Environ. Sci. Technol. 2002, 36, 2783-2788
Evidence of Perchlorate (ClO4-) Reduction in Plant Tissues (Poplar Tree) Using Radio-Labeled 36ClO4BENOIT VAN AKEN AND JERALD L. SCHNOOR* Department of Civil and Environmental Engineering, 4112 Seamans Center, The University of Iowa, Iowa City, Iowa 52242
Phytoremediation of perchlorate (ClO4-) by poplar trees Populus deltoide x nigra was investigated using small cuttings growing in hydroponic Hoagland solution and plant tissue cultures, consisting of spherical photosynthetic cell aggregates (i.e. nodules) developing in Murashige and Skoog culture medium. Both plants and nodules were grown under a 16 h/8 h photoperiod cycle and under sterile conditions. Degradation experiments, performed by the incubation of pregrown plants and nodules in the presence of 36Cl radio-labeled ClO4- (25 mg L-1), showed a reduction of the initial ClO4- concentration in the solution of about 50% after 30 d of incubation. Analysis of the distribution of radioactivity in different plant fractions indicated that 27.4% of the total was translocated to the leaves, while 66.9% remained in the solution. Very little radioactivity (less than 3.0%) was detected in the other parts of the plants. 32.0% of the radioactivity recovered in the solution was shown to consist of 36Cl- and 68.0% of nontransformed 36ClO4-. The radioactivity recovered in the leaf extracts was distributed as chloride (36Cl-) (1.6% of the total), chlorite (36ClO2-) (2.4%), chlorate (36ClO3-) (4.8%), nontransformed 36ClO4- (21.6%), and an unidentified organic compound (1.4%). The radioactivity recovered in the solution containing submerged nodules consisted of 36Cl(6.4% of the total), 36ClO3- (1.3%), and nontransformed 36ClO - (51.5%). Radioactivity detected in the nodule extracts 4 was distributed as 36Cl- (2.0% of the total), 36ClO2(5.2%), 36ClO3- (6.4%), 36ClO4- (22.7%), and an unidentified organic compound (0.5%). These results provide evidence of perchlorate reduction inside poplar tree tissues. 36ClO4is partially reduced to 36ClO3-, 36ClO2-, and 36Cl-.
Introduction Since the mid-1990s, bioremediation of contaminated soils, groundwater, and surface water has entered a new fields namely phytoremediationsinvolving plants and plantmicrobe systems. Phytoremediation encompasses a range of processes and may be best described as a plant-assisted remediation. Phytoremediation may include rhizofiltration (i.e. removal by roots), phytotransformation (i.e. transformation in plant tissues), phytovolatilization (i.e. volatilization through leaves), phytostabilization (i.e. binding to soil particles or plant tissues), and rhizodegradation (i.e. biodegradation by rhizosphere associated microbes) (1). The * Corresponding author phone: (319)335-5649; fax: (319)335-5660; e-mail:
[email protected]. 10.1021/es020560t CCC: $22.00 Published on Web 05/11/2002
2002 American Chemical Society
idea of using plants for cleaning up polluted soils and water began with applications for heavy metals, which are not susceptible to chemical degradation and require exposed plants to be harvested and burned or landfilled, a process referred to as phytoextraction (2-4). However, besides uptake and storage, plants are known to metabolize a range of organic pollutants, including pesticides, nitro-substituted explosives, organochlorides, polychlorinated biphenyls (PCB), polyaromatic hydrocarbons (PAH), etc. (3, 5, 6). The green liver model describes the plant metabolism of organic contaminants as a detoxification mechanism (7, 8). Ammonium perchlorate (NH4ClO4) has been used extensively as an oxidizer and a primary component in explosives and solid missile propellants. Perchlorate anion (ClO4-) has also been found in certain fertilizers and in water treatment chemicals (9, 10). Recently, perchlorate has been observed in groundwater at numerous hazardous waste sites in North America (11, 12). Perchlorate has also been detected in surface water (Colorado River, Lake Mead, AZ, NV) at levels up to 1700 µg L-1 and in water supplies (Las Vegas, NV) at levels up to 280 µg L-1 (13). While perchlorate is not listed under the National Primary Drinking Water Regulation (NPDWR), the U.S. Environmental Protection Agency (USEPA) has