Environ. Sci. Technol. 2003, 37, 754-760
XAS Speciation of Arsenic in a Hyper-Accumulating Fern SAMUEL M. WEBB,† J E A N - F R A N C¸ O I S G A I L L A R D , * , † LENA Q. MA,‡ AND CONG TU‡ Department of Civil and Environmental Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3109 and Department of Soil and Water Science, University of Florida, Gainesville, Florida 32611-0290
The coordination environment and the redox speciation of arsenic in a newly discovered arsenic hyper-accumulating fern (Pteris vittata L.) were investigated by X-ray absorption spectroscopy. This method allowed us to probe arsenic directly, i.e., with minimal sample preparation. The results indicate that arsenic is predominantly accumulated as As(III) in the leaves. XANES and EXAFS results show that As(III) in the leaves is primarily present as aqueous arsenite species. The plant actively maintains arsenic in this reduced oxidation state, because after sample collection and subsequent aging and drying of the plant material, As(III) is gradually oxidized to As(V). We think that these arsenite species are sequestered in vacuoles. At extremely high As concentrations (ca. 1% As per dry weight) arsenic in the fern leaves is coordinated to a significant degree by sulfur in addition to oxygen. This spectral signature indicates that thiol-rich compounds are implicated in the biochemical transformations of arsenic within the plant.
Introduction Arsenic contamination of drinking water and soils has become an environmental problem that has received increasing attention in recent years. The National Research Council (1) and the U.S. Environmental Protection Agency (2) have suggested that the arsenic levels in drinking water should be reduced. This has led to the EPA lowering the maximum contaminant level standard for arsenic in drinking water in the United States from the current value of 50 µg L-1 to 10 µg L-1 as of February 2002. Arsenic occurs naturally in the environment at elevated levels in specific geological formations where it is present in a wide variety of minerals (3, 4). The dissolution of these minerals as a result of weathering (5) or geothermal activity (6, 7) results in increased concentrations levels of arsenic in aquatic systems. In addition, numerous anthropogenic sources, including mine wastes (8), coal fly ash (9), and arsenical pesticides (10) contribute significantly to arsenic mobilization. Arsenic is a redox-sensitive element whose toxicity, bioavailability, and mobility vary depending on its chemical speciation and oxidation state. Arsenate (H3AsO4) is frequently found under oxic conditions, whereas arsenite (H3AsO3) and arsenic sulfides are commonly found in anoxic environments (11). In general, arsenite is considered the more toxic and * Corresponding author phone: (847) 467-1376; fax: (847) 4914011; e-mail:
[email protected]. † Northwestern University. ‡ University of Florida. 754
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 4, 2003
bio-available form of arsenic because it is present at ca. neutral pH as an uncharged species that can easily permeate biological membranes (12). Therefore, determination of the oxidation state and of the coordination of arsenic is key for assessing its fate and impact in the environment. Arsenic readily undergoes metabolic conversions mediated by microorganisms, plants, and animals (13). Although inorganic forms of arsenic tend to dominate the speciation of arsenic in many plants (14, 15), numerous species of arsenic, including methylated arsenic compounds (16), arsenosugars (14, 17), and arsenobetaine (18), have been detected in a variety of organisms. Bioaccumulation of toxic species by hyper-accumulating plants or conversion to less toxic forms may be an efficient and cost-effective method for the remediation of contaminated soils and waters (19, 20). Phytoremediation of sites contaminated by elements such as arsenic and lead have been examined in mine wastes and other contaminated soils (21-24). The Chinese Brake Fern (Pteris vittata L.) has been discovered recently to be an extremely effective hyperaccumulator of arsenic (25). The fern leaves accumulate arsenic at a concentration up to 200 times that of the soil, and consequently concentrations in excess of 20 000 µg/g of dry weight can be observed. In contrast, the concentration of arsenic in the roots is generally much lower, reaching a maximum of approximately 300 µg/g. This shows that the fern has a highly efficient root to shoot transport mechanism for arsenic, leading to high ratios of metalloid in the fronds vs the roots, typical of hyperaccumulators. Additionally, plant growth and biomass production are stimulated by the presence of arsenic over a control soil. The Chinese brake fern is also widely cultivated, can tolerate many soil environments, and grows relatively quickly. These properties all point to a high potential for the Chinese brake fern to be an effective plant for the phytoremediation of arsenic contaminated soils. However, to use phytoremediation effectively and efficiently, it is important to obtain a detailed understanding of the biochemical pathways and mechanisms that operate to translocate As species from the soil to the shoots. Thus, the chemical environment of arsenic inside the plant must be studied. Because of its ability to provide information on short-range order and its elemental selectivity, X-ray absorption spectroscopy (XAS) has been used to determine the local coordination environment of elements such as As in both laboratory (26-36) and natural settings (37-42). In addition, the chemical speciation of various elements in diverse plant species has been investigated recently by XAS (e.g., 20, 4348). To this end, XAS provides an ideal spectroscopic probe for investigating directly the coordination environment of arsenic in the Chinese brake fern.
