Arsenic speciation and distribution in an arsenic hyperaccumulating plant
The Science of the Total Environment 300 (2002) 167–177 0048-9697/02/$ - see front matter ᮊ 2002 Elsevier Science B.V. All rights reserved. PII: S0048-9697 Ž 02 . 00165-1 Arsenic speciation and distribution in an arsenic hyperaccumulating plant Weihua Zhang , Yong Cai *, Cong Tu , Lena Q. Ma * aa, bb,1, Department of Chemistry and Southeast Environmental Research Center, Florida International University, Miami, FL 33199, a USA Soil and water Sciences Department, University of Florida, Gainesville, FL 32611, USA b Received 5 October 2001; accepted 25 March 2002 Abstract Arsenic-contaminated soil is one of the major arsenic sources for drinking water. Phytoremediation, an emerging, plant-based technology for the removal of toxic contaminants from soil and water, has been receiving renewed attention. Although a number of plants have been identified as hyperaccumulators for the phytoextraction of a variety of metals, and some have been used in field applications, no hyperaccumulator for arsenic had been previously reported until the recent discovery of Brake fern (Pteris vittata), which can hyperaccumulate arsenic from soils. This finding may open a door for phytoremediation of arsenic-contaminated soils. Speciation and distribution of arsenic in the plant can provide important information helpful to understanding the mechanisms for arsenic accumulation, translocation, and transformation. In this study, plant samples after 20 weeks of growth in an arsenic-contaminated soil were used for arsenic speciation and distribution study. A mixture of methanolywater (1:1) was used to extract arsenic compounds from the plant tissue. Recoveries of 85 to 100% were obtained for most parts of the plant (rhizomes, fiddle heads, young fronds and old fronds) except for roots, for which extraction efficiency was approximately 60%. The results of this study demonstrate the ability of Brake fern as an arsenic hyperaccumulator. It transfers arsenic rapidly from soil to aboveground biomass with only minimal arsenic concentration in the roots. The arsenic is found to be predominantly as inorganic species; and it was hypothesized that the plant uptakes arsenic as arsenate w As(V) x and arsenate was converted to arsenite w As(III) x within the plant. The mechanisms of arsenic uptake, translocation, and transformation by this plant are not known and are the objectives of our on-going research. ᮊ 2002 Elsevier Science B.V. All rights reserved. Keywords: Arsenic; Phytoremediation; Pteris vittata; Hyperaccumulator; Speciation *Corresponding author. Tel.: q1-305-348-6210; fax: q1- 305-348-3772. E-mail address: cai@fiu.edu (Y. Cai), lqma@ufl.edu (L.Q. Ma). Co-corresponding author. Tel.: q1 352 392 9063 1 1. Introduction Arsenic, ranking 20th in abundance in the earth’s crust, is a toxic element widely encountered in the environment and organisms (Cullen and Reimer, 1989). Arsenic can enter terrestrial and aquatic environments through both natural forma- 168 W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 tion and anthropogenic activity. Natural pathways of arsenic include weathering, biological activity, and volcanic activity. The primary anthropogenic input derives from combustion of municipal solid waste, fossil fuels in coal- and oil-fired power plants, release from metal smelters, and direct use of arsenic-containing herbicides by industry and agriculture. There are a number of ways by which the human population can become exposed to arsenic. The most important one is probably through ingestion of arsenic in drinking water or food (National Research Council, 1999; Le et al., 2000; US EPA, 2001a). Arsenic species are bioactive and toxic. Long- term exposure to low concentrations of arsenic in drinking water can lead to skin, bladder, lung, and prostate cancer. Non-cancer effects of ingesting arsenic at low levels include cardiovascular dis- ease, diabetes, and anemia, as well as reproductive, developmental, immunological and neurological effects. Short-term exposure to high doses of arsenic can cause other adverse health effects (US EPA, 2001a). A recent report by the National Academy of Sciences concluded that the previous arsenic standard of 50 mgyl in drinking water does not achieve US EPA’s goal of protecting public health and should be lowered as soon as possible (National Research Council, 1999). EPA has recently decreased the drinking water standard to 10 mgyl in October 2001 to more adequately protect public health (US EPA, 2001b). The increase in the public awareness of the toxicity and the environmental impact of arsenic contami- nation and the possible implementation of new regulations limiting arsenic in drinking water have resulted in a growing interest in the study of the