included it on its Drinking Water Contaminant Candidate List (CCL) (14). In addition, four states have currently drinking water action levels for perchlorate, i.e., the California Department of Health Services (DHS) and the Nevada Division of Environmental Protection (DEP) have established Health Based Action Levels of 18 µg L-1 (1997), the Texas Natural Resource Conservation Commission (TNRCC) has established a Interim Action Level of 22 µg L-1 (January 1999), and the Arizona Department of Environmental Quality (ADEQ) has established a Health Based Guidance Level of 14 µg L-1 (May 2000) (14). Finally, based on a previously determined Provisional NonCancer Reference Dose (RfD), the USEPA has calculated a Guidance Level of groundwater cleanup of 4-18 µg L-1 (14). The health hazard of perchlorate is mainly related to its capacity of blocking iodine uptake by the thyroid, which impedes synthesis of thyroid hormones (L-triiodothyronine T3 and L-tetraiodotyrosine T4), leading to cretinism, an iodine-deficient mental disease observed among isolated populations around the world (15). From a physical and chemical viewpoint, perchlorate salts are extremely water soluble, and perchlorate anion, although highly oxidized, is stable in the aqueous environment, even under reducing conditions (16). Given the increasing threat of perchlorate contamination, typical physical and chemical water treatment technologies have been tested (e.g. ion exchange, air stripping, carbon adsorption, etc.) with no costeffective satisfactory results (16). Until recently, only anaerobic bacteria have been reported to reduce perchlorate into harmless chloride (16, 17). Several facultative anaerobic bacterial strains have been shown to use chlorate and perchlorate as terminal electron acceptors under anaerobic conditions (16-20). Perchlorate-degrading bacteria are dissimilatory (per)chlorate-reducing strains, able to perform the enzymatic stepwise reduction of perchlorate to chloride (Cl-) (18, 21). In a recent report, Nzengung et al. (22) have shown that woody plants (Salix spp., Populus ssp., and Eucalyptus cineria) are able to decontaminate perchlorate-polluted water. The phytoremediation process involves two successive phases: the first one including uptake and partial reduction of perchlorate in plant tissues (22) and the second one attributed to perchlorate-degrading microbes developing in the root zone. Similarly, Susarla et al. (23) have described the VOL. 36, NO. 12, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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capacities of six plant species, including two tree species (Liquidambar styraciflua, S. nigra), two herbaceous wetland species (Allenrolfea occidentalis, Polygonum punctatum), and two herbaceous aquatic species (Nymphea odorata, Spirodela polyrhyza), to take up and to partially reduce perchlorate inside the plant tissues. Uptake of perchlorate without efficient transformation results in storage inside plant tissues. Sooner or later, accumulated perchlorate will return to the soil or enter the food chain. Strictly speaking, uptake and storage of perchlorate inside the plant only results in a pollution transfer. Validation of the phytoremediation process requires transformation to innocuous products and a further understanding of the fate of perchlorate inside the plant organism. The objective of this study is to demonstrate that reduction of perchlorate to chloride occurs within poplar tissues and to determine the actual capacity of plant tissues for transformation. Using poplar tree cuttings and tissue cultures under sterile conditions, the pure action of plant cells (i.e. without the presence of any microbial contamination) in the transformation of perchlorate has been investigated. Working with 36Cl radio-labeled perchlorate, it has been possible to track reduced metabolites and to determine with certainty their perchlorate origin. Experiments with tissue cultures were based on an original material consisting of spherical photosynthetic cell aggregatessreferenced as nodules (24)swhich were shown to constitute a useful model system, quite promising for the investigation of any phytoremediation system at the laboratory level.
