Optimization of the extraction for the determination of arsenic species in plant materials by high-performance liquid chromatography coupled with hydride generation atomic fluorescence spectrometry

A study has been conducted for the separation and the determination of arsenic species in plants using high-performance liquid chromatography–hydride generation atomic fluorescence spectrometry with emphasis on sample extraction procedures. Various extraction solvents have been applied to extract arsenic species from plants in order to investigate the uptake, transfer and accumulation processes of arsenic. The method was optimized with respect to the selection of extraction solvent, extraction time and the number of extraction steps. The analytical procedure has been validated by analyzing standard reference material GBW 82301 (peach leaves) and successfully used for the arsenic speciation in plants grown on contaminated soil near an arsenic mine. Inorganic arsenic, especially arsenate (As(V)) appears to be the major component in plants and organic arsenic species of monomethylarsenic acid and dimethylarsenic acid were detected at low concentrations.     

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.     

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.     

Extraction and speciation of arsenic in plants grown on arsenic contaminated soils

A sequential arsenic extraction method was developed that yielded extraction efficiencies (EE) that were approximately double those using current methods for terrestrial plants. The method was applied to plants from two arsenic contaminated sites and showed potential for risk assessment studies. In the method, plants were extracted first by 1:1 water-methanol followed by 0.1 M hydrochloric (HCl) acid. Total arsenic in plant and soil samples collected from contaminated sites was mineralized by acid digestion and detected by inductively coupled plasma-atomic emission spectrometry (ICP-AES) and hydride generation-atomic absorption spectrometry (HG-AAS). Arsenic speciation was done by high performance liquid chromatography coupled with HG-AAS (HPLC-HGAAS) and by HPLC coupled with ICP-mass spectrometry (HPLC-ICP-MS). Spike recovery experiments with arsenite (As(III)), arsenate (As(V)), methylarsonic acid (MA) and dimethylarsinic acid (DMA) showed stability of the species in the extraction processes. Speciation analysis by X-ray absorption near edge spectroscopy (XANES) demonstrated that no transformation of As(III) and As(V) occurred due to sample handling. Dilute HCl was efficient in extracting arsenic from plants; however, extraction and determination of organic species were difficult in this medium. Sequential extraction with 1:1 water-methanol followed by 0.1 M-HCl was most useful in extracting and speciating both organic and inorganic arsenic from plants. Trace amounts of MA and DMA in plants could be detected by HPLC-HGAAS aided by the process of separation and preconcentration of the sequential extraction method. Both organic and inorganic arsenic compounds could be detected simultaneously in synthetic gastric fluid extracts (GFE) but EEs by this method were lower than those of the sequential method. The developed sequential method was shown to be reliable and applicable to various terrestrial plants for arsenic extraction and speciation.     

Uptake of lead and arsenic in food plants grown in contaminated soil from Barber Orchard, NC

Elevated levels of heavy metals in soil may allow uptake of these toxic species in food plants. Barber Orchard, Haywood County, NC has been designated a U.S. EPA Superfund site, primarily because of elevated levels of lead and arsenic. In this work, carrots, lettuce, and tomatoes were cultivated in a greenhouse in control soil and soil obtained from Barber Orchard. The resulting samples were then analyzed for lead and arsenic using inductively coupled plasma optical emission spectrometry (ICP-OES). Except for carrot roots grown in the contaminated soil, the concentrations of lead and arsenic in the plants were below the ICP-OES detection limit. The concentration of lead in carrot roots was 20 ± 11 μg/g, which represents a bioconcentration factor (BCF) of 0.03.     