biogeochemical cycling of arsenic and the devel- opment of arsenic decontamination technologies. Arsenic-contaminated soil is one of the major sources of arsenic in drinking water (Nriagu, 1994; National Research Council, 1999; Welch et al., 2000; Kim and Nriagu, 2000). The concentration of arsenic in cereals, vegetables and fruits is directly related to the level of arsenic in contami- nated soil. Although the remediation of arsenic- contaminated soil is an important and timely issue, cost-effective remediation techniques are not cur- rently available. Phytoremediation, an emerging, plant-based technology for the removal of toxic contaminants from soil and water is a potentially attractive approach (US EPA, 2000; Terry and Banuelos, 1999; Raskin and Ensley, 2000; Dah- mani-Muller et al., 2000). This technique has received much attention lately as a cost-effective alternative to the more established treatment meth- ods used at hazardous waste sites. It is often the only way to remediate soils contaminated with metals without affecting their biological function. A number of plants have been identified as hyper- accumulators for the phytoextraction of a variety of metals including Cd, Cr, Cu, Hg, Pb, Ni, Se and Zn, and some of these plants have been used in field applications (Dobson et al., 1997; Brooks, 1998; Terry and Banuelos, 1999; Reeves et al., 2001). Recently, Brake fern (Pteris vittata), an effica- cious arsenic hyperaccumulating fern plant, has been discovered in an abandoned wood-treatment site in central Florida (Ma et al., 2001). This fern can tolerate arsenic concentration as high as up to 1500 mgyg in soil, and has a bioconcentration factor of 193. The arsenic concentration in the plant can reach as high as 2.3% (dried weight). The toxicity and bioavailability of arsenic are closely associated with its oxidation state and species. The determination of total arsenic in a sample is insufficient to assess its environmental risk (Koch et al., 2000). Speciation of arsenic in plant samples can provide important information helpful to understanding the mechanisms for arsen- ic accumulation, translocation, transformation and detoxification by Brake fern. It has been found that a large amount of the arsenic in marine organisms is in organic forms such as arsenosugars in algae, and arsenobetaine and arsenocholine in fish, mollusks, and crustaceans (Maeda, 1994; Francesconi et al., 1994). The chemical structures for these organoarsenic compounds in many marine organisms have been reported (Maeda, 1994; Francesconi et al., 1994). However, little is known about arsenic speciation in freshwater aquatic plants or those in terrestrial environments (Koch et al., 2000). Based on the limited infor- mation available, it appears that, in contrast to marine organisms, inorganic arsenic is the predom- inant form of arsenic found in some freshwater 169W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 and terrestrial plants (Helgesen and Larsen, 1998; Koch et al., 1999, 2000). Uptake, accumulation and translocation of arsenic in both arsenic-tolerant and non-tolerant plants have been studied although those plants are not arsenic hyperaccumulators (Meharg and Mac- nair, 1990, 1991a,b; Sneller et al., 1999; Schmoger ¨ et al., 2000; Pickering et al., 2000). Brake fern as an arsenic hyperaccumulator not only has the potential for phytoremediation of arsenic contam- inated soil, but also provides an excellent oppor- tunity to investigate plant detoxification mechan- isms for arsenic. Following our identification of this plant as an efficient hyperaccumulator of arsenic, we investigated the speciation and distri- bution of arsenic in the plant. This paper summa- rizes the results from these studies. 2. Experimental 2.1. Reagents and standards The inorganic arsenic standard and other indi- vidual stock solutions of internal standards used for inductively coupled plasma mass spectrometry (ICPyMS) analysis (ICP grade, 1000 mgyl) were purchased from GFS Chemicals, Inc. (Powell, OH). Arsenic standards for speciation analysis were obtained as sodium hydrogenarsenate heptah- ydrate, Na HAsO Ø7H O (Aldrich, Milwaukee, 242 WI); sodium metaarsenite, NaAsO (Aldrich); and 2 cacodylic acid, (CH ) AsO(OH)(Sigma, St. Lou- 32 ise, MO). These standards were dissolved in dis- tilled, deionized water to make 1000 ppm stock solutions of arsenate w As(V) x , arsenite w As(III) x , and dimethylarsinic acid (DMA), respectively. A stock solution of monomethylarsonic acid (MAA) (1000 mgyl) was provided by P.S. Analytical (Kent, UK). The standards were used as received without further purification. Fresh calibration stan- dards were prepared every week or as needed by diluting these commercial standards or stock solu- tions either in 5% nitric acid (for total arsenic analysis by