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.     

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.     

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.     

Organoarsenic compounds in plants and soil on top of an ore vein

Plants and soil collected above an ore vein in Gasen (Austria) were investigated for total arsenic concentrations by inductively coupled plasma mass spectrometry (ICP‐MS). Total arsenic concentrations in all samples were higher than those usually found at non‐contaminated sites. The arsenic concentration in the soil ranged from ∼700 to ∼4000 mg kg⁻¹ dry mass. Arsenic concentrations in plant samples ranged from ∼0.5 to 6 mg kg⁻¹ dry mass and varied with plant species and plant part. Examination of plant and soil extracts by high‐performance liquid chromatography–ICP‐MS revealed that only small amounts of arsenic (<1%) could be extracted from the soil and the main part of the extractable arsenic from soil was inorganic arsenic, dominated by arsenate. Trimethylarsine oxide and arsenobetaine were also detected as minor compounds in soil. The extracts of the plants (Trifolium pratense, Dactylis glomerata, and Plantago lanceolata) contained arsenate, arsenite, methylarsonic acid, dimethylarsinic acid, trimethylarsine oxide, the tetramethylarsonium ion, arsenobetaine, and arsenocholine (2.5–12% extraction efficiency). The arsenic compounds and their concentrations differed with plant species. The extracts of D. glomerata and P. lanceolata contained mainly inorganic arsenic compounds typical of most other plants. T. pratense, on the other hand, contained mainly organic arsenicals and the major compound was methylarsonic acid. Copyright © 2002 John Wiley & Sons, Ltd.     

Determination of Arsenic in Algae – Results of an Interlaboratory Trial: Determination of Arsenic Species in the Water-Soluble Fraction

Seven algae samples, five purchased from food stores and two reference algae (BCR 279 Sea Lettuce) were distributed as blind samples to 13 laboratories from which five labs attempted a full characterisation of the water-soluble fraction with respect to their arsenic species. The extraction efficiency is largely dependant on the algae and varied from 3% to 96%. Besides inorganic arsenic (mainly as As^(V)) DMA^(V) and, in particular, several arsenosugars were identified in all samples. From the five labs, three labs gave agreeable results in respect of the arsenic species identification and its quantification, although different chromatographic methods were used. Different Hijiki samples seem to contain largely different arsenic concentration (67–113 mg As/kg) which may also have an influence on the distribution of inorganic arsenic and arsenosugars.     

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.     

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.     

Ion chromatography–inductively coupled plasma mass spectrometry determination of arsenic species in marine samples

Arsenic speciation analysis in marine samples was performed using ion chromatography (IC) with inductively coupled plasma mass spectrometry (ICP‐MS) detection. The separation of eight arsenic species, viz. arsenite, monomethyl arsonic acid, dimethylarsinic acid, arsenate, arsenobetaine, tetramethylarsine oxide, arsenocholine and tetramethylarsonium ion was achieved on a Dionex AS4A (weaker anion exchange column) by using a nitric acid pH gradient eluent (pH 3.3 to 1.3). The entire separation was accomplished in 12 min. The detection limits for the eight arsenic species by IC–ICP‐MS were in the range 0.03–1.6 µ g l^?1, based on 3σ of the blank response (n = 6). The repeatability and day‐to‐day reproducibility were calculated to be less than 10% (residual standard deviation) for all eight species. The method was validated by analyzing a certified reference material (DORM‐2, dogfish muscle) and then successfully applied to several marine samples, e.g. oyster, fish muscle, shrimp and marine algae. The low power microwave digestion was employed for the extraction of arsenic from seafood products. Copyright © 2004 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.     

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.     

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.     