Materials and Methods Fern Growth Conditions. An uncontaminated soil, collected in central Florida, was used to grow the fern. Detailed information on soil properties are presented elsewhere (49). A 2-kg sample of soil was spiked with 100 mg of As, as NaAsO4, and was thoroughly mixed with 2.0 g of Osmocote extendedtime-release fertilizer as base fertilizer. The soil mixture was then placed in a 2.5-L plastic pot. After equilibration for one week, one fern seedling with 5-6 fronds was planted in the pot. The plants were allowed to grow for 30 weeks in a greenhouse where the temperature ranged between 14 and 30 °C; and where the mean photosynthetically active radiation was 825 µEinsteins m-2 s-1. Two arsenic amendments of 100 mg As per kg of soil were applied to the soil at 6-week intervals after 12 weeks of planting in order to produce a fern biomass 10.1021/es0258475 CCC: $25.00
2003 American Chemical Society Published on Web 01/18/2003
containing high arsenic levels. The concentrations of arsenic in the plant were determined by graphite furnace atomic absorption spectrophotometer (Perkin-Elmer SIMMA 6000, Norwalk, CT) after digestion of the plant following the protocol given in the USEPA method 3051. XAS Experiments. Leaves were harvested fresh and sealed immediately between 2 layers of Kapton tape. The encapsulated samples were then stored under liquid nitrogen conditions to quench further reactions and minimize any chemical changes between the time the samples were prepared and exposed to the beam. The typical storage time never exceeded 3 days to ensure the freshness of the plant samples. X-ray absorption measurements were performed at the Advanced Photon Source, Argonne National Laboratory, on the Dupont Northwestern Dow Collaborative Access Team (DND-CAT) bending magnet beam-line. All the spectra were acquired in the continuous scanning mode (CS-XAS) to improve signal-to-noise ratio, determine experimental errors, and ensure that the high photon flux of the beam did not induce changes in the oxidation state of arsenic during the measurements. Because the scans are obtained rapidly (120 s/scan), any radiation-induced changes in the oxidation state of arsenic can be determined by comparing the individual data scans over the time of the data collection, assuming that the oxidation takes more than 120 s to complete. A detailed description of the CS-XAS experimental setup can be found elsewhere (50). Briefly, a Si(111) piezo-driven double crystal monochromator was used to vary the energy of the X-ray photons produced by the bending magnet from 200 eV below to 1000 eV above the absorption K-edge of As (11 868 eV). The monochromator was kept detuned at all energies to approximately 50% of the maximum intensity to minimize harmonic interferences. The incident intensity, I0, and transmitted intensity, IT, were measured using ionization chambers. The fluorescence signal, IF, was measured with a Stern-Heald “Lytle” detector (EXAFS Co) that was filled with xenon. The outputs of the fluorescent detector and the two ionization chambers were connected to current amplifiers (Stanford Research System SRS 570) that were continuously sampled at 12.5 kHz by a 16-bit analog-to-digital converter. Data were collected while the monochromator was continuously slewed between the initial and final energies. Nine successive scans were recorded for each sample at a rate of 120 s per scan. XAS Data Collection and Analysis. The spectroscopic data were analyzed using a series of codes that were developed in-house specifically for the interpretation of the formatted data structure as described elsewhere (51). Briefly, the original data were binned and averaged over 0.5-eV increments in the edge region and over 0.05 Å-1 in the EXAFS region. The experimental errors for each channel were calculated using the standard deviations over each bin. To improve the signalto-noise ratio of an experiment, multiple scans of each sample were averaged. Using these values, transmitted and fluorescent X-ray absorption signals, µT and µF, were determined together with their associated experimental errors. The data were then normalized, following conventional procedures, and the EXAFS signal was extracted after background removal using the AUTOBK routine (52). To carry over the calculation of the errors during each of these steps, a pseudo-Monte Carlo method was performed using a Gaussian distribution with a 2 σ standard deviation to sample the errors. The simulation consisted of n ) 1000 trials, which was determined to be a reasonable number of repetitions without affecting the numerical results (42). To determine the speciation of arsenic, spectral decompositions were performed on the samples’ XANES or EXAFS signals using a basic set of selected standard reference compounds. The decomposition was performed using quadratic linear programming to solve the problem
datasample )
∑ Φ data i
standard-i
i
under the constraints that
∑ Φ ) 1; Φ g 0 i
i
i
where φi is the fraction of the metal corresponding to standard i. This simply states that the sample spectrum can be represented as the sum of each of the standard spectra times their respective contribution to the sample. Constraints exist so that the spectral contribution of the standard must be nonnegative and all contributions must sum to one. The estimates of the associated experimental errors on the predicted fraction, φi, were then calculated by repeating a Monte Carlo process. The error reported in this manner is the experimental error that arises from the counting statistics of the EXAFS spectra. The coordination environment of arsenic in the plants was also calculated using ab initio parameters determined using the computer code FEFF8 (53). Single scattering paths in model structure were calculated up to a distance of 8 Å, with a cutoff limit of 2% in amplitude with respect to the most intense scattering path. EXAFS fitting was performed using the standard EXAFS formula (54) as utilized by the IFEFFIT fitting routine (55). EXAFS refinements were carried out in k-space and in R-space to the imaginary and magnitude portions of the k3-weighted Fourier transformed data for consistency. Data ranges used in fitting included in k-space from 2 to 13 Å-1 and in R-space from 1 to 3 Å. In the fitting procedure, the passive electron reduction factor, S02, was determined by the EXAFS fitting of model compounds for each type of coordination environment (either As-O or AsS). Fitting of the arsenic environment in the plant samples involved minimizing the residuals between the experimental data and fitted spectrum by varying the coordination number (CN), the bond length (R), and the relative mean square displacement around the equilibrium bond length, or the Debye-Waller parameter (σ2) of each coordination shell.