ICPyMS) or in water (for speciation analysis). Trace metal grade hydrochloric acid, nitric acid, and HPLC grade methanol were obtained from Fisher Scientific (Pittsburgh, PA). All other chemicals used were of analytical grade or better. Distilled deionized water was prepared using a Barnstead Fistream II Glass Still System (Barnstead Thermolyne Corp., Dubuque, Iowa) and was used in all standard and sample prepara- tions. High purity grade (99.99%) argon for ICPy MS was purchased from Air Products (Allentown, PA). All glass and plastic ware was cleaned prior to use by soaking in 5% nitric acid overnight, rinsing with water and storing clean. The procedural blank produced after these cleaning steps has been found to contain negligible amount of arsenic. 2.2. Sampling and sample preparation Brake fern (Pteris vittata) samples used in this study were collected from an arsenic contaminated soil, following 20 weeks of growth in a green- house. The surface layer (0–15 cm) of arsenic- contaminated soil (sandy, siliceous, hyperthermic, grossarenic paleudult), which contained 97 mgyg of arsenic, was collected from an abandoned chro- mated–copper–arsenate (CCA) wood preservation site in Central Florida (Ma et al., 2001). Air-dried soil of 1.5 kg was weighed into each plastic pot with a diameter of 15 cm (2.5 l). The soil was thoroughly mixed with 1.5 g of Osmocote ᭨ extended time-release fertilizer as a base fertilizer (18-6-12)(Scotts–Sierra Horticultural Products Co., Marysville, OH). A petri dish was placed under each pot to collect potential leachate during the experiment. After a one-week equilibrium under moist conditions, each pot was transplanted with one healthy fern with 5 to 6 fronds. The plants were watered daily or as necessary. During the experiment, the average temperature in the greenhouse ranged from 14 (night) to 30 8C (day), with an average photosynthetically active radiation (PAR) of 825 mmol m Øs . After 12 weeks of y2 y1 transplanting, additional fertilizers containing 50 mg N kg in the form of NH NO and 25 mg P y1 43 kg of KH PO were applied to all ferns. After y1 24 harvest, the plants was washed with water, and then separated into 5 different groups for samples collected from greenhouse (roots, rhizomes, fiddle heads, young fronds, and old fronds). The samples were freeze-dried, ground to fine powder using a 170 W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 ceramic mortar and pestle, and stored in 20 ml plastic vials at room temperature until use. For total arsenic analysis, a digestion procedure previously developed for arsenic analysis in sea- grass by ICPyMS was adapted (Cai et al., 2000). Briefly, 10 mg samples were digested in open vessels with 10 ml of nitric acid for 1 h using a sand bath (150 8C). Then, 1 ml hydrogen peroxide was added into the sample vessel and the sample was allowed to digest for an additional 30 min. After cooling, the samples were transferred into a 100-ml volumetric flask, and brought to volume with water. These solutions were diluted by a factor of 10 with 5% nitric acid prior to analysis using ICPyMS. For arsenic speciation, 10 mg samples were ultrasonically extracted with 5 ml 1:1 methanoly water for 2 h. The samples were then centrifuged; the supernatant was decanted into a 100-ml volu- metric flask. The procedure was repeated with the residual pellet and the two extracts were combined. The residue was rinsed three times with 5 ml of water (5ml=3), and all supernatants were com- bined. The extract was then diluted to the 100 ml mark with water and then filtered using 0.45 PTFE syringe filters (Gelman). The filtrate was directly subjected to HPLC, or diluted by a factor of 10 with water for speciation analysis. For total arsenic analysis, the filtrate was diluted by a factor of 10 with 5% nitric acid. 2.3. Sample analysis Total arsenic analysis was carried out on a Model HP 4500 plus ICPyMS instrument (Hew- lett–Packard Co., Wilmington, DE) equipped with a Babington-type nebulizer and an ASX-500 auto- sampler (Cetac Technologies Inc., Omaha, NE). The instrumental configuration and general exper- imental conditions can be found elsewhere (Cai et al., 2000). Arsenic standard solutions prepared in 5% nitric acid were used for the calibration curves. Internal standard ( Y as internal standard) method 89 was used for quantitative determination of total arsenic in the nitric acid-digested samples (Cai et al., 2000) whereas the method of standard addi- tions was applied to the methanolywater-extracted samples. Speciation analysis of arsenic in the methanoly water-extracted samples was performed using both high performance liquid chromatography (HPLC) coupled with hydride generation atomic fluores- cence spectrometry (HPLC-HG-AFS) and HPLC– ICPyMS. The HPLC-HG-AFS instrument used was a P S