Materials and Methods Hybrid Poplar Trees. Poplar tree (genus Populus, family Salicaceae) is one of the most studied woody plants, due to its potential for pulp and paper production and its particular usefulness in phytoremediation, e.g. fast growth, large transpiration flux, and regrowth from cut stems (25-27). Small trees used for in vivo degradation experiments were produced as previously described (25). In short, 8-in. dormant cuttings of hybrid poplar trees (Populus deltoides x nigra DN34) were grown in hydroponic solution, under a 16-h/8-h (light/dark) photoperiod. The growth medium consisted of half-strength modified Hoagland nutrient solution at pH 7.0 (28). In Vivo Degradation Experiments. After about 2 weeks, the prerooted cuttings, possessing both shoots and roots, were transferred individually into bioreactors consisting in 250-mL conical flasks. Before the transfer, the lower parts of the plant (lower stem and roots in contact with the solution) were sterilized by immersion successively for 30 s in 70% aqueous ethanol and for 30 s in 1.0% commercial bleaching solution (5.25% initial sodium hypochloride, NaClO) containing 0.1% Tween 80, before being rinsed for 5 min in sterilized distilled water. Each flask contained 200 mL of the growth medium described above and was supplemented with 25 mg L-1 radio-labeled perchlorate (36ClO4-, sodium salt) exhibiting a specific activity of 400 000 dpm mg-1. The bioreactors were sterilized by autoclaving 20 min at 121 °C, 1 atm before introducing the plant. They were closed with a drilled Teflon-lined septum and screw cap fitting with the stem passing through. The septum was sealed to the wood segment with Teflon tape and silicon caulk. Conical flasks possessed lateral tubing in the upper part, which was stoppered with a cotton plug, allowing gas exchanges with external air. The conical flasks, housing the submerged parts of the plants, were wrapped with aluminum foil in order to prevent light infiltration. The flasks were refilled every 1-3 days with sterilized distilled water, to compensate for transpiration by the leaves. The transpiration volumes were recorded. The concentrations of ClO4- and its reduction derivatives, i.e., chlorate (ClO3-), chlorite (ClO2-), and Cl-, 2784
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in the nutrient solution were monitored by removing periodically 1-mL aliquots. Samples were frozen at -40 °C overnight and filtered on 0.22-µm Acrodisk filters (Pall Gelman Laboratory, Ann Arbor, MI) before being analyzed by ion chromatography (IC). Manipulations involving opened flasks were performed under a sterile laminar flow hood. After 4 weeks, the cultures were sacrificed in order to determine the distribution of ClO4- and potential metabolites in the different plants fractions (22). Plant material was extracted as follows: The different plant fractions, i.e., roots, lower stems (i.e. submerged stems), upper stems (i.e. aerial stems), and leaves were washed with distilled water, wiped, and weighed, before being dried overnight at 105 °C. Plant fractions were ground mechanically. The resulting raw powdered material was then mixed with a defined volume (25-75 mL) of sodium hydroxide (NaOH, 0.1 M) and homogenized for 2 min. The suspension was introduced in centrifugation tubes and sonicated overnight. The samples were centrifuged at 15 000 rpm for 30 min. Then, the supernatant was frozen overnight, filtered (0.22-µm), and stored at 4 °C for further analysis. Two further extractions on remaining pellets did not allow the recovery of more than 1.0% of the ClO4- extracted in the first step, indicating that one extraction procedure was sufficient. Bioreactors supplemented with ClO4-, but containing a plant whose stem had been cut off at the surface of the screw cap, were used as controls. Another control consisted of