Results XANES spectra for arsenic reference compounds are shown in Figure 1. In Figure 1a, the shift in the K-edge of arsenic can be seen as a function of oxidation state and the type of coordination. In Figure 1b, the XANES spectra show that there is a significant change in the white line portion of the spectrum when the sodium salt of arsenic is hydrated. Once the salts are dissolved in water, the white lines become much stronger, increasing nearly fifty percent in intensity. This effect is present for both the As(III) and the As(V) salts. These differences make it easy to distinguish aqueous arsenic from solid arsenic forms. Figure 2 shows the XANES spectra for Chinese brake fern leaves containing approximately 10 000 µg/g arsenic by dry weight. In each case, the sample from the first scan was identical to the last, indicating that the beam did not induce As chemical change. Using the quadratic programming approach to fit the XANES region, the arsenic speciation in the leaves show 94 ( 2% aqueous As(III) and 6 ( 2% As2S2. The six percent of arsenic sulfide is significant, as there is a shift in the K-edge of the sample toward lower energies with respect to As(III), suggesting that the sample contains a mixture of As(III) and sulfidic coordination. Figure 3 shows XANES data from ferns with an arsenic concentration of about 1000 µg/g. Leaves were examined which were young, old, half-dried, and fully dried fronds. In actively growing plants, the arsenic exists primarily as As(III). In contrast to the ferns with high arsenic concentrations (∼10 000 µg/g), these fronds had no detectable concentraVOL. 37, NO. 4, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
755
FIGURE 3. Arsenic K-edge XANES data of Pteris vittata leaves at various stages of drying. The shift in oxidation state from As(III) to As(V) can be seen as the aging process progresses. Arsenic concentration is approximately 1000 µg/g.
TABLE 1. Spectral Deconvolution of K-edge Arsenic XANES Spectra for Fern Sections Derived from Plants Containing 1000 µg/g Arsenica samples
As(III) %
As(V) %
leaves
FIGURE 1. Arsenic K-edge XANES data reference compounds. (a) XANES spectra for As2S2, aqueous As(III), dimethyl arsenic acid (DMA), and aqueous As(V). (b) XANES spectra showing the increase in intensity of the white line from the sodium salts of arsenic to the salts in aqueous solution.
young mature half dry dry spore
100 93.8 ( 0.8 51.9 ( 1.7 0 20.2 ( 0.8
0 6.2 ( 0.8 48 ( 1.7 100 79.7 ( 2.7
young mature half dry dry
stems 92.3 ( 4.9 95.1 ( 4.9 85.6 ( 3.7 0
7.7 ( 4.9 4.9 ( 4.9 14.4 ( 3.7 100
a The arsenite and arsenate fractions represented in the fit are aqueous ions. No fractions of solid forms of arsenic were detected.
