Environ. Sci. Technol. 2004, 38, 5744-5749
Fate of Carbon Tetrachloride during Phytoremediation with Poplar under Controlled Field Conditions XIAOPING WANG,† MICHAEL P. DOSSETT,‡ MILTON P. GORDON,‡ AND S T U A R T E . S T R A N D * ,† University of Washington, College of Forest Resources, and Department of Biochemistry, Seattle, Washington 98195
The fate of carbon tetrachloride (CT) during phytoremediation with poplar was assessed by examining the transpiration of CT from leaves, diffusion from soil, tree trunks, and surface roots, and accumulation of chloride ion in soil and plant tissues. Feedwater containing 12-15 mg/L CT was added to the field test beds planted with poplar, and over 99% of the CT was removed. No significant amount of CT was transpired or diffused into the air, and no significant amount of CT-chlorine accumulated in the tree tissues. Chloride ion accumulated in the soil accounted for all of the CT-chlorine removed. When soils from the root zones were compared to unvegetated soils, microbial mineralization of CT was not enhanced in soils from the root zones as compared to unvegetated soils. Thus, we conclude that uptake and dechlorination of CT by plant tissues is likely the primary mechanism for phytoremediation by poplar.
Introduction Carbon tetrachloride (CT) was widely used in industry and in nuclear weapons manufacture (1), and it is frequently encountered as a groundwater contaminant (2). Since CT is a systemic poison and a carcinogen (3), its fate in the environment is of concern. Conventional pump-and-treat remediation of groundwater contaminated with CT is costly. There are no known microbial pathways for CT metabolism under aerobic conditions, and microbial degradation of CT in anaerobic environments often results in the accumulation of toxic metabolites such as chloroform (CF) (4, 5). Phytoremediation of chlorinated hydrocarbons has potential economic, aesthetic, and ecological advantages as compared to other remediation approaches. Several plants have been tested for their uptake and transformation of chlorinated solvents, such as trichloroethylene (TCE) (6-8), and for possible enhancement of mineralization of volatile chlorinated hydrocarbons (VCHCs) in the rhizosphere or root zone (9-13). Poplar cells have been shown to mineralize CT (14), suggesting that phytoremediation may provide a new way to degrade CT aerobically without significant accumulation of toxic products. The current study was undertaken to determine the potential for phytoremediation of CT using poplar trees grown under field conditions. Two hypotheses were tested in the study: (1) mineralization is the dominant fate of CT in phytoremediation and (2) CT mineralization is due to plant * Corresponding author. Phone: (206)543-5350. Fax: (206)5433254. E-mail:
[email protected]. † College of Forest Resources. ‡ Department of Biochemistry. 5744
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metabolic activity, rather than microbial metabolism in the root zone. The field study was performed using artificial aquifer cells as described in Newman et al. (13). To provide a mass balance of CT and its dechlorination products, the following components were determined: (1) total mass of CT applied to and removed from the artificial aquifers, (2) the amount of CT transpired by the leaves, (3) the amount of CT diffused from soil, trunks, and roots exposed in the surface soil, (4) the accumulation of inorganic and organic chloride in the plant tissues, and (5) the accumulation of chloride ion in the soil of the test bed. This is the first time that diffusion removal has been included in a mass balance study of phytoremediation of volatile chlorinated hydrocarbons.