bioreactors containing a whole tree incubated without ClO4-. Experiments were performed with three replicates. To ensure the absence of microbial contamination during the degradation experiments, 0.1-mL aliquots of the nutrient solutions were plated both on nutrient agar 2.3% (Difco, Sparks, MD) and on yeastextract agar 2.0% (Difco). Inoculated Petri dishes were incubated for 1 week at 37 °C prior to observation. Production of Nodules. Explants from a hybrid poplar tree (Populus deltoides x nigra DN34), consisting of 1-cm length pieces of young stems or 1-cm2 pieces of young leaves, were sterilized by immersion while stirring successively in 70% aqueous ethanol (30 s), in 5.0% bleaching solution containing 0.1% Tween 80 (15 min), in 0.05% mercuric chloride (HgCl2) (15 min), and rinsed several times in sterilized distilled water. Sterilized explants were grown on a solid medium, Murashige and Skoog (29) culture medium (MS) at pH 5.8 supplemented with 30 g L-1 of sucrose, 0.9 g L-1 of agar, and 5.0 mg L-1 of 2,4-dichlorophenoxyacetic acid (2,4D), and 1.0 mg L-1 of 6-furfurylaminopurine (kinetin) as growth regulators. After about 1 month of growth under a 16-h/8-h photoperiod, cell calli developed actively. About 1-cm3 callus material was introduced in 500-mL conical flasks containing 300 mL MS liquid medium (without agar) supplemented with 30 g L-1 sucrose and the same combination of growth regulators. The flasks were then incubated under agitation at 125 rpm on an orbital shaker and under a 16-h/8-h photoperiod. After about 1 month, the callus material developed in the form of a cell suspension (free cells and small aggregates) containing 20-50 spherical green plant cell aggregates of about 2-20 mm diameter, referred as nodules (24). Cell suspension and nodules were separated by filtration on cheesecloth. Nodules were shown to be photosynthetic. Microscopic observation revealed a radial cell organization and a chaotic vascularization. Manipulations were performed under strict sterile conditions. In Vitro Degradation Experiments. About 10 cm3 nodule material (3-4 pieces) was introduced into 250-mL conical flasks stoppered with a cotton plug and containing 100 mL of MS liquid medium supplemented with 30 mg L-1 of sucrose, 5 mg L-1 of 2,4-D, 1 mg L-1of kinetin, and 25 mg L-1 of 36ClO4- (sodium salt, 400 000 dpm mg-1). Flasks were incubated under agitation at 125 rpm under a 16-h/8-h photoperiod. One-milliliter aliquots of medium were col-
lected periodically and treated as described above. Nodules were separated from a light cell suspension by filtrating the solution on cheesecloth. Nodule material was extracted as described above for the plant fractions. Control experiments were conducted under the same conditions but contained either autoclaved nodules in the presence of ClO4- or living nodules without ClO4-. Experiments were performed in triplicate. Analysis. Analysis of ClO4- and reduction metabolites, i.e., ClO3-, ClO2-, and Cl-, were performed by ion chromatography (IC) using a Dionex DX-500 ion chromatograph (Dionex Co., Sunnyvale, CA) equipped with a Dionex ASRS suppressor operating in external water mode. Twenty to 200 µL injections were made using a Dionex AS50 autosampler. Separation was achieved on a Dionex AS11 column maintained at 40 °C using a NaOH eluent flowing at 1 mL min-1. For the quantitative determination of ClO4- alone, the injection volume was 50 µL, and the eluent was an isocratic solvent system consisting of 25 mM NaOH. For the simultaneous determination of ClO4- and ClO4- metabolites, the injection volume was 20 µL, and the eluent was a gradient solvent system. The initial solvent composition was 100% H2O, a linear gradient up to 2 mM NaOH was run over 20 min, and then a linear gradient up to 25 mM NaOH was run over the next 5 min and held for 5 min. Finally, a linear