Biodegradation of arsenosugars in marine sediment

In the marine environment, arsenic accumulates in seaweed and occurs mostly in the form of arsenoribofuranosides (often called arsenosugars). This study investigated the degradation pathways of arsenosugars from decaying seaweed in a mesocosm experiment. Brown seaweed (Laminaria digitata) was placed on top of a marine sediment soaked with seawater. Seawater and porewater samples from different depths were collected and analysed for arsenic species in order to identify the degradation products using high‐performance liquid chomatography–inductively coupled plasma mass spectrometry. During the first 10 days most of the arsenic found in the seawater and the shallow sediment is in the form of the arsenosugars released from the seaweed. Dimethylarsenoylethanol (DMAE), dimethylarsinic acid (DMA(V)) and, later, monomethylarsonic acid (MMA(V)) and arsenite and arsenate were also formed. In the deeper anaerobic sediment, the arsenosugars disappear more quickly and DMAE is the main metabolite with 60–80% of the total arsenic for the first 60 days besides a constant DMA(V) contribution of 10–20% of total soluble arsenic. With the degradation of the soluble DMAE the solubility of arsenic decreases in the sediment. The final soluble degradation products (after 106 days) were arsenite, arsenate, MMA(V) and DMA(V). No arsenobetaine or arsenocholine were identified in the porewater. Copyright © 2005 John Wiley & Sons, Ltd.     

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.     

Conversion of arsenite and arsenate to methylarsenic and dimethylarsenic compounds by homogenates prepared from livers and kidneys of rats and mice

Pooled livers and pooled kidneys from rats or mice were homogenized and spiked with arsenite or arsenate in the concentration range 1.3–20 μmol dm^?3. Methylarsenic and dimethylarsenic compounds were determined by the hydride generation technique in the homogenates after a 90 min incubation at 37°C. The rat homogenates methylated arsenite and arsenate more efficiently than the mouse homogenates. Monomethylated arsenic was present in larger amounts than dimethylated arsenic in the rat homogenates. In the absence of reduced glutathione (GSH), no methylation occurred. Addition of GSH promoted monomethylation and dimethylation, whereas dithiothreitol and mercaptoethanol (10 mmol dm^?3) fostered only monomethylation. The amounts of monomethylated arsenic in the rat liver homogenates increased with increasing arsenite concentration (1.3–20 μmol dm^?3) however, the percentage of arsenic that had been methylated decreased. A similar trend, but with much less monomethylarsenic formed, was observed for arsenate-spiked homogenates. Rat kidney homogenates methylated arsenite and arsenate to a much smaller extent than rat liver homogenates. The K_m values for the monomethylation in rat liver homogenates were found to be 5.3 μmol dm^?3 for arsenite and 59 μmol dm^?3 for arsenate.     

Determination of arsenic species and arsenosugars in marine samples by HPLC–ICP–MS

Arsenic-speciation analysis in marine samples was performed by high-pressure liquid chromatography (HPLC) with ICP–MS detection. Separation of eight arsenic species—As^III, MMA, DMA, As^V, AB, TMAO, AC and TeMAs⁺—was achieved on a C₁₈ column with isocratic elution (pH 3.0), under which conditions As^III and MMA co-eluted. The entire separation was accomplished in 15 min. The HPLC–ICP–MS detection limits for the eight arsenic species were in the range 0.03–0.23 μg L⁻¹ based on 3σ for the blank response (n=5). The precision was calculated to be 2.4–8.0% (RSD) for the eight species. The method was successfully applied to several marine samples, e.g. oysters, fish, shrimps, and marine algae. Low-power microwave digestion was employed for extraction of arsenic from seafood products; ultrasonic extraction was employed for the extraction of arsenic from seaweeds. Separation of arsenosugars was achieved on an anion-exchange column. Concentrations of arsenosugars 2, 3, and 4 in marine algae were in the range 0.18–9.59 μg g⁻¹. This paper was presented at the European Winter Conference 2005     

Effects of arsenobetaine,a major organic arsenic compound in seafood,on the maturation and functions of human peripheral blood monocytes,macrophages and dendritic cells