Materials and Methods Site Description. The field test beds used in this study were described in Newman et al. (13). The beds were approximately 1.5 m deep by 3.0 m wide by 5.7 m long and were lined with double-walled 1.5 mm polyethylene. On May 26, 1995, 15 rooted hybrid poplar H11-11 cuttings (Populous trichocarpa x P. deltoides) were planted in the bed, with a spacing of approximately 1 m between trees. The number of trees was reduced to 10 in 2001. The beds had an independent water delivery system and an extraction well for sampling and removal of excess water. Effluent water was treated on the site by carbon filtration and reused. Makeup water was supplied by a municipal water company. CT Dosing and Sampling. Starting from 1996, the bed received influent water containing CT during the growing season of each year from May to October, 1996-2002. CT solutions were prepared using stock solutions of CT-saturated water, diluted to 12-15 mg L-1 CT immediately prior to introduction into the bed. Water sampling and analysis was performed as described in Newman et al. (13). Samples of both influent and effluent water were analyzed for the parent compound and its dechlorinated product, CF, as well as for other volatile chlorinated hydrocarbons following EPA Analytical Method 8260A. The amount of volatile chlorinated hydrocarbons was determined on a Perkin-Elmer gas chromatograph Autosystem XL with Tekmar 3000 purge and trap concentrator and Tekmar Precept II autosampler. A Supelco 2-5381 column (60 m × 0.53 mm) was used with oven temperature starting at 40 °C for 2 min, ramping 10 °C/min to 180 °C, and holding for 10 min. The concentration of chloride ion in influent and effluent water was also analyzed using ion chromatography as described next. Accumulation of Chloride in Soil. To calculate the total chloride accumulation in the soil of the test bed during the mass balance period in the growing season of 2002, soils were sampled on May 16, 2002 and October 17, 2002 using a 1.75 cm diameter soil corer. Soil samples were taken from four horizontal positions between the influent and the effluent ends of the bed. At each position, soil samples were collected at four depths (0-0.25, 0.25-0.5, 0.5-0.75, and 0.76-1.0 m). The soil samples were frozen for analysis. Prior to analysis, the soil samples were baked at 100 °C for 24 h to remove moisture and ground and filtered to remove gravel and fine roots. An aliquot (1.0 g) from each sample was weighed into a 50 mL conical vial and extracted in 5 mL of water. Vials were placed on a shaker for 30 min and centrifuged for 15 min at 4000 rpm. A portion of the supernatant (0.5 mL) was placed in sampling vials and injected on a Dionex DX-120 ion chromatograph with an AS14 column using 3.5 mM Na210.1021/es0499187 CCC: $27.50
2004 American Chemical Society Published on Web 09/23/2004
FIGURE 1. Schematic of diffusion trap for measuring flux of volatile organic compounds from trunks of poplar in test beds. Similar traps were used to determine flux of VCHCs from soil surface. CO3 and 1 mM NaHCO3 as eluent. Concentrations were determined from peak areas using external standards. Transpiration of CT. The amount of CT transpired by the plants was measured during the growing season. The method is described in detail in Newman et al. (13). Briefly, the amount of CT transpired from a singe leaf was determined by enclosing a leaf in a Teflon bag (Norton Performance Plastics), loosely fitted around the petiole to allow passage of air into the bag. CT transpired by the leaf was trapped on activated carbon and then extracted and analyzed by GC-ECD. CT Concentrations in Tree Trunk Cores. An increment borer (Haglof, Sweden) was used to obtain samples of tree trunk tissues on October 17, 2002. Cores were approximately 4.2 mm diameter by 75 mm long. Cores were collected in duplicate from the trunks of three trees at soil surface and at three different heights (0.6, 1.2, and 1.8 m) above the ground. For analysis of CT and CF in the trunk tissue, the cores were broken into two pieces and placed in 2 mL of 0.2 M NaHSO4 in 40 mL vials with Teflon-lined septa. The vials were loaded into the autosampler of a Perkin-Elmer Autosystem XL with purge and trap using the solids mode as described previously. Concentrations were calculated by comparison to a series of external standards. Spiked recoveries were greater than 90% by this method. Measurement of CT Diffusion from Tree Trunks and Soil. Gases diffusing from the tree trunks were sampled using concave glass traps firmly attached to the trunks, as shown in the schematic in Figure 1. The traps were sealed against the trunks with plumber’s putty (mixture of limestone, mineral and vegetable oils, talc, and clay, Oatey, Inc., Cleveland, OH) and strapped firmly in place with elastic bands placed around the trunk. The area of the opening of the cup placed against the tree trunk was 26.4 cm2. Two sampling ports, located on opposite sides of the glass traps, were fitted with activated carbon tubes for collecting volatile chlorinated hydrocarbons (Anasorb CSC, ST charcoal 200/240, SKC, Inc.). Air was pulled through the activated carbon tubes and the glass sampling trap by means of a vacuum pump connected to a flow meter. The airflow was maintained at 2 L min-1 for 1 h. The influent carbon tube was used to remove contaminants from the outside air, and the effluent carbon tube was used for sampling of chlorinated hydrocarbons diffused from