gradient came back to the initial solvent of 100% H2O, and the column was equilibrated for 5 min. Intermediates and products were identified by comparison of their retention times with authentic standards. The total 36Cl radioactivity in the solutions and in the extracts was determined by mixing 100 µL of sample and 900 µL of H2O with 9 mL of scintillation cocktail (Ultima Gold XR, Packard Instrument Co., Meriden, CA) and by analyzing the radioactivity (dpm) using a Beckman liquid scintillation counter (LSC) LS 6000IC (Beckman Coulter Inc., Fullerton, CA). The total radioactivity, expressed in dpm, was processed using the Auto DPM method (Beckman). Distribution of the radioactivity in the samples was analyzed by IC using the gradient system described above. The injection volume was a 200 µL sample, and 1-mL fractions of outflow were collected every min on a fraction collector Dynamax FC-1 (Rainin Instrument Inc., Woburn, MA). Onemilliliter fractions were mixed with 9 mL of scintillation cocktail before being assayed by LSC as described above. Synthesis of 36Cl Radio-Labeled Perchlorate. 36Cl radiolabeled ClO4- was synthesized from 36Cl radio-labeled sodium chloride (Na36Cl) (DuPont NEN, Boston, MA) by electrochemical oxidation of chloride (36Cl-) according to a modified method described by Walton (30). In short, Na36Cl (1.0 mCi mL-1) was mixed with nonlabeled NaCl in aqueous solution to a concentration of 165 mg L-1 NaCl (i.e. 100 mg L-1 Cl-) and a radioactivity of 2.2 × 108 dpm mL-1. The solution was slightly acidified with a few drops of 0.1 N HCl. Three milliliters of the reaction mixture underwent electrolysis in a stirred 10-mL sealed cell. Electrodes were platinum (Pt) wires, and the system worked at a fixed current intensity of 0.025 A for 2 days, increased to 0.05 A on the third day. The potential difference was about 3 V. Samples were collected periodically to monitor the reaction advancement, i.e., the stepwise oxidation of Cl- into hypochlorite (ClO-), ClO2-, ClO3-, and ClO4-. After 2 weeks, the reaction yield was 100% formation of ClO4-. The reaction mixture was further purified by recrystallization and treatment on OnGuard-Ag cartridge (Dionex). Labeled and nonlabeled ClO4- were mixed to obtain a stock solution with a final concentration of 5.0 mg mL-1 of ClO4- and a specific radioactivity of 2.0 × 106 dpm mL-1 (i.e. 400 000 dpm mg-1 of ClO4-). Chemicals. All chemicals were of analytical grade and purchased from Fluka (Ronkonkoma, NY) or Sigma (St. Louis, MO). Plant growth regulators were from Sigma. 36Cl radio-
FIGURE 1. Uptake of ClO4- (initial concentration 25 mg L-1) by poplar trees as measured by the ClO4- concentration (ion chromatography) and by the radioactivity (LSC) remaining in solution.
FIGURE 2. Uptake of ClO4- (initial concentration 25 mg L-1) by plant nodules as measured by the ClO4- concentration (ion chromatography) and by the radioactivity (LSC) remaining in solution. labeled sodium chloride (Na36Cl) was purchased from DuPont NEN.
Results Both trees and nodules grew actively during the time of the experiments (30 d). The average growth of the trees exposed to ClO4-, measured by the increase of fresh biomass, was 0.24 ( 0.07 g d-1 (or 0.61 ( 0.22% of the initial weight d-1) and did not show any significant differences with the controls grown without ClO4- (0.24 ( 0.09 g d-1 or 0.74 ( 0.34% d-1). Similarly, the cumulated transpiration volumes over the time of the experiment were not significantly different for the plants growing either in the presence (23.4 ( 3.3 mL g-1 fresh weight) or in the absence of ClO4- (23.9 ( 1.4 mL g-1 fresh weight). The increase of nodule biomass in the presence of ClO4- was 0.23 ( 0.03 g d-1 (or 3.03 ( 1.31% of the initial weight d-1), which was comparable to the increase of the biomass observed in the controls without ClO4- (0.25 ( 0.08 g