FIGURE 2. Arsenic K-edge XANES spectra of Pteris vittata leaves containing 10 000 µg/g arsenic. Data points are shown with the associated amount of experimental error. The solid line is the best fit line to the sample, giving a composition of 94 ( 2% As(III) and 6 ( 2% As2S2. tions of sulfur coordination. As the plants dried, a distinct shift in the absorption edge can be seen, representing a change in oxidation state from As(III) to As(V). When compared to arsenic standards, the spectrum of the arsenic in the fresh leaves resembles most closely the spectrum of the aqueous arsenite ion (AsO3-). Other As(III) compounds, such as sodium salts and oxides have distinctly smaller white lines at the absorption edge of arsenic as described above and shown in Figure 1. Table 1 shows the arsenic speciation as determined through XANES fitting for the fern fronds containing 1000 µg/g arsenic as a function of their drying age. The EXAFS of arsenic was also examined in these samples. Figure 4 shows the arsenic K-edge EXAFS spectra of several 756
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 4, 2003
FIGURE 4. Raw EXAFS data (k3-weighted) for fern leaves and arsenic model compounds. Error bars represent the experimental error as carried through the background removal procedures. model compounds and samples from the fern fronds. Figure 5 shows the corresponding Fourier transforms (radial distribution functions, RDFs) of each of these spectra. The RDFs show that arsenic is coordinated with oxygen in most samples at a distance that is similar to that of As(III). When the fronds are dried, this distance shifts slightly to one that more closely matched As(V). Additionally, the fronds that contained high concentrations of arsenic (10 000 µg/g) show a second distinct coordination shell corresponding to sulfur.
FIGURE 5. Radial distribution functions, uncorrected for phase shifts, for fern leaves and arsenic model compounds.
FIGURE 7. Best fit of EXAFS data from ab initio calculations to fern fronds containing 10 000 µg/g total arsenic. Fitting was performed in k-space. (a) Both the real portion and the magnitude of the Fourier transform of the data and fit. (b) Fit to the data of the isolated first shell EXAFS in k-space. Parameters determined as a result of the fitting are displayed in Table 2. the data. Table 2 presents the calculated coordination numbers, bond lengths, and fitting parameters.
Discussion
FIGURE 6. Best fit of EXAFS data from ab initio calculations to fern fronds containing 1000 µg/g total arsenic. Fitting was performed in k-space. (a) Both the real portion and the magnitude of the Fourier transform of the data and fit. (b) Fit to the data of the isolated first shell EXAFS in k-space. Parameters determined as a result of the fitting are displayed in Table 2. EXAFS fitting of the data was performed using ab initio calculations from FEFF8 (53) and FEFFIT (55). These results (Figure 6) show that the arsenic in the plant fronds is dominated by a local environment characterized by the presence of three oxygen ligand atoms. The calculated bond distances, as well as the 3-fold coordination, support the XANES results, suggesting that the arsenic is present as the arsenite ion. Figure 7 shows the calculated fit to the fronds that contained 10 000 µg/g arsenic. In this spectra, the fitting resolves both the As(III) oxygen coordination as well as the As(III) sulfur coordination. These fitting parameters for the EXAFS of these coordination shells were adjusted simultaneously, and the fitted results show good agreement with
The data presented in previous work showed that arsenic is stored preferentially in the leaves of the Chinese brake fern fronds (25). The X-ray absorption spectroscopy results indicate that arsenic in the fern leaves is present primarily as aqueous arsenite ion As(III), which is consistent with data obtained using high performance liquid chromatography coupled with hydride generation atomic fluorescence spectrometry (49). In an arsenic speciation experiment, Tu and others determined the impacts of arsenic species in soil on arsenic species in Chinese brake fern (56). They found that, regardless of the arsenic species (inorganic or organic, arsenate or arsenite) fed to the plant, As(III) is the predominant species in the fern biomass. For a typical soil, most of the As is provided to the plant as As(V), which requires a reduction step that most likely occurs within the plant after arsenic uptake. In a manner similar to that of nonhyperaccumulating plants (57), arsenate from the soil is transported into P. vittata through the phosphate uptake system, as shown by Wang and others (58). It is important to note that the hyperaccumulation of arsenic is not directly related to this transport system, as the ferns do not hyperaccumulate phosphorus. It is possible that other forms of arsenic, such as arsenite and organic species that can be accumulated in the fern, undergo alteration in the rhizosphere before uptake as hypothesized by Meharg and Hartley-Whitaker (57). Arsenic in the stems of the fronds (Table 1) show that most of the arsenic at this point has already been reduced, showing only a small fraction of detectable arsenate by X-ray spectroscopy. The speciation VOL. 37, NO. 4, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
757
TABLE 2. Results of Fitting K-edge EXAFS Data to ab Initio Parameters for Plants and Model Compoundsa
a
sample
CN
type
R (Å)
σ2
R-value
arsenite arsenate As2S2 leaf, 1 k leaf, 1 k, half-dry leaf, 1 k, dry leaf, 10 k
3 4 3 3.1 ( 0.2 3.1 ( 0.3 4.2 ( 0.4 2.4 ( 0.1 0.63 ( 0.13
As-O As-O As-S As-O As-O As-O As-O As-S
1.786 ( 0.009 1.692 ( 0.005 2.284 ( 0.007 1.776 ( 0.005 1.821 ( 0.008 1.784 ( 0.010 1.777 ( 0.002 2.272 ( 0.005
0.007 ( 0.002 0.003 ( 0.001 0.004 ( 0.001 0.003 ( 0.001 0.004 ( 0.001 0.009 ( 0.001 0.003 ( 0.001 0.001 ( 0.001
0.005 0.005 0.006 0.005 0.010 0.008 0.0005
The coordination number (CN), interatomic distance (R), and Debye-Waller factor (σ2), and the goodness-of-fit (R-value) are presented.