Analytical Millennium Excalibur system (PSA 10.055, P.S Analytical, Kent, UK) coupled to an HPLC system from Spectra-Physics Analyt- ical, Inc. (Fremont, CA). The Millennium Excali- bur system is an integrated atomic fluorescence system incorporating vapor generation, gas–liquid separation, moisture removal and atomic fluores- cence stages. Data were acquired by a real-time chromatographic control and data acquisition sys- tem. The HPLC system is comprised of a P4000 pump and an AS 3000 autosampler with a 100-ml injection loop. A strong anion exchange column (PRP X-100, 250=4.6 mm, 10 mm particle size, Hamilton) was used for separation. Potassium phosphate (0.015 M for both K HPO and 24 KH PO ) at pH of 5.9 and flow rate of 1 mlymin 24 was used as mobile phase. For HPLC–ICPyMS, the outlet of the analytical column was connected to the nebulizer of the ICPyMS system by a 40 cm=0.25 mm i.d. PTFE tube. The HPLC condi- tions used were the same as for HPLC-HG-AFS. 3. Results and discussion 3.1. Total arsenic concentration and distribution The total amounts of arsenic and its distribution in the Brake fern in the greenhouse experiments are illustrated in Fig. 1. Brake fern rapidly and efficiently accumulated a large amount of arsenic from the moderately contaminated soil (97 ppm). Arsenic distribution in the plant varied significant- ly in different parts of the plant with concentrations of 3894; 2610; 2336; 728; and 168 mgyg for old fronds, young fronds, fiddle heads, rhizomes and roots, respectively. It is interesting to note that arsenic concentration in Brake fern roots was the lowest (-168 mgyg), whereas those in fronds were substantially greater with the old fronds having the highest arsenic level. It is estimated that )95% of arsenic taken up by the plant was concentrated in the aboveground biomass. This is 171W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 Fig. 1. Total concentrations of arsenic in different parts of the Brake fern (Pteris vittata) obtained using ICPyMS for plants from greenhouse and field. The soils used for greenhouse experiments contained 97 mgyg of arsenic, while those in the field where fern grew contained 153 mgyg. Rhizome apart from field samples was not analyzed. in good agreement with our previous observations (Ma et al., 2001). Arsenic concentrations in dif- ferent parts of the plants collected from the field are also included in Fig. 1 for comparison. Similar distribution patterns can be found for plants from both greenhouse experiments and field. High arsenic tolerant plants have been reported previously (Porter and Peterson, 1975; Meharg and Macnair, 1991a,b; Bech et al., 1997; Helgesen and Larsen, 1998; Brooks, 1998; Sneller et al., 1999; Koch et al., 1999, 2000; Pickering et al., 2000; Schmoger et al., 2000). Depending on plant spe- ¨ cies, arsenic tolerance may result from two strate- gies: arsenic exclusion and arsenic accumulation (Baker, 1987; Dahmani-Muller et al., 2000). The exclusion strategy involves avoidance of arsenic uptake or restriction of arsenic transport to the shoots. Typha latifolia, found abundantly at arsen- ic-contaminated sites, appears to be one example of this (Dushenko et al., 1995; Koch et al., 1999). The accumulation strategy consists of strong con- centration of arsenic in plant tissue. Several terres- trial plants found on mine tailings have been observed to contain high levels of arsenic. Arsenic concentrations of up to 3470 mgyg (dry weight) have been reported for Agrostis tenuis (Porter and Peterson, 1975). However, in order for the plant to accumulate these high levels of arsenic, the soil must contain an extremely high concentration of arsenic (as high as 26500 mgyg). The metal accumulation efficiency in plants can be evaluated using the bioconcentration factor (BF), which is defined as the ratio of metal concentration in the plant biomass to metal concentration in the soil. Hyperaccumulating plants are those that have a BF)1 (Brooks, 1998). Although Agrostis tenuis can accumulate arsenic to concentrations as high as 3470 mgyg, it has a BF much less than one. The BF for Brake fern can be as high as 193 (Ma et al., 2001), indicating an efficient accumulation of arsenic from soil by this plant. Plants that accumulate arsenic may either store arsenic in the roots or translocate it to the above- ground biomass. These differences in storage of 172 W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 arsenic suggest different processes for arsenic accumulation and transport mechanisms within different plants. Arsenic accumulation in root cells, such as those observed in tomato root systems can be related to an exclusion strategy (Carbonell- Barrachina et al., 1997; Dahmani-Muller et al., 2000). When high arsenic concentrations are pres- ent in shoots but not in roots an