Speciation of arsenic in a contaminated soil by solvent extraction

Soil collected from a disused cattle dip in northern New South Wales was studied with the aim of developing an inexpensive, yet effective method for quantitative determination of arsenic(III), arsenic(V) and total organic arsenic in a contaminated soil. Hydrochloric acid extractions were used as a method for removal of the arsenic from the soil in a form suitable for speciation. It was found that the extraction efficiency varied with the ratio of soil to acid, and the concentration of the acid. Arsenic(III), as arsenic trichloride, was selectively extracted into chloroform from a solution highly concentrated in hydrochloric acid. This was followed by back-extraction of the arsenic into water. Total inorganic arsenic was determined in a similar manner after the reduction of arsenic(V) to the trivalent state with potassium iodide. Arsenic(V) was determined by the difference between the results for arsenic(III) and total inorganic arsenic. All analyses for the various arsenic species were performed by hydride generation-atomic absorption spectroscopy; concentrations of total arsenic in the soil were confirmed using X-ray fluorescence spectrometry. It was found that all the arsenic in the soil was present as inorganic arsenic in the pentavalent state. This reflects the ability of arsenic to interchange between species, since the original species in cattle dipping solution is arsenic(III).     

Speciation and determination of bioavailable arsenic species in soil samples by one‐step solvent extraction and high‐performance liquid chromatography with inductively coupled plasma mass spectrometry

A new analytical method was developed to determine the bioavailable arsenic species (arsenite, arsenate, monomethylarsonic acid, and dimethylarsonic acid) in soil samples using high‐performance liquid chromatography with inductively coupled plasma mass spectrometry. Bioavailable arsenic was extracted with ammonium phosphate buffer by a simplified one‐step solvent extraction procedure. To estimate the effect of variables on arsenic extraction, a two‐level Plackett–Burman factorial design was conducted to screen the significant factors that were further investigated by a separate univariate approach. The optimum conditions were confirmed by compromising the stability of arsenic species and the extraction efficiency. The concentration of arsenic species was determined in method blank and soil‐certified reference materials both spiked with standard solutions of arsenic species. All the target arsenic species were stable during the whole extraction procedure. Furthermore, the proposed method was applied to release bioavailable arsenic from contaminated soil samples, showing that the major arsenic species in soil samples were inorganic arsenic: arsenite and arsenate, of which the latter was dominant.     

CZE for the speciation of arsenic in aqueous soil extracts

We developed two separation methods using CZE with UV detection for the determination of the most common inorganic and methylated arsenic species and some phenylarsenic compounds. Based on the separation method for anions using hydrodynamic sample injection the detection limits were 0.52, 0.25, 0.27, 0.12, 0.37, 0.6, 0.6, 1.2 and 1.0 mg As/L for phenylarsine oxide (PAO), p-aminophenylarsonic acid (p-APAA), o-aminophenylarsonic (o-APAA), phenylarsonic acid (PAA), 4-hydroxy-3-nitrobenzenearsonic acid (roxarsone), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenite or arsenious acid (As(III)) and arsenate (As(V)), respectively. These detection limits were improved by large-volume sample stacking with polarity switching to 32, 28, 14, 42, 22, 27, 26 and 27 microg As/L for p-APAA, o-APAA, PAA, roxarsone, MMA, DMA, As(III) and As(V), respectively. We have applied both methods to the analysis of the arsenic species distribution in aqueous soil extracts. The identification of the arsenic species was validated by means of both standard addition and comparison with standard UV spectra. The comparison of the arsenic species concentrations in the extracts determined by CZE with the total arsenic concentrations measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) indicated that CZE is suited for the speciation of arsenic in environmental samples with a high arsenic content. The extraction yield of phenylarsenic compounds from soil was derived from the arsenic concentrations of the aqueous soil extracts and the total arsenic content of the soil determined by ICP-AES after microwave digestion. We found that 6-32% of the total amount of arsenic in the soil was extractable by a one-step extraction with water in dependence on the type of arsenic species.     

Occurrence of methylated arsenic species in parts of plants growing in polluted soils

Arsenic compounds were determined in extracts of branches, leaves and roots from plants growing in a mining contaminated area. The selected species were Dryopteris filix-max, Quercus pubescens, Dipsacus fullonum, Alnus glutinosa, Buxus sempervirens and Brachythecium cf. reflexum. Total arsenic content in the subsamples was analysed by ICPMS after acidic digestion. In general, concentrations in the plant parts followed the gradient roots?>?branches?>?leaves indicating that they are arsenic-resistant plants. Arsenic species were determined in water/methanol (9?+?1, v/v) extracts by HPLC-ICPMS. Different levels of organoarsenicals were found depending on plant part and plant species. Higher percentages of organoarsenic compounds were recorded in branches and leaves (up to 35% in the boxtree sample), than in roots (0.7–5.2% in the same plant species). The absence of organic arsenic species in the soil where the plants were collected and the low levels of organoarsenicals found in the roots, indicate that the studied plants have the ability to accumulate or synthesise organoarsenic compounds in relatively high percentages, and this information contributes to enlarge the knowledge of arsenic uptake and speciation in plants.     

