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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Biotransformation of arsenate to arsenosugars by Chlorella vulgaris

Chlorella vulgaris was cultivated in a growth medium containing arsenate concentration of <0.01, 10, 100 and 1000 mg l^?1. Illumination was carried out in 12 h cycles for 5 days. The health status of the culture was monitored by continuous pH and dissolved oxygen (DO) readings. Destructive sampling was used for the determination of biomass, chlorophyll, total arsenic and arsenic species. The chlorophyll a content, the DO and pH cycles were not significantly different for the different arsenate concentrations in the culture. In contrast, biomass production was significantly (p < 0.05) increased for the arsenic(V) treatment at 1000 mg l^?1 compared with 100 mg l^?1. The arsenic concentration in the algae increased with the arsenate concentration in the culture. However, the bioconcentration factor decreased a hundred‐fold with increase of arsenate from the background level to 1000 mg l^?1. The arsenic species were identified by using strong anion‐exchange high‐performance liquid chromatography–inductively coupled plasma mass spectrometry analysis after methanol/water (1 : 1) extraction. The majority (87–100%) of the extractable arsenic was still arsenate; arsenite was found to be between 1 and 6% of total extractable arsenic in the algae. In addition to dimethylarsinic acid, one unknown arsenical (almost co‐eluting with methylarsonic acid) and three different arsenosugars have been identified for the first time in C. vulgaris growing in a culture containing a mixture of antibiotics and believed to be axenic. The transformation to arsenosugars in the algae is not dependent on the arsenate concentration in the culture and varies between 0.2 and 5% of total accumulated arsenic. Although no microbiological tests for bacterial contamination were made, this study supports the hypothesis that algae, and not associated bacteria, produce the arsenosugars. Copyright © 2003 John Wiley & Sons, Ltd.     

Arsenic accumulation by arsenic-tolerant freshwater blue-green alga (Phormidium sp.)

Accumulation, biomethylation and excretion of arsenic by the arsenic-tolerant freshwater blue–green alga, Phormidium sp., which had been isolated from an arsenic-polluted environment, were investigated. The cellular growth curves were in fair agreement with a ‘logistic curve’ equation. The growth increased with an increase in the surrounding arsenic concentration up to 100 m?g g^?1. The cells survived even at 7000 m?g g^?1. The arsenic concentration of the cells increased with an increase of the surrounding arsenic concentration up to 7000 m?g g^?1. Phosphorus concentrations in the medium affected the growth and arsenic accumulation. No arsenic was accumulated by cells killed by ethanol. The arsenic was methylated to the extent of 3.2% of the total arsenic accumulated. When the cells were transferred into an arsenic-free medium, 85% of the arsenic accumulated was excreted; 58% of the excreted arsenic was in methylated form implying extensive methylation in the arsenic-free medium.     

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.     

Disruption of <Emphasis Type="BoldItalic">Chlorella vulgaris</Emphasis> Cells for the Release of Biodiesel-Producing Lipids: A Comparison of Grinding,Ultrasonication, Bead Milling,Enzymatic Lysis,and Microwaves

A comparative evaluation of different cell disruption methods for the release of lipids from marine Chlorella vulgaris cells was investigated. The cell growth of C. vulgaris was observed. Lipid concentrations from different disruption methods were determined, and the fatty acid composition of the extracted lipids was analyzed. The results showed that average productivity of C. vulgaris biomass was 208 mg L⁻¹ day⁻¹. The lipid concentrations of C. vulgaris were 5%, 6%, 29%, 15%, 10%, 7%, 22%, 24%, and 18% when using grinding with quartz sand under wet condition, grinding with quartz sand under dehydrated condition, grinding in liquid nitrogen, ultrasonication, bead milling, enzymatic lysis by snailase, enzymatic lysis by lysozyme, enzymatic lysis by cellulose, and microwaves, respectively. The shortest disruption time was 2 min by grinding in liquid nitrogen. The unsaturated and saturated fatty acid contents of C. vulgaris were 71.76% and 28.24%, respectively. The extracted lipids displayed a suitable fatty acid profile for biodiesel [C16:0 (~23%), C16:1 (~23%), and C18:1 (~45%)]. Overall, grinding in liquid nitrogen was identified as the most effective method in terms of disruption efficiency and time.     

Accumulation of arsenic(III) by chlorella vulgaris

Arsenic occurs naturally in the environment and also through agricultural and industrial pollution. Since arsenic species show different toxicities, it is important to be able to separate them. Methods using microorganisms are being applied increasingly to remove metal ions and different metal species from aqueous solutions. Accumulation of As(III) by Chlorella vulgaris algae was studied, including various factors that influence on accumulation capacity, e.g. pretreatment of the algae (live, dry and lyophilized algae), temperature (4, 22, 37 and 100 °C), pH and exposure time of the algae to arsenic solutions. The pH appears to be the most critical factor, probably due to the species presenting different charges with pH variation. For arsenic species determination, hydride generation atomic absorption spectrometry (HG–AAS) was employed. Copyright © 1999 John Wiley & Sons, Ltd.     

Differential preconcentration of arsenic(III) and arsenic(V) with thionalide loaded on silica gel

2-Mercapto-N-2-naphtylacetamide (thionalide) on silica gel is used for differential preconcentration of μg l^?1 levels of arsenic(III) and arsenic(V) from aqueous solution. In batch experiments, arsenic(III) was quantitatively retained on the gel from solutions of pH 6.5–8.5, but arsenic(V) and organic arsenic compounds were not retained. The chelating capacity of the gel was 5.6 μmol g^?1 As(III) at pH 7.0. Arsenic retained on teh column was completely eluted with 25 ml of 0.01 M sodium borate in 0.01 M sodium hydroxide containing 10 mg l^?1 iodine (pH 10). The arsenic was determined by silver diethyldithiocarbamate spectrophotometry. Arsenic(V) was subsequently determined after reduction to arsenic(III) with sulphite and iodide. Arsenic(III) and arsenic(V) in sea water are shown to be < 0.12 and 1.6 μg l^?1, respectively.     