of arsenic in the roots of the fern could not be determined by XAS because its concentration was too low to yield interpretable spectra. However, considering arsenic uptake is performed as arsenate via the phosphate uptake system, and all forms inside the fern so far have been shown to be dominantly As(III), it is reasonable to hypothesize that the initial reduction of arsenic occurs immediately after uptake and at least before transport into the shoot and leaf biomass. This immediate reduction of arsenic after uptake is also supported by observations made by Sutton and others who showed using X-ray microtomography that a greater ratio of As(III) to As(V) existed on the inside of roots of a cattail compared to the outside (59). However, Ma and others (25) observed that only 8.3% of the arsenic present in the roots was As(III); this suggests that in addition to this immediate reduction step in the roots arsenic can also be reduced in the leaves. At this time, our understanding of the process by which As is reduced remains unclear and awaits further research. Because the thermodynamically stable oxidation state of arsenic under oxic conditions is As(V) (11), the Chinese brake fern must continually supply reductants in order to maintain arsenic in the observed As(III) state. This is supported by the observation that once the fronds are harvested and allowed to slowly dry out, the arsenic slowly oxidizes back to As(V) as shown in Table 2. Although the abiotic oxidation of arsenic by oxygen is relatively slow, as suggested by half-lives of As(III) in natural waters ranging from 20 min to more than 100 days (60, 61), there must still be a process that maintains the arsenic in the reduced form in the fern fronds. This suggests that there is an active mechanism in the brake fern, linked to its metabolic activity, that reduces arsenic. Such arsenic reduction has been observed in many bacterial assemblages in both aerobic and anaerobic settings (6265), as well as in higher plants, such as the Indian mustard (Brassica juncea) (20). At higher arsenic concentrations in the fern leaves, As displays a significant degree of coordination by sulfur atoms. Because the X-ray absorption data provide information about the average coordination of arsenic in the sample illuminated by the beam, it is difficult to extract precise information about a pure compound in a mixture. At best, we can suggest that As is present as a mixture of arsenite-oxygen and arsenitesulfur coordinated compounds. Inorganic forms of As-S, such as realgar and orpiment, are not likely because there is little to no secondary As-As or As-S shell structure in the EXAFS. As(III) has a strong affinity for thiols and thus it is likely that a thiol group is responsible for the observed arsenite-sulfur coordination. Although there is no direct evidence of As-PC binding, previous work has shown that As stimulates the production of thiol rich proteins such as phytochelatins (PCs) in Holcua lanatus (66, 67). Other works have shown clearly that As(III) is complexed entirely by thiols in plants in an As(III)-tris-glutathione compound in the Indian mustard (20) and by phytochelatins (PC) in the cells of Rauvolfia serpentina (68). Because the plant must supply 758
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 4, 2003
reductants to keep the arsenic in the observed As(III) oxidation state, these thiol compounds are strong candidates for being involved in the reduction pathway of As. In the case of the fern, these reductants may form a complex with the arsenic. Similar thiol complexes were hypothesized to be the possible reductants for As(V) in Indian mustard, as observed by Pickering and others (20). Such thiols have also been shown to reduce As(V) to As(III) in aqueous solutions (69, 70). In the case of the brake fern, the complex does appear to be either a transient or an intermediate species in comparison to the dominant aqueous arsenite species. Further analysis can also be performed to quantify the type of sulfur coordination in the plants. Considering the bond distance of the As-O shell is supportive of an aqueous As(III) ion, we can assume from fundamental coordination chemistry principles that the As-O component of the mixture will have a coordination number of three, typical of As(III) ions (71). Thus, we can calculate that approximately 80% of the As exists in this state to lower the observed coordination of As-O of 2.4. Assuming that the only other major detectable species is the As-S compound, we can determine its coordination number by dividing the observed coordination of 0.63 by its percentage in the sample (20%) to get a coordination number of 3.15. This suggests that the As-S coordination in the plant, on average, has a coordination number of effectively 3. It is important to note that since this result is based on the coordination number determinations of the EXAFS the estimated error of this value is approximately 30%. Based on the assumption above that the sulfur group that is coordinating arsenic is a thiol group of some type (i.e., glutathione, phytochelatin) this As compound in the plant exists as a sulfur tris-thiolate complex. However, these XAS results cannot distinguish in this case if the binding of the thiol groups results from three distinct monodentate thiol containing ligands or if bidendate or tridentate binding is occurring. The difference between the composition of the As-thiol contribution in the EXAFS fitting and XANES spectral deconvolution is most likely due to the fact that the XANES used As2S2 as a model compound. This compound, although it maintains a relative edge position similar to that of thiol groups, may be significantly different in its other XANES characteristics (e.g., magnitude, resonances) than the arsenicthiol complex. In addition, the XANES edge at these types of concentrations should be used only to suggest that an As-S phase is present and not to quantitatively determine or identify it. To resolve the true identity of the As-S shell, one needs to isolate this compound to perform XAS experiments together with other arsenic-thiol reference compounds in the future. A further limitation of the XAS technique is that it will detect only the major species of As in the plant. It is likely that there are several other minor species that exist in the plant tissues that are not detected below the 5% level. A major difference between P. vittata and the observations in nonhyperaccumulators such as the Indian mustard is that the thiol-coordinated arsenic compound is the minority
species in the case of the fern. This speciation difference may play an important role in understanding the hyperaccumulating nature of P. vittata. Because As(III) has a high affinity for thiol groups and is phytotoxic, any As(III) must be either complexed in a nontoxic form, such as by PCs, or stored away from metabolic activity in vacuole. The former appears to occur in many resistant, yet nonhyperaccumulating plants (20, 67, 68). However, in order for this to be the case with hyperaccumulating plants, the fixed complexation of arsenite into tris-sulfur compounds would be a major drain of resources. Zhao et. al. infer that up to 60 to 100% of the observed sulfur in P. vittata fronds would need to be accounted for the synthesis of the complexing agent (72). A possible alternative is that arsenite is complexed by thiol-containing PCs and transported from the roots to the shoots and fronds and stored in vacuoles. The PCs are then recycled by the plant for further translocation of arsenic (72, 73). Attempts to directly detect As-PC compounds by HPLCICPMS have been unsuccessful (58, 74), but this may be due to instabilities of the complexes in extraction or subsequent HPLC separation (68). The appearance of putative As-thiol coordination observed in this study may be the first evidence in support of PC shuttling. However, the sulfur bound arsenic was observed only in plants that had large concentrations of arsenic. This may be due to the fact that the As-PC species is transient and is only observed above detection levels under high arsenic levels. In the other cases, the background As(III) as arsenite in vacuoles masks any arsenic present in thiolate complexes. Because measurements were performed without any alteration of the plant material, this gives an inital insight into the native speciation of arsenic in hyperaccumulators. Further spatially resolved XAS experiments need to be performed to determine whether the As(III) in the plant fronds is actually present in vacuoles.
Acknowledgments Funding for this work was provided through the U.S. National Science Foundation, Grant BES-0132114. This work was performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron Research Center located at Sector 5 of the Advanced Photon Source. DNDCAT is supported by the E.I. DuPont de Nemours & Co., The Dow Chemical Company, the U.S. National Science Foundation through Grant DMR-9304725, and the State of Illinois through the Department of Commerce and the Board of Higher Education Grant IBHE HECA NWU 96. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research under Contract W-31-102-Eng-38. We thank I. Pickering and three anonymous reviewers for providing helpful comments that improved the manuscript.
Literature Cited (1) National Research Council. Arsenic in Drinking Water; National Academy Press: Washington, DC, 1999. (2) Pontius, F. W.; Brown, K. G.; Chen, C. J. J. Am. Water Works Assoc. 1994, 86, 52-63. (3) Nriagu, J. O. In Advances in Environmental Science and Technology; Nriagu, J. O., Ed.; Wiley-Interscience: New York, 1994; Vol. 26, p 430. (4) Smedley, P. L.; Kinniburgh, D. G. Appl. Geochem. 2002, 17, 517568. (5) Huang, Y.-C. In Arsenic in the Environment, Part I: Cycling and Characterization; Nriagu, J. O., Ed.; John Wiley: New York, 1988. (6) Welch, A. H.; Lico, M. S.; Hughes, J. L. Groundwater 1988, 26, 333-347. (7) Hering, J. G.; Wilkie, J. A. Environ. Sci. Technol. 1998, 32, 657662. (8) Bowell, R. J. Appl. Geochem. 1994, 9, 279-286. (9) Wang, C. F.; Chang, C. Y.; Chin, C. J.; Men, L. C. Anal. Chim. Acta 1999, 392, 299-306.