Speciation of As(III), As(V), MMA and DMA in contaminated soil extracts by HPLC-ICP/MS

A method to separate and quantify two inorganic arsenic species As(III) and As(V) and two organic arsenic species, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), by HPLC-ICP/MS has been developed. The separation of arsenic species was achieved on the anionic exchange column IonPac AS11 (Dionex) with NaOH as mobile phase. The technique was successfully applied to analyze extracts of two contaminated soils, sampled at a former tannery site (soil 1) and a former paint production site (soil 2). The soils were extracted at pH values similar to the natural environment. Extractions were performed at different pH values with 0.3 M ammonium oxalate (pH = 3), milli-Q water (pH = 5.8), 0.3 M sodium carbonate (pH = 8) and 0.3 M sodium bicarbonate (pH = 11). No organically bound arsenic was found in the extracts. As(V) was the major component. Only up to 0.04% of the total arsenic contained in soil 1 were mobilized. The highest amount of extracted arsenic was found at the highest pH. In the milli-Q water extract of soil 1 As(III) and As(V) were found. High amounts of As(V) were found in the extracts of soil 2. Up to 20% of the total arsenic bound to soil 2 constituents were released. The results show that the mobilization of arsenic depended on the pH value of the extraction solution and the kind of extracted soil. Dramatic consequences have to be expected for pH changes in the environment especially in cases where soils contain high amounts of mobile arsenic.     

Evaluation of extraction procedures for the ion chromatographic determination of arsenic species in plant materials

The determination of arsenic species in plants grown on contaminated sediments and soils is important in order to understand the uptake, transfer and accumulation processes of arsenic. For the separation and detection of arsenic species, hyphenated techniques can be applied successfully in many cases. A lack of investigations exists in the handling (e.g., sampling, pre-treatment and extraction) of redox- and chemically labile arsenic species prior to analysis. This paper presents an application of pressurized liquid extraction (PLE) using water as the solvent for the effective extraction of arsenic species from freshly harvested plants. The method was optimized with respect to extraction time, number of extraction steps and temperature. The thermal stability of the inorganic and organic arsenic species under PLE conditions (60-180 degrees C) was tested. The adaptation of the proposed extraction method to freeze-dried, fine-grained material was limited because of the insufficient reproducibility in some cases.     

A systematic study on extraction of total arsenic from down-scaled sample sizes of plant tissues and implications for arsenic species analysis

An easily feasible, species-conserving and inexpensive protocol for the extraction of total arsenic and arsenic species from terrestrial plants was designed and applied to the investigation of accumulation and metabolization of arsenite (As(III)), arsenate (As(V)), monomethylarsonate (MMA(V)), and dimethylarsinate (DMA(V)) by the model plant Tropaeolum majus. In contrast to existing extraction methods hazardous additives and elaborate procedures to enhance the extraction yields were omitted. The proposed protocol is suited to down-scale the sample sizes used for the extractions and to promote a compartmentally resolved analysis of the arsenic distribution within individual leaves, leaf stalks, and stems instead of the conventional extraction of pooled samples. In a two-step extraction, the high extraction efficiencies (85-92%) for arsenic achieved by phosphate buffer from larger amounts (200mg) of homogenized leaf material in a one-step extraction, could be enhanced to 94-100% in a second extraction step. A strong dependence of the arsenic extractability on the type of arsenic species accumulated in the tissue as well as on the type of the tissue (leaf, leaf stalk, stem) was found. For the extraction of 5mm long segments cut from individual leaves without previous homogenization of the plant parts yields between 75 and 93% depending on arsenic species prevailing in the cells were obtained using 1 or 10mM phosphate buffer. The total extraction and analysis protocol was validated using a standard reference material as well as by spiking experiments. The arsenic species analysis by IC/ICPMS revealed a number of nine unidentified metabolites in the plant extracts in addition to the species MMA(V), DMA(V), As(III), and As(V) that were provided to the plants during their growth phase.     