Arsenic Compounds in Terrestrial Organisms II: Arsenocholine in the Mushroom Amanita muscaria

Arsenic compounds were identified and quantified in the mushroom Amanita muscaria, collected close to a facility that had roasted arsenic ores. The powdered dried mushrooms were extracted with methanol/water (9:1), the extracts were concentrated and the concentrates were dissolved in water. The resulting solutions were chromatographed on anion-exchange, cation-exchange and reversed- phase columns. Arsenic was detected on-line with an ICP–MS detector equipped with a hydraulic high-pressure nebulizer. Arsenite, arsenate, dimethylarsinic acid and the tetramethylarsonium cation were minor arsenic compounds (∼2% each of the total 22 mg kg⁻¹ dry mass), and arsenobetaine, arsenocholine (∼15% each) and several unidentified arsenic compounds (∼60%) were the major arsenic compounds in Amanita muscaria. The presence of arsenocholine (detected for the first time in a terrestrial sample) was ascertained by matching retention times in the anion-exchange, cation- exchange and reversed-phase chromatograms with the retention time of synthetic arsenocholine bromide and chromatographing extracts spiked with arsenocholine bromide. © 1997 John Wiley & Sons, Ltd.     

Extraction and Spectrophotometric Determination of Arsenic in the Environment

Abstract

A simple, sensitive and selective method for the determination of microamounts of arsenic (III) in the environment is described. Arsenic forms a yellow coloured complex with N-phenylbenzo-hydroxamic acid (PBHA) at pH 4.5-5.2 which can be extracted from chloroform. The effective molar absorptivity of As-PBHA extract is 1.1 × 1mol^?1cm^?1 at 410 nm. Many common ions associated with arsenic do not interfere. The effect of pH, reagent concentration and solvent is described. The arsenic in trace quantities is estimated in the industrial effluents, soil and glass samples.     

Speciation analysis to unravel the soil-to-plant transfer in highly arsenic-contaminated areas in Cornwall (UK)

Two areas near derelict calciners in Cornwall (UK) were chosen to study the uptake of arsenic from arsenic-contaminated soil into indigenous plants (heather, Calluna vulgaris; blackberry, Rubus ulmifulmus; gorse, Ulex europaeus). With total arsenic concentrations in soil ranging from 1240 to 2860 mg kg^?1 at Site 1 (Tuckingmill), no adverse effects on the growth of the plants studied were observed. Very low soil-to-plant transfer factors (0.01 to 0.03) were found although they were much higher when the extractable soil arsenic concentrations were taken into account (0.1 to 1.1). In the central dump area at Site 2 (Bissoe, 9.78% [w/w] arsenic in soil), the only plant to grow was heather, although it was severely impaired. However, heather was thriving at the edge of the dump where higher soil arsenic concentrations were found (10.32% [w/w]), indicating that arsenic is not a growth-limiting factor in itself. Soil-to-plant transfer factors in the range 2 × 10^?5–9 × 10^?4 confirm that arsenic is indeed effectively excluded from uptake, even taking into account extractable soil arsenic concentrations (9 × 10^?4–1.2 × 10^?2).

Extraction of bioavailable arsenic from soil using 0.05 mol L^?1 ammonium sulphate yielded recoveries from 1.18 to 3.34% of the total arsenic, predominantly in the form of arsenate. Extraction of arsenic and its metabolites from plants was achieved with water or a water/methanol mixture yielding recoveries up to 22.4% of the total arsenic, with arsenite and arsenate the predominant arsenic species and a minor fraction consisting of methylarsonic acid, dimethylarsinic acid and trimethylarsine oxide. The identity of the remainder of the non-extractable arsenic species still has to be revealed. Although the data suggest that higher plants synthesise organoarsenic compounds it cannot be excluded that symbiotic organisms have synthesised these compounds.     

Urinary arsenic species in an arsenic‐affected area of West Bengal,India (part III)

Arsenic contamination of groundwater has long been reported in the Mushidabad district of West Bengal, India. We visited 13 arsenic‐affected families in the Makrampur village of the Beldanga block in Mushidabad during 18–21 December 2001 and collected five shallow tubewell‐water samples used general household purposes, four deep tubewell‐water samples used for drinking and cooking purposes, and 44 urine samples from those families. The arsenic concentrations in the five shallow tubewell‐water samples ranged from 18.0 to 408.4 ppb and those in the four deep tubewell‐water samples were from 5.2 to 9.6 ppb. The average arsenite (arsenic(III)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA) and arsenate (arsenic(V)) in urine were 28.7 ng mg^?1, 168.6 ng mg^?1, 25.0 ng mg^?1 and 4.6 ng mg^?1 creatinine respectively. The average total arsenic was 227.0 ng mg^?1 creatinine. On comparison of the ratio of (MMA + DMA) to total arsenic, the average proportion was 86.7 ± 9.2% (mean plus/minus to residual standard deviation, n = 43). The exception was data for one boy, whose proportion was 8.0%. One woman excreted the highest total arsenic, at 2890.0 ng mg^?1 creatinine. When using 43 of the urine samples (the exception being the one sample obtained from the boy) there were significantly positive correlations (p < 0.01) between arsenic(III) and MMA, between arsenic(III) and DMA and between MMA and DMA. Copyright © 2005 John Wiley & Sons, Ltd.