efficient root-to- shoot transport system may be important for arsenic tolerance and account for hyperaccumula- tion as in Brake fern. The results shown in Fig. 1 for plants grown under greenhouse conditions and those of fern samples taken from the field indicate arsenic concentration in old fronds is greater than those in young fronds. The transport of arsenic from roots to fronds is most likely carried out through the xylem sap. The differences in arsenic concentration in young and old fronds may suggest that a larger cumulative amount of transpiration stream has been probably passed through the old fronds over time. Translocation of metals from roots to the aging leaves has been considered as a detoxification process to assist removal of arsenic from the plant as the old leaves senesce and eventually fall off the plant (Dahmani-Muller et al., 2000; Perronnet et al., 2000). In an experiment with Brake fern taken from the CCA site, arsenic content in a naturally dried (dead) frond was found to be relatively low (84 and 428 mgyg for the young and old fronds, respectively), whereas that in the living fronds taken from the same plant was found to be high (4893 and 7575 mgyg, for the young and old frond parts, respectively). It is postulated that low concentrations of arsenic in the dead leaves resulted either from being washed out by rain after break up of the plant cell or by translocation of arsenic to the living parts before abscission in a manner similar to that of plant nutrients (Goodwin and Mercer, 1983). 3.2. Speciation and transformation of arsenic In order to obtain speciation information on arsenic present in the plant, arsenic was extracted with a 1:1 mixture of methanolywater. The recov- ery of arsenic using this extraction procedure with respect to the total arsenic concentration obtained using nitric acid digestion was evaluated. Recov- eries ranged from 85 to 100% for most parts of the plant (rhizomes, fiddle heads, young fronds and old fronds) except for the roots, where extrac- tion efficiency was approximately 60%. These recoveries were higher than those reported for other plants using the same extraction method (Koch et al., 1999, 2000). Chromatograms of arsenic species in living plant parts obtained using HPLC-HG-AFS and HPLC- ICPyMS are shown in Fig. 2. The HPLC-HG-AFS technique determines only the hydride-forming arsenic species (As(III),As(V), MMA, and DMA), whereas the HPLC-ICPyMS method can provide extra information on other species of arsenic. The results shown in Fig. 2 clearly indicate that the extractable arsenic species in the Brake fern consisted of only inorganic arsenic species, As(III) and As(V). In order to confirm the absence of other arsenic species, which may not be deter- mined by either HPLC-HG-AFS or HPLC-ICPy MS, the methanolywater extract was directly analyzed by ICPyMS without HPLC separation. The method of standard addition was used for quantification in order to compensate for matrix effects. It was found that the total concentration of arsenic obtained in each part of the plant with ICPyMS was in good agreement with the sum of As(III) and As(V) obtained using HPLC-HG-AFS (Fig. 3). This result suggests that the stable orga- noarsenic compounds (e.g. methylated species, arsenosugars) are not present in the living plant in any significant quantity. This, however, does not rule out the presence of intermediary organoarsenic compounds such as arsenic-biomolecule complex- es, which may decompose into simple inorganic arsenic species during the course of the extraction andyor separation. In fact, such complexation may be necessary to enable the plant to accumulate extremely high concentrations of arsenic while at the same time avoiding high concentration of free arsenic in cytoplasm, which cause disruption of cell function and even cellular death. Phytochela- tins (PCs), a family of peptides with the general structure (g-GluCys) -Gly, have been reported to n be induced upon exposure to arsenic in some plants (Grill et al., 1987; Maitani et al., 1996; Sneller et al., 1999, 2000; Schmoger et al., 2000). ¨ Complexation and detoxification of arsenic by the 173W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 Fig. 2. Chromatograms of arsenic species extracted with a 1:1 methanolywater mixture. a: analyzed by HPLC-HG-AFS, and b: analyzed by HPLC-ICPyMS. induced PCs has been confirmed using different techniques in some research (Schmoger et al., ¨ 2000). However, other research failed to demon- strate the formation of arsenic-PC complexes (Maitani et al., 1996) although PCs were indeed induced in plants upon exposure to arsenic. The 174 W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 Fig. 3. Total concentration of arsenic obtained by ICPyMS and the sum of As(III) and As(V) obtained by HPLC-HG-AFS