fatty-acid (C_18:1, C_18:3) and water-extractable carbohydrate content in the cells. Arsenic accumulation also rose steadily with an increase in the nitrogen concentration in the medium.     

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.     

Determination of arsenic species in a freshwater crustacean Procambarus clarkii

The arsenic species present in samples of the crayfish Procambarus clarkii caught in the area affected by the toxic mine‐tailing spill at Aznalcóllar (Seville, Southern Spain) were analyzed. The total arsenic contents ranged between 1.2 and 8.5 µg g^?1 dry mass (DM). With regard to the different species of arsenic, the highest concentrations were for inorganic arsenic (0.34–5.4 µg g^?1 DM), whereas arsenobetaine, unlike the situation found in marine fish products, was not the major arsenic species (0.16 ± 0.09 µg g^?1 DM). Smaller concentrations were found of arsenosugars 1a (0.18 ± 0.11 µg g^?1 DM), 1b (0.077 ± 0.049 µg g^?1 DM), 1c (0.080 ± 0.089 µg g^?1 DM), and 1d (0.14 ± 0.13 µg g^?1 DM). The presence of two unknown arsenic species was revealed (U1: 0.058 ± 0.058 µg g^?1 DM; U2: 0.12 ± 0.12 µg g^?1 DM). No significant differences were seen with respect to the total arsenic contents between the sexes. However, significant differences in the total arsenic contents were revealed between the area affected by the spill and the area not affected, the contents being greater in the affected area. Copyright © 2002 John Wiley & Sons, Ltd.     

Methylation of inorganic arsenic by arsenic-tolerant freshwater algae

Five arsenic-resistant freshwater algae which had been isolated from an arsenic-polluted environment were studied for the biotransformation of arsenic compounds accumulated by them from the aqueous phase. The algal cells bioaccumulating arsenic were digested by 2 mol dm^?3 NaOH at 95°C, the As? C bonds except for As? CH₃ were cleaved by the treatment and the methylated arsenic compounds were reduced to the corresponding arsines by sodium borohydride (hydride generation). The arsines were chromatographically separated on the basis of their boiling-point difference and determined by atomic absorption spectrophotometry. Methylated arsenic compounds were found in all algal cells. The predominant arsenic species in the cells, however, were non-methylated arsenic compounds which were mainly present in the residue of a chloroform–methanol extract. The non-methylated arsenic compounds were found to be not present in the free inorganic arsenic substrate and to be bound strongly with proteins or polysaccharides in the cells. Methylated arsenic compounds were found mainly in the lipid-soluble fractions and the major form was a dimethylarsenic compound. Trimethyl- and monomethyl-arsenic compounds were detected but at very low level. The dimethylarsinic acid was not present in the free form in the lipid-soluble fraction and should be bound with a lipid molecule. It was also found that the accumulation of arsenic by Nostoc occurred only in living cells.     

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).     

Distribution of arsenic in the natural environment with emphasis on rocks and soils

Arsenic is ubiquitous in the environment. Although the average arsenic concentrations in rocks (～2 mg kg^?1), soils (～2 mg kg^?1), freshwater (～1 m?g dm^?3), seawater (～2 m?g dm^?3) and organisms is generally low, high arsenic concentrations in limited areas are not uncommon. Whereas terrestrial organisms appear not to accumulate arsenic, marine organisms effectively concentrate arsenic to levels thousand of times higher than in ocean waters. The geochemical cycle and mineralogy of arsenic are reviewed with some emphasis towards Japanese locations and arsenic concentrations (averages, ranges) found in samples from the lithosphere, pedosphere, hydrosphere and biosphere are tabulated and discussed.     

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30?g?g^?1) and productivity (0.19?g?L^?1?h^?1). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3?g?L^?1 resulted in high biomass production. The highest biomass concentration (21?g?L^?1), yield (0.45?g?g^?1) and productivity (0.31?g?L^?1?h^?1), as well as ribonucleotide production (13.13?mg?g^?1), were observed at 700?rpm and 0.5?vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.     