d-1 or 3.27 ( 1.45% d-1). Over the 30-d experiment, trees were shown to reduce continuously the ClO4- concentration in the hydroponic solution to 51.6 ( 1.2% of the initial 25 mg L-1, which corresponds to a specific reduction rate of 1.9 ( 0.2 ng d-1 g-1 of fresh plant biomass in average over the period. At the same time, the radioactivity in solution decreased to 67.2 ( 2.3% of the initial concentration after 30 d (Figure 1). Neither the ClO4- concentration nor the radioactivity decreased significantly in the control solutions with excised trees, i.e., consisting only of lower stem and roots. The submerged plant nodules were shown to reduce the initial ClO4- concentration from the liquid medium to 50.4 ( 5.8% of the initial 25 mg L-1 after 30 d. Radioactivity in VOL. 36, NO. 12, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Distribution of ClO4- (Initial Concentration 25 mg L-1) in Different Plant Fractions after 30 d of Exposure fraction
leaves
radioactivity 36Cl (% of the initial)
roots
27.35 ( 4.01
hydroponic solution
balance
Plants Exposed to 36ClO41.27 ( 0.32 0.84 ( 0.33 0.44 ( 0.19
66.93 ( 2.03
96.83 ( 2.48
Controls Exposed to 36ClO42.53 ( 1.10
100.81 ( 6.49
103.34 ( 7.37
radioactivity 36Cl (% of the initial)
upper stems
lower stems
TABLE 2. Distribution of ClO4- (Initial Concentration 25 mg L-1) in Different Fractions of Nodule Cultures after 30 d of Exposure fraction
nodules
free cells 36ClO 4
nutrient solution
balance
radioactivity 36Cl (% of the initial)
Nodules Exposed to 43.00 ( 2.04 0.00 ( 0.00
53.13 ( 7.00
96.13 ( 8.19
radioactivity 36Cl (% of the initial)
Controls Exposed to 36ClO41.37 ( 0.23 0.00 ( 0.00
101.14 ( 7.33
102.51 ( 7.41
TABLE 3. Distribution (Percent of the Total) of the Radioactivity Originating from the Initial 36ClO4- (25 mg L-1) among Reduced Metabolites in Different Extracts of the Plants after 30 d of Exposure fraction
ClO4-
solution t ) 0 100.00 solution t ) 30 d 41.75 leaf extract 21.59 control solution 102.30 t ) 30 d
ClO3- ClO20.00 0.00 4.81 0.00
Cl-
0.00 0.00 0.00 19.67 2.42 1.64 0.00 0.0
unidentified
total
0.00 0.00 1.37 0.00
100.00 61.42 31.83 102.30
solution decreased to 53.1 ( 7.0% of the initial concentration over the same period of time (Figure 2). The average specific rate of ClO4- reduction was 2.6 ( 0.3 ng d-1 g-1 of fresh biomass. No significant change of ClO4- concentration or radioactivity was recorded in the control solutions containing autoclaved nodules. Analysis of the distribution of the radioactivity in the different parts of the plants showed that 27.4 ( 4.0% of the initial radioactivity was translocated to the leaves, which represents 91.5 ( 13.4% of the total radioactivity taken up by the plants. Very little radioactivity (2.6 ( 0.8% of the initial) was detected in the other parts of the plants (i.e. roots, lower stems, and upper stems) (Tables 1 and 2). Looking at the chemical speciation of radioactive 36Cl revealed that 32.0% of the radioactivity recovered in the solution after 30 d (i.e. 19.7% of the total) consisted of 36Cl- and 68.0% (i.e. 41.8% of the total) of nontransformed 36ClO4-. On the other hand, the radioactivity recovered in the leaf extracts was distributed into 36Cl- (1.6% of the total), 36ClO2- (2.4% of the total), 36ClO3(4.8% of the total), 36ClO4- (21.6% of the total), and an unidentified, likely organic compound (1.4% of the total) (Table 3 and Figure 3). The radioactivity recovered in the solution containing submerged nodules consisted of 36Cl- (6.4% of the total), 36ClO - (1.3% of the total), and nontransformed 36ClO - (51.5% 3 4 of the total). The radioactivity detected in the nodule extracts was distributed into 36Cl- (2.0% of the total), 36ClO2- (5.2% of the total), 36ClO3- (6.4% of the total), 36ClO4- (22.7% of the total), and an unidentified organic compound (0.5% of the total) (Table 4 and Figure 4).