in different parts of the Brake fern ( Pteris vittata). role played by PCs in the accumulation and detox- ification of arsenic and other metals in plants is still in debate (De Knecht et al., 1992; Leopold et al., 1999). Our results suggest that the formation of stable arsenic-PC complexes does not occur in the Brake fern in any significant quantity. Research on heavy metal hyperaccumulating plants indicates that some organic and amino acids (histine and proline) and polyhydroxy phenolic compounds may also be involved in heavy metal detoxification in plants (Kramer et al., 1996). The role played ¨ by organic and amino acids in the accumulation and detoxification of arsenic in plants is currently unknown. The conversion of As(V) to As(III) within the plant is interesting to note. Fig. 4 shows the percentages of As(III) with respect to the total arsenic content obtained by ICPyMS in different parts of Brake fern. Approximately 60–74% of the arsenic in the fronds was present as As(III) compared to only 8.3% in the roots. Note that As(V) is the predominant species in the roots. In a recent study, soils were spiked with 50 mgAs gasAs(III),As(V), dimethylarsinic acid y1 (DMA), or methylarsonic acid (MMA). After 18 weeks, arsenic in soil was mainly present as arsenate with little detectable organic species or arsenite regardless of arsenic species added to the soil (Tu et al., 2002). It is conceivable from these results that arsenic was taken up by Brake fern roots primarily as arsenate from soil using the phosphate uptake system. Arsenic competes with phosphate as a substrate for the phosphate uptake system in a wide variety of species (e.g. Wells and Richardson, 1985; Macnair and Cumbes, 1987; Meharg and Macnair, 1990, 1991a). It has been reported that in both arsenic tolerant and non- tolerant Holcus lanatus L., arsenic uptake uses phosphate uptake system (Macnair and Cumbes, 1987; Meharg and Macnair, 1990, 1991a). It was further proposed that the uptake of arsenic in the arsenic-tolerant H. lanatus is restricted by the altered phosphate uptake system, yet the tolerant plants were capable of accumulating arsenic to high concentration over longer time periods (Meharg and Macnair, 1991a). It seems unlikely 175W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 Fig. 4. Percentage of As(III) found in different parts of the plant with respect to the total As content obtained by ICPyMS. that the restricted uptake of arsenic by plant roots is a proper hypothesis for Brake fern since arsenic is taken up by this hyperaccumulator to an extremely high level in a very short time period (Ma et al., 2001). The effect of phosphate on the arsenic uptake by Brake fern is a topic of further study. In the fronds, As(III) is the major species. Consider that total arsenic level in the roots is much smaller that that in fronds, As(III) is the predominant species in the Brake fern. Terrestrial plants do not have arsenic detoxification system of algae by methylation of arsenic, and this is perhaps the reason why inorganic arsenics species are predominant in terrestrial plants (Helgesen and Larsen, 1998; Koch et al., 2000; Mattusch et al., 2000). It seems likely that reduction of As(V) to As(III) is an essential process for arsenic detoxi- fication in Brake fern, although As(III) is generally believed to be more toxic than As(V) to organisms. Under the reducing environment of plant cells, it is postulated that As(V) is readily reduced to As(III). Organic ligands such as thiols, induced probably by the exposure of the plant to arsenic, should be able to complex arsenic to avoid the damage of the plant cells by free As(III). The presence of this type of organic ligands (chelators) and their role in the arsenic accumulation and tolerance by Brake fern is currently being investigated. In summary, the discovery of the arsenic hyper- accumulating plant opens a door for phytoreme- diation of arsenic-contaminated soils (Ma et al., 2001) and also provides a unique research oppor- tunity to understand arsenic uptake, translocation, transformation, and detoxification. The present study demonstrates that (1) the plant can accu- mulate a large amount of arsenic from soils and transfer it to the aboveground biomass; (2) the plant contains predominately inorganic arsenic spe- cies; and (3) conversion of As(V) to As(III) occurs during the course of arsenic translocation with 60–74% of arsenic in the fronds as As (III) compared to only 8.3% in the roots. Further studies are currently underway to address the mechanisms of arsenic uptake and transformation. 176 W. Zhang et al. / The Science of the Total Environment 300 (2002) 167–177 Acknowledgments This research is partially supported by NSF grant (BES-0086768). We thank John T. 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