We examine the in vitro immunotoxicity of synthetically pure arsenobetaine [AsBe; trimethyl (carboxymethyl) arsonium zwitterion], which is a major organic arsenic compound in seafood, on various human immune cells, such as peripheral blood monocytes, monocyte‐derived macrophages and monocyte‐derived dendritic cells (DCs). In particular, we examine the differentiation of monocytes into macrophages or DCs by comparing the effects of AsBe with those pentavalent inorganic arsenate. AsBe neither enhanced nor inhibited the differentiation of human monocytes into macrophages or DCs, and also did not affect their various immune functions. Furthermore, AsBe had no cytolethality in monocyte‐derived macrophages or DCs even at a concentration of 5 mmol l⁻¹. In contrast, inorganic arsenate showed strong cytolethality in these human immune cells in vitro at micromolar concentrations. These data indicate that the organic arsenic compound AsBe in seafood has no in vitro immunotoxicity in human immune cells. Copyright © 2004 John Wiley & Sons, Ltd.     

Effects of iron on the degradation of triphenyltin by pyoverdins isolated from Pseudomonas chlororaphis

The yellow compound pyoverdin was isolated from the bacteria Pseudomonas chlororaphis, isolated from mud in Japan. A study of the effects of iron, phosphorus, manganese and zinc on degradation of triphenyltin (TPT) by pyoverdin (20 mg) was carried out in distilled water (30 ml) containing 6 µg l^?1 concentration of TPT at 20 °C for 48 or 96 h. The organotins in water were analyzed by gas chromatograph–mass spectrometry in the selected ion mode. The degradation of TPT by pyoverdin decreased with increase of phosphorus at 0–35 mg l^?1 and Fe‐EDTA at 0–2 mg l^?1 concentrations. Also, degradation of diphenyltin by pyoverdin decreased with increase of Mn‐EDTA at 0–1 mg l^?1 and Zn‐EDTA at 0–1 mg l^?1. On the other hand, degradation of TPT by pyoverdin was found to be unaffected by manganese and zinc in water. Copyright © 2002 John Wiley & Sons, Ltd.     

Transformation of arsenic(V) by the fungus Fusarium oxysporum melonis isolated from the alga Fucus gardneri

A fungus isolated from the macroalga Fucus gardneri was identified by using 28S rDNA sequence analysis, 99% similarity match, as Fusarium oxysporum meloni. The fungus was exposed to arsenic(V) (500 ppb) in artificial seawater to investigate the possibility that the fungus is the source of the metabolic activity that results in the presence of arsenosugars in the macroalga. High‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry was used to identify the arsenic species in the fungus, and in the growth medium. The fungus was able to accumulate arsenic(V) and an increase in arsenite and dimethylarsinate was also observed. Some reduction of arsenate led to a small increase of arsenite in the growth medium. The fungus does not seem to be involved with the accumulation of arsenosugars by the Fucus. Copyright © 2002 John Wiley & Sons, Ltd.     

Comparison of mild extraction procedures for determination of plant-available arsenic compounds in soil

In this work three mild extraction agents for determination of plant-available fractions of elements in soil were evaluated for arsenic speciation in soil samples. Pepper (Capsicum annum, L.) var. California Wonder was cultivated in pots, and aqueous solutions of arsenite, arsenate, methylarsonic acid, and dimethylarsinic acid, at a concentration of 15 mg As kg^–1 soil, were added at the beginning of the experiment. Control pots (untreated) were also included. Deionized water, 0.01 mol L^–1 CaCl₂, and 0.05 mol L^–1 (NH₄)₂SO₄ were used to extract the plant-available fraction of the arsenic compounds in soil samples collected during the vegetation period of the plants. Whereas in control samples the extractable arsenic fraction did not exceed 1% of total arsenic content, soil amendment by arsenic compounds resulted in extraction of larger amounts, which varied between 1.4 and 8.1% of total arsenic content, depending on soil treatment and on the extracting agent applied. Among arsenic compounds determined by HPLC–ICPMS arsenate was predominant, followed by small amounts of arsenite, methylarsonic acid, and dimethylarsinic acid, depending on the individual soil treatment. In all the experiments in which methylarsonic acid was added to the soil methylarsonous acid was detected in the extracts, suggesting that the soil bacteria are capable of reducing methylarsonic acid before a further methylation occurs. No significant differences were observed between analytical data obtained by using different extraction procedures.     

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.