(10) Peryea, F. J.; Creger, T. L. Water Air Soil Pollut. 1994, 78, 297306. (11) Cullen, W. R.; Reimer, K. J. Chem. Rev. 1989, 89, 713-764. (12) Korte, N. E.; Fernandao, Q. Crit. Rev. Environ. Control 1991, 21, 1-39. (13) National Research Council. Arsenic; National Academy Press: Washington, DC, 1977. (14) Koch, I.; Wang, L.; Ollson, C. A.; Cullen, W. R.; Reimer, K. J. Environ. Sci. Technol. 2000, 34, 22-26. (15) Francesconi, K.; Visoottiviseth, P.; Sridokchan, W.; Goessler, W. Sci. Total Environ. 2002, 284, 27-35. (16) Larsen, E. H.; Hansen, M.; Gossler, W. Appl. Organomet. Chem. 1998, 12, 285-291. (17) Madsen, A. D.; Goessler, W.; Pedersen, S. N.; Francesconi, K. A. J. Anal. Atomic Spectrosc. 2000, 15, 657-662. (18) Edmonds, J. S.; Francesconi, K. A.; Stick, R. V. Nat. Prod. Rep. 1993, 10, 421-428. (19) de Haro, A.; Pujadas, A.; Polonio, A.; Font, R.; Velez, D.; Montoro, R.; del Rio, M. Fresenius Environ. Bull. 2000, 9, 275-280. (20) Pickering, I. J.; Prince, R. C.; George, M. J.; Smith, R. D.; George, G. N.; Salt, D. E. Plant Physiol. 2000, 122, 1171-1177. (21) Wong, H. K. T.; Gauthier, A.; Nriagu, J. O. Sci. Total Environ. 1999, 228, 35-47. (22) Chen, H. M.; Zheng, C. R.; Tu, C.; Z.G., S. Chemosphere 2000, 41, 229-234. (23) Cunningham, S. D.; Shann, J. R.; Crowley, D. E.; Anderson, T. A. In Phytoremediation of Soil and Water Contaminants; Kruger, E. L., Anderson, T. A., Coats, J. R., Eds.; American Chemical Society: Washington, DC, 1997; Vol. 664, pp 2-17. (24) Cooper, E. M.; Sims, J. T.; Cunningham, S. D.; Huang, J. W.; Berti, W. R. J. Environ. Qual. 1999, 28, 1709-1719. (25) Ma, L. Q.; Komar, K. M.; Tu, C.; Zhang, W.; Cai, Y.; Kennelley, E. D. Nature 2001, 409, 579. (26) Fendorf, S. E.; Lamble, G. M.; Stapleton, M. G.; Kelley, M. J.; Sparks, D. L. Environ. Sci. Technol. 1994, 28, 284-289. (27) Fendorf, S.; Eick, M. J.; Grossl, P.; Sparks, D. L. Environ. Sci. Technol. 1997, 31, 315-320. (28) Manning, B. A.; Fendorf, S. E.; Goldberg, S. Environ. Sci. Technol. 1998, 32, 2383-2388. (29) Korshin, G. V.; Frenkel, A. I.; Stern, E. A. Environ. Sci. Technol. 1998, 32, 2699-2705. (30) Bargar, J. R.; Reitmeyer, R.; Davis, J. A. Environ. Sci. Technol. 1999, 33, 2481-2484. (31) Thompson, H. A.; Brown, G. E., Jr.; Parks, G. A. Am. Mineralog. 1997, 82, 483-496. (32) Templeton, A. S.; Ostergren, J. D.; Trainor, T. P.; Foster, A. L.; Traina, S. J.; Spormann, A.; Brown, G. E., Jr. J. Synchrotron Radiat. 1999, 6, 642-644. (33) Stern, E. A.; Heald, S. M. In Handbook on Synchrotron Radiation; Koch, E., Ed.; North-Holland Publishing: New York, 1983; Vol. 1B, pp 955-1014. (34) Chisholm-Brause, C. J.; P. A., O. D.; Brown, G. E., Jr. Nature 1990, 348, 528-530. (35) Brown, G. E., Jr. In Mineral-Water Interface Geochemistry; Hochella, M. F., White, A., Eds.; Mineralogical Society of America: Washington, DC, 1990; Vol. 23, pp 314-384. (36) Foster, A. L.; Brown, G. E.; Parks, G. A. Environ. Sci. Technol. 1998, 32, 1444-1452. (37) Welterm, E.; Calmano, W.; Mangold, S.; Tro¨ger, L. Fresnius’ J. Anal. Chem. 1999, 364, 238-244. (38) Manceau, A.; Boisset, M. C.; Sarret, G.; Hazemann, R. L.; Mench, M.; Cambier, P.; Prost, R. Environ. Sci. Technol. 1996, 30, 15401552. (39) O’Day, P. A.; Carroll, S. A.; Waychunas, G. A. Environ. Sci. Technol. 1988, 32, 943-955. (40) Ostergren, J. D.; Brown, G. E., Jr.; Parks, G. A.; Tingle, T. N. Environ. Sci. Technol. 