Speciation of As(III), As(V), MMA and DMA in contaminated soil extracts by HPLC-ICP/MS

A method to separate and quantify two inorganic arsenic species As(III) and As(V) and two organic arsenic species, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), by HPLC-ICP/MS has been developed. The separation of arsenic species was achieved on the anionic exchange column IonPac^®AS11 (Dionex) with NaOH as mobile phase. The technique was successfully applied to analyze extracts of two contaminated soils, sampled at a former tannery site (soil 1) and a former paint production site (soil 2). The soils were extracted at pH values similar to the natural environment. Extractions were performed at different pH values with 0.3 M ammonium oxalate (pH = 3), milli-Q water (pH = 5.8), 0.3 M sodium carbonate (pH = 8) and 0.3 M sodium bicarbonate (pH = 11). No organically bound arsenic was found in the extracts. As(V) was the major component. Only up to 0.04% of the total arsenic contained in soil 1 were mobilized. The highest amount of extracted arsenic was found at the highest pH. In the milli-Q water extract of soil 1 As(III) and As(V) were found. High amounts of As(V) were found in the extracts of soil 2. Up to 20% of the total arsenic bound to soil 2 constituents were released. The results show that the mobilization of arsenic depended on the pH value of the extraction solution and the kind of extracted soil. Dramatic consequences have to be expected for pH changes in the environment especially in cases where soils contain high amounts of mobile arsenic.     

Phytoremediation of arsenic by two hyperaccumulators in a hydroponic environment

Phytofiltration involves the use of plants to remove toxic compounds from water. Arsenic is an element of considerable environmental and toxicological interest because of its potential deleterious effects upon human health. In this research, a laboratory-constructed hydroponic system was employed to characterize phytofiltration for the uptake of arsenic and macronutrients by two arsenic hyperaccumulators, Pteris cretica cv Mayii (Moonlight fern) and Pteris vittata (Chinese brake fern). Arsenic was shown to preferentially accumulate in the leaves and stems of P. cretica cv Mayii compared to roots. The amounts of the macronutrients calcium and phosphorous absorbed were compared for control plants (growth solution) and plants exposed to arsenic(III) (growth solution and arsenic(III)). Significant differences in the concentration levels of the macronutrients were observed in roots, stems, and leaves between the control and arsenic-exposed plants. The arsenic contents of entire P. vittata plants exposed to hydroponic solutions containing arsenic(III) and arsenic(V) were compared, and no significant difference was observed.     

Profile distribution of As(III) and As(V) species in soil and groundwater in Bozanta area

The profile distribution of arsenic(III) and arsenic(V) species in soil and groundwater was investigated in the samples collected in 2005 from a hand-drilled well, in the Bozanta area, Baia Mare region, Romania. The total content of arsenic in the soil was in the range of 525–672 mg kg⁻¹ exceeding 21–27 times the action trigger level for sensitive soil. 0.9–11.3 % of the total content was soluble in water, 83.0–92.6 % in 10 mol dm⁻³ HCl and 2.6–13.3 % was the residual fraction. Arsenic(V) was the dominant arsenic species in the soil in the range of 405–580 mg kg⁻¹. The distribution and mobility of arsenic species was governed by soil pH and contents of Al, Fe, and Mn. The mobility of arsenic(V) decreased with depth, while that of arsenic(III) was high at the surface and in the proximity of groundwater. The total concentration of arsenic in groundwater was (43.40 ± 1.70) μg dm⁻³, which exceeded the maximum contaminant level of 10 μg dm⁻³. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

Environmental bioremediation for organometallic compounds: Microbial growth and arsenic volatillization from soil and retorted shale