Bioaccumulation of Antimony by Chlorella vulgaris and the Association Mode of Antimony in the Cell

The bioaccumulation and excretion of antimony by the freshwater alga Chlorella vulgaris , which had been isolated from an arsenic-polluted environment, are described. When this alga was cultured in a medium containing 50 μg cm⁻³ of antimony(III) for 14 days, it was found that Chlorella vulgaris bioaccumulated antimony at concentrations up to 12 000 μg Sb g⁻¹ dry wt after six days' incubation. The antimony concentration in Chlorella vulgaris decreased from 2570 to 1610 μg Sb g⁻¹ dry wt after the cells were transferred to an antimony-free medium. We found that the excreted antimony consists of 40% antimony(V) and 60% antimony(III). This means that the highly toxic antimony(III) was converted to the less toxic antimony (V) by the living organism. Antimony accumulated in living Chlorella vulgaris cells was solvent-fractionated with chloroform/methanol (2:1), and the extract residue was fractionated with 1% sodium dodecyl sulfate (SDS). Gel-filtration chromatography of the solubilized part showed that antimony was combined with proteins whose molecular weight was around 4×10⁴ in the antimony-accumulated living cells. © 1997 by John Wiley & Sons, Ltd.     

Blue light induces arsenate uptake in the protist Thraustochytrium

The effects of light on arsenic accumulation of Thraustochytrium CHN‐1 were investigated. Thraustochytrium CHN‐1, when exposed to blue light from light‐emitting diodes (LEDs), accumulated arsenate added to its growth medium to a much greater extent than Thraustochytrium cells exposed to fluorescent or red light, or when cultured in the dark. Arsenic compounds in Thraustochytrium CHN‐1 were analyzed by high‐performance liquid chromatography, with an inductively coupled plasma mass spectrometer serving as an arsenic‐specific detector. Arsenate, arsenite, monomethylarsonic acid (MMAA), dimethylarsinic acid (DMAA) and arsenosugar were identified. The order of arsenic species in Thraustochytrium CHN‐1 was arsenic(V)> arsenic(III)> MMAA > DMAA at an arsenic concentration of 10 mg dm^?3 in the medium in blue LED light. As it is known that blue light induces the synthesis of certain metabolites in plants and microorganisms, this indicates that the accumulation of arsenic is an active metabolic process. Copyright © 2005 John Wiley & Sons, Ltd.     

Accumulation of Arsenic by Rhaphydophyceae Chattonella antiqua (Hada) Ono

Rhaphydophyceae Chattonella antiqua (Hada) Ono was grown in seawater containing an arsenic concentration up to 50 mg dm⁻³, and survived even at 200 mg dm⁻³. The arsenic content increased with an increase of the surrounding arsenic, iron and manganese concentrations. However, arsenic accumulation was unaffected by phosphorus concentration. Also, arsenic content in C. antiqua decreased at a selenium concentration of up 20 mg dm⁻³, and was reduced by the addition of antimony. In the living cells, about 52% of the arsenic which accumulated in each cell was found in the intracellular fraction, 27% in the lipid fraction, and 21% in the cell wall fraction.     

Observations on toxicologically relevant arsenic in urine in adult offspring of families with Balkan Endemic Nephropathy and controls by batch hydride generation atomic absorption spectrometry

The aim of this study was to develop a method for the characterization of internal exposure to arsenic, which is thought to play a role in the development of a kidney disease, known as Balkan Endemic Nephropathy, typical for a district in Bulgaria, and to investigate whether the As body burden differs in the offspring versus control individuals. For this case study, an analytical procedure for the determination of toxicologically relevant arsenic (the sum of arsenite, arsenate, monomethylarsonate, and dimethylarsinate) in urine by batch-type hydride generation atomic absorption spectrometry was developed. Optimization experiments for levelling off the sensitivity of inorganic arsenic and its mono- and dimethylated species in dilute HCl–L-cysteine medium were performed. The limit of detection for hydride forming arsenic fraction was 0.5?ng As, i.e. 0.25?µg?L^?1 in 10?mL of 1?+?4 v/v diluted urine. The relative standard deviation was typically 1.5–1.8% for aqueous solution and 2–6% for urine samples at 1.0?µg?L^?1 As. The sample throughput rate was 15?h^?1. No statistical correlation and cross-correlation between individuals case-control and sex at 95% confidence were found: controls (n?=?99), mean 3.5?±?2.1 (SD), range 0.9–10.4, median 3.0?µg?L^?1 As and cases (n?=?102), mean 3.6?±?2.2 (SD), range 0.5–11.0, median 3.2?µg?L^?1 As. On the basis of this study, arsenic can be excluded as a factor involved in BEN development.