1999, 33, 1627-1636. (41) Sarret, G.; Manceau, A.; Spadini, L.; Roux, J.-C.; Hazemann, J.-L.; Soldo, Y.; Eybert-Berard, L.; Menthonnex, J.-J. Environ. Sci. Technol. 1998, 32, 1648-1655. (42) Webb, S. M. Ph.D. Thesis, Northwestern University, Evanston, IL, 2001. (43) Pilon-Smits, E. A. H.; de Souza, M. P.; Lytle, C. M.; Shang, C.; Lugo, T.; Terry, N. J. Exp. Bot. 1998, 49, 1889-1892. (44) Polette, L. A.; Gardea-Torresdey, J. L.; Chianelli, R. R.; George, G. N.; Pickering, I. J.; Arenas, J. Microchem. J. 2000, 65, 227236. (45) Lytle, C. M.; Lytle, F. W.; Smith, B. N. J. Environ. Qual. 1996, 25, 311-316. (46) Lytle, C. M.; Lytle, F. W.; Yang, N.; Qian, J. H.; Hansen, D.; Zayed, A.; Terry, N. Environ. Sci. Technol. 1998, 32, 3087-3093. (47) Zayed, A.; Lytle, C. M.; Terry, N. Planta 1998, 206, 284-292. VOL. 37, NO. 4, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
759
(48) Zayed, A.; Lytle, C. M.; Qian, J. H.; Terry, N. Planta 1998, 206, 293-299. (49) Tu, C.; Ma, L. Q. J. Environ. Qual. 2002, 31, 641-647. (50) Quintana, J. P. G. Synchrotron Radiat. Instrum. 2000, 194-197. (51) Gaillard, J.-F.; Webb, S. M.; Quintana, J. P. G. J. Synchrotron Radiat. 2001, 8, 928-930. (52) Newville, M.; Livins, P.; Yacobi, Y.; Stern, E. A.; Rher, J. J. Phys. Rev. B 1993, 47, 14126-14131. (53) Ankudinov, A. L.; Ravel, B.; Rehr, J. J.; Conradson, S. D. Phys. Rev. B 1998, 58, 7565. (54) Rehr, J. J.; Albers, R. C.; Zabinsky, S. I. Phys. Rev. Lett. 1992, 69, 3397. (55) Newville, M. J. Synchrotron Radiat. 2001, 8, 322-324. (56) Tu, C.; Ma, L. Q.; Zhang, W.; Cai, Y. Environ. Pollut. 2002, Accepted for publication. (57) Meharg, A. A.; Hartley-Whitaker, J. New Phytol. 2002, 154, 2943. (58) Wang, J.; Zhao, F. J.; Meharg, A. A.; Raab, A.; Feldmann, J.; McGrath, S. P. Plant Physiol. 2002, In press. (59) Sutton, S. R.; Newvile, M.; Rivers, M.; Eng, P.; Hansel, C.; Fendorf, S. Presented at the 222nd National Meeting of the American Chemical Society, Chicago, IL, 2001. (60) Pettine, M.; Campanella, L.; Millero, F. J. Geochim. Cosmochim. Acta 1999, 63, 2727-2735. (61) Kim, M. Y.; Nriagu, J. O. Sci. Total Environ. 2000, 247, 71-79. (62) Myers, D. J.; Heimbrook, M. E.; Osteryoung, J.; Morrison, S. M. Environ. Lett. 1973, 5, 53.
760
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 4, 2003
(63) (64) (65) (66) (67) (68) (69) (70) (71)
(72) (73) (74)
Johnson, D. L. Nature 1972, 240, 44. Forsberg, C. W. Can. J. Microbiol. 1978, 24, 36. Freeman, M. C. N. Z. J. Freshwater Res. 1985, 19, 277. Hartley-Whitaker, J.; Ainsworth, G.; Vooijs, R.; Bookum, W. T.; Schat, H.; Meharg, A. A. Plant Physiol. 2001, 126, 299-306. Hartley-Whitaker, J.; Woods, C.; Meharg, A. A. New Phytol. 2002, 155, 219-225. Schmoger, M. E. V.; Oven, M.; Grill, E. Plant Physiol. 2000, 122, 793-801. Delnomdedieu, M.; Basti, M. M.; Otvos, J. D.; Thomas, D. J. Chem.-Biol. Interact. 1994, 90, 139-155. Carter, D. E. Environ. Health Perspect. 1995, 103, 17-19. Cotton, F. A.; Wilkinson, G.; Murillo, C. A.; Bochmann, M. Advanced Inorganic Chemistry, 6th ed.; John Wiley: New York, 1999. Zhao, F. J.; Dunham, S. J.; MacGrath, S. P. New Phytol. 2002, 156, 27-31. Meharg, A. A. New Phytol. 2002, 156, 1-8. Fransconi, K.; Visoottiviseth, P.; Sridokchan, W.; Goessler, W. Sci. Total Environ. 2002, 284, 27-35.
Received for review June 4, 2002. Revised manuscript received November 27, 2002. Accepted December 10, 2002. ES0258475