Discussion Our results have shown that both trees in hydroponic solution and nodules in liquid suspension were able to remove perchlorate (ClO4-) from the nutrient solution. Since the uptake rates did not seem to slow significantly at the end of the experiment, it may be assumed that the uptake process 2786
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was still active. No toxic effect of ClO4- occurred at the concentration applied (25 mg L-1). The increase in biomass of plants and nodules growing either in the presence or in the absence of ClO4- did not show significant differences. Similarly, the transpiration volumes of trees grown in the presence of perchlorate were comparable to those recorded in controls without ClO4-, which further suggests that there was no toxic effect of ClO4- at the concentration tested. This is consistent with previous observations reporting no phytotoxic effect of ClO4- in the solution below 2500 mg l-1, i.e., 2 orders of magnitude higher than the concentration applied in our experiments (22, 23). While plant cell cultures are known to grow slowly, the nodule cultures showed a biomass growth rate similar to that of whole plants. This surprising result could be explained by the presence of sucrose in cell culture nutrient solution, supporting the growth of nodules in addition to the natural photosynthetic process. The initial concentration of ClO4- was reduced not only by one-half both by trees and nodules over the time of the experiment, suggesting an active uptake by the roots, but also by the photosynthetic tissue in the periphery of nodules. This suggests that besides the specialized root tissue, photosynthetic tissue, mimicking leaf cells, possesses ion transporters able to take up ClO4-. Using willow trees growing in diluted Hoagland solution, Nzengung et al. (22) have shown an almost complete disappearance of the initial 22 mg L-1 ClO4- over 30 d. However, the transformation kinetics showed two distinct phases: the first one slower (disappearance of about 20% in 15 d) and the second one faster (disappearance of the remaining 80% in 15 d). Since the experiments were not performed under sterile conditions, the first phase has been described as the result of a plant-mediated uptake and the second as the result of a plant-assisted microbial transformation of ClO4- (i.e. rhizodegradation). The first 2 weeks would correspond to the lag phase required for the adaptation of ClO4- -reducing bacteria (31)srecently reported to be ubiquitous in the environment (17)sand/or to the development of anaerobic conditions in the root zone (22). In a separate report, sweet gum trees growing in sand were shown to reduce the initial 20 mg L-1 ClO4- to undetectable levels, while willow trees, growing both in sand and in hydroponic solution, were shown to reduce the same concentration by about 50% over the same period of time (23). Even though poplar trees were reported to be poor at removing ClO4- by comparison to other species, our results showed that they are at least midway in the range of performances reported using other terrestrial trees (23). Poplar trees continue therefore to constitute a good model for phytoremediation studies and a potential candidate for cleaning up ClO4- -contaminated environments. More ex-
FIGURE 3. Distribution of the radioactivity (percent) originating from the initial 36ClO4- (25 mg L-1) among reduced metabolites in different extracts of plant cultures after 30 d of exposure. tensive screening of species and environmental conditions are required before an objective plant selection can be made. Because our experiments were performed under sterile conditions and because no contamination was detected, it can be considered that the reduction of perchlorate concentration observed in the solution was the result of a pure plant-mediated process (i.e. uptake and phytotransformation). Most of the radioactivity recovered from the plant tissues at the end of the experiment was located in the leaves (more than 90%), while insignificant amounts were extracted from other fractions. Using willow trees growing in diluted Hoagland solution and in sand, Nzengung et al. (22) observed similarly that the major part of ClO4- taken up by the plants after 26 d of exposure was located in the leaves (46%), even though significant proportions were also recovered from other organs (33% and 17% in the upper and lower stems, respectively). In experiments using sweet gum trees growing in sand, Susarla et al. (23) have also observed larger titers of ClO4- -metabolites in leaf tissues (355 mg kg-1 fresh material) than in roots (191 mg kg-1) or stems (83 mg kg-1). However, using willow trees in the same experiments, only 51 mg kg-1 of ClO4- metabolites were extracted from the leaves, while 102 and 127 mg kg-1 were extracted from the roots and the stems, respectively. The large variations reported in the distribution of ClO4- and metabolites between the different plant organs may be explained by the use of different plant species and different experimental conditions, e.g. the time
FIGURE 4. Distribution of the radioactivity (percent) originating from the initial 36ClO4- (25 mg L-1) among reduced metabolites in different extracts of nodule cultures after 30 d of exposure.