Nutrient effects on microbial growth and arsenic volatilization from retorted oil shale and soil were evaluated in a laboratory study. Dimethylarsinic acid (DMAA), methanearsonic acid (MAA) and sodium arsenate amendments were used with added nutrients, or with retort process water added to simulate possible co-disposal conditions. In experiments with soil and retorted shale, dimethylarsinic acid showing the highest cumulative arsenic releases, in comparison with added inorganic sodium arsenate (SA). Low but detectable amounts of innate arsenic present in retorted shale could be volatilized with added organic matter. In soil, arsenic volatilization showed a direct relationship to nutrient levels and microbial growth. With shale, in comparison, a threshold response to available nutrients was evident. Distinct increases in fungal community development occurred with nutrients available at a level of 2.5% w/v, which also allowed incresed arsenic volatization. Codisposal of retort process waters with shale allowed arsenic volatilization without the addition of other nutrients. The presence of retort process water limited arsenic volatilization from the added organometallic compounds DMAA and MAA, but not from SA or innate arsenic. These differences should be useful in the definition of permissive and non-permissive environmental conditions for arsenic volatilization in bioremediation programs.     

Phytoremediation of lead with green onions (Allium fistulosum) and uptake of arsenic compounds by moonlight ferns (Pteris cretica cv Mayii)

Phytoremediation has been investigated as an alternative to excavation to remediate contamination in soil. In this work, Allium fistulosum (green onions) and Pteris cretica cv Mayii (moonlight ferns) were investigated for phytoremediation. Green onions were planted in lead-spiked soil, and chelating agents were introduced to enhance the uptake of lead by the plants. Lead uptake was low in the absence of chelating reagents. Ethylenediaminetetraacetic acid (EDTA) significantly enhanced the concentration of lead in the stems of green onions, while propylenediaminetetraacetic acid (PDTA) did not induce lead absorption.Moonlight ferns (P. cretica cv Mayii) were planted in a hydroponic system to which arsenic (III), arsenic (V), and monomethylarsenate (MMA) were added with hydroponic solution. Ferns exposed to arsenic (III) showed the highest extraction of arsenic followed by ferns exposed to arsenic (V). The extraction of arsenic by the ferns was higher when arsenic (III) was mixed with arsenic (V) than the combination of arsenic (III) and MMA. These results suggest that inorganic arsenic is phytoextracted preferentially to MMA.     

砷污染对植物和人体健康的影响及防治对策

讨论了土壤环境背景值及来源，地植物和人体健康的影响，砷的环境质量标准，国内外砷对环境的情况，以及砷污染防治对策。。     

Arsenic Speciation Governs Arsenic Uptake and Transport in Terrestrial Plants

Arsenic is a carcinogenic metalloid that occurs in the environment in a variety of chemical forms, showing different mobility, bioavailability and toxicity. Terrestrial plants may accumulate large amounts of arsenic. To understand how terrestrial plants take up, transport and metabolise these arsenic species, it is essential to characterise arsenic speciation in plant tissues. Given that As species can be transformed from one form to another during sample preparation and the measurement process, arsenic speciation in biological extracts needs to be performed with great care. This paper describes the methods used to measure arsenic speciation in plant tissue and assesses the role of As speciation in As metabolism in higher plants.     

Evaluation of extraction methods for arsenic speciation in polluted soil and rotten ore by HPLC-HG-AFS analysis

Three extraction systems including shaking, ultrasonic and microwave-assisted extraction were evaluated. Water and phosphate buffer were tested for the extraction of arsenic compounds in polluted soil, describing the water-soluble or plant-available fraction. The stabilities and recoveries of various arsenic species indicated that no obvious changes of species occurred during the extraction process. The raw extracts were cleaned up by C₁₈ cartridge before analysis. Having optimized the extraction conditions, the arsenic species in polluted soil and ore from the different pollution sources were extracted by microwave-assisted extraction with 0.5 M phosphate buffer as extractant. Arsenic species were quantitatively determined by high performance liquid chromatography on-line coupled with hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS). As(III) and As(V) were the major arsenic species in the polluted soil samples resulting from irrigation by waste water. As^V was the only form found in the rotten ore sampled in mining area. During the extraction process, the recoveries of spiked As(III), As(V), DMA(V) and MMA(V) were 85.4 ± 7.2%, 80.2 ± 6.7%, 101.6 ± 6.7% and 98.8 ± 9.1%, respectively, showing that most water-soluble arsenic could be measured.