TABLE 4. Distribution (Percent of the Total) of the Radioactivity Originating from the Initial 36ClO4- (25 mg L-1) among Reduced Metabolites in Different Extracts of Nodule Cultures after 30 d of Exposure fraction
ClO4-
ClO3- ClO2-
solution t ) 0 100.00 0.00 solution t ) 30 d 51.51 1.31 nodule extract 22.67 6.41 control solution 93.13 0.00 t ) 30 d
0.00 0.00 5.15 0.00
Cl-
unidentified
total
0.00 6.64 1.97 0.00
0.00 0.00 0.52 0.00
100.00 57.52 36.71 93.13
of exposure, which seems to correlate with the proportion of ClO4- and ClO4- metabolites accumulated in the leaves. Both small trees growing in hydroponic solution and nodule cultures have been shown to reduce the concentration of 36ClO4-sas measured by ICsfrom the solution by about 50% in 30 d. However, the radioactivity recovered in the solutions corresponded to 67 and 53% for the experiments using trees and nodules, respectively. This suggests that a part of the 36Cl in the solutions was not accounted for by 36ClO - but was a different 36Cl species. Analysis of the 4 distribution of the radioactivity detected in the solutions after 1 month showed that a significant proportion (about 33% and 10% in experiments with trees and nodules respectively) was recovered as chloride, 36Cl-. On the other hand, the radioactivity extracted from the leaves and from the nodules was shown to distribute between nontransformed ClO4VOL. 36, NO. 12, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(about 66%) and various proportions of reduced metabolites (i.e. ClO3-, ClO2-, and Cl-). Because experiments were performed under sterile conditions, these results show unambiguously that plant tissues are able to achieves qualitatively speakingsthe complete reduction of ClO4into harmless Cl-. The identified metabolites inside the plant tissues suggest a stepwise reduction of ClO4- to Cl-, likely through a pathway similar to the microbial metabolism of ClO4- involving (per)chlorate reductases and chlorite dismutases (18, 21):
Unlike microbial (per)chlorate-reducing enzymes, corresponding plant biocatalysts tolerate oxygen, since they are likely located in photosynthetic tissues where oxygen is produced in situ. The relatively limited transformation of ClO4- performed by plants in comparison to anaerobic bacteria might reflect the penalty for reductive enzymes working under aerobic conditions. These results are in accordance with the previous reports about phytoremediation of ClO4-. Based on the comparison of ClO4- concentrations extracted from the leaves of willow trees in short term (60 d) experiments, Nzengung et al. (22) concluded that there was first accumulation of ClO4- in leaf tissues followed by its stepwise reduction into Cl-. Other ClO4- reduction metabolites, in variable proportions, were also extracted from tissues of sweet gum and willow trees. Contrary to our results, large proportions of reduced metabolites (i.e. ClO3-, ClO2-, and Cl-) were recovered not only from the leaves but also from the other parts of the plant (23), suggesting either a translocation of the metabolites upon their production in leaves or their transformation by cells located in other plant organs. Because no transformation can take place in the sterile nutrient medium, both the large proportion of 36Cl detected in the solution as Cl- and the corresponding low 36Cl- titers extracted from the leaves suggest a release of Cl- in the solution after its reduction in leaf tissues. Similarly, Nzengung et al. (22) have reported an increase of Cl- concentration in the solution following the reduction of the ClO4- concentration but attributed it to a microbial-mediated rhizodegradation of ClO4- inside the solution. The absence of Clobserved elsewhere in the leaf extracts from willow and sweet gum trees supports this hypothesis (23). Similarly, Newman et al. (32) have reported release of chloride ions from the roots of poplar trees exposed to trichloroethylene (TCE). The authors suggested that TCE, after uptake, underwent dechlorination in plant tissues, with a subsequent excretion of chloride by the roots. ClO4- has been shown to be taken up, to accumulate, and to be slowly reduced inside poplar plant tissues. Our results, obtained using radio-labeled 36ClO4- and working under sterile conditions, provide conclusive evidence that stepwise reductionsand subsequent detoxificationsof ClO4-, previously believed to be the exclusive property of facultative anaerobic microorganisms, can also be achieved by higher plants.
Acknowledgments We thank the Operations Support Command of the U.S. Army for financial support, Cyril Onewokae, grant administrator, Award No. DAAA 09-00-C-0016. The support of a postdoctoral fellowship from FSR (Special Research Fund, Catholic University of Louvain, Belgium) to Benoıˆt Van Aken is gratefully acknowledged. We benefited from discussion and help in the laboratory from Craig Just, Melissa Mezzari, Ester Mu ¨ eller, and Joshua Shrout. 2788
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Received for review January 28, 2002. Revised manuscript received April 9, 2002. Accepted April 11, 2002. ES020560T