the tree trunks. Each of the carbon tubes contained two sections of activated carbon. The second, downstream section served as a control. If no chlorinated hydrocarbons were observed in the second section, it was assumed that all chlorinated hydrocarbons in the effluent gas were trapped in the first section. Trunk diffusive fluxes were sampled on Sept. 23-25, 2002 on two trees, one at the influent and the other at the effluent end of the test bed. Each tree was sampled at ground level, 0.25, 0.4, and 0.57 m height above the ground. Three measurements of flux were taken at each height. To minimize forced diffusion, the vacuum in the diffusive trap was kept low, 0.032 atm, as determined by the manometer. Gases from exposed roots in the test beds were measured with the same sampling devices, which were sealed against the roots with plumber’s putty and held firmly in place with weights. Removal of CT from the soil surface was measured using a diffusion trap similar to that used to measure trunk fluxes, except that the side of the cup was 15 cm longer to allow greater penetration into the soil. After sampling, the activated carbon tubes were capped, sealed in a jar with activated carbon in the bottom, and refrigerated until extracted. For analysis of trapped chlorinated hydrocarbons, activated carbon in the two sections of the trapping tubes was separately removed and placed in 4 mL vials with 3 mL of the hexane and acetone mixture (50:50). The vials were capped with Teflon-lined rubber septa, vortexed for 5 min, and stored at room temperature for 24 h. A total of 1.5 mL of the supernatant was pipetted into 2 mL vials, sealed with Teflon-lined rubber septa crimp caps, and injected into a Perkin-Elmer Autosystem XL gas chromatograph with a 30 m × 0.32 mm Supelco 24214 column. Oven temperature started at 40 °C for 1 min, ramped at 4 °C/min to 100, then ramped at 35 °C/min to 200 °C, and held for 1 min. Peak areas were integrated and quantified using external standards. In control tests, more than 90% of CT added to activated carbon tubes was extracted using this procedure. Inorganic and Organic Chloride in Tree Tissues Exposed to CT. Two test beds planted with hybrid poplar H11-11 were used for this portion of the study, which was conducted in the summer of 1996. One bed received water containing 1215 mg L-1 CT, and the other received only water. Both beds received the same volume of water. In September 1996, one tree was harvested from the dosed and control beds separately, and leaf, branch, and root samples were collected. Chloride ions and organic halides in plant tissues were determined using the methods described in Newman et al. (13). Oxidation of CT in Root Zone Soil. Soil samples from 0 to 0.4, 0.4 to 0.8, and 0.8 to 1.1 m depths were collected from the CT-dosed test bed in September 2000. Unvegetated soil from the test bed site was collected in June 2000 for use as an unvegetated control soil and was dosed daily with 1000 mL of water containing 15 mg L-1 CT for three months before the experiment started. Soil (5 g of either vegetated or control soils) was placed into 40 mL bottles (microcosms) sealed with septum valve caps (Mininert). Soils collected from the 0.8-1.1 m depth were incubated under anaerobic conditions produced by flushing the bottles with N2. Soils collected from 0 to 0.4 and 0.4 to 0.8 m depths were incubated with air in the headspace. All incubations were set up in triplicate. For both vegetated and unvegetated soil, 5 µL of radiolabeled CT (dissolved in methanol with a specific activity of 4.0 mCi/ mmol, New England Nuclear) and 35 µL of unlabeled saturated CT was injected through the septum valves, providing a total CT concentration of 5.5 µg (g soil)-1. The soil microcosms were incubated in the dark at room temperature for 5 days. At the end of the incubation, 1 mL of 0.2 N NaOH and 2 mL of water were injected into the VOL. 38, NO. 21, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Daily influent and effluent masses of aqueous carbon tetrachloride and chloroform applied to and recovered from the poplar test bed during the 2002 season.
FIGURE 3. Concentration of carbon tetrachloride and chloroform in the influent and effluent water of the poplar test beds.
TABLE 1. Total Removal of CT, CF, and Water in the Poplar Test Bed total mass of CT, mol total mass of CF, mol total volume of water, L
influent
effluent
% removed
4.17 0.43 48181
0.01 0.01 590
100 99 99
microcosms, which were shaken at 175 rpm overnight to trap CO2. Then the bottles were opened and shaken at 175 rpm overnight in a vent hood to allow the remaining CT to volatilize. NaOH (0.25 mL, 1N) was added to 1 mL hanging cups (Supelco), the handle of the cups attached tightly to a shallow hole drilled in the underside of the septum valve caps, and the bottles sealed using the septum valve caps with the hanging cups. Two mL of 1.2 N H3PO4 was injected into the microcosms through septum valve caps, carefully avoiding introduction of acid into the cups. After the bottles were shaken at 175 rpm for 5 days, the caps were removed, and the NaOH solution in the cups was transferred to 30 mL vials and diluted to a total volume of 3 mL. To eliminate the possibility of 14C-labeled volatiles being mistaken for CO2, 1 mL of the NaOH solution was used to count total 14C activity (designated value A), and 1 mL was used to count the 14C activity after precipitation of 14C-CO32- as BaCO3. To precipitate 14CO32- in the NaOH solution, 1 mL of the solution was transferred to a 1.5 mL centrifuge tube. Solutions of 25 µL of 0.12 M Na2CO3 and 0.1 mL of 2 N BaCl2 were added to the tubes to precipitate 14C-carbonate. After centrifugation at 10 000 rpm for 10 min, the supernatant was removed for 14C counting (designated value B). The difference between the two counts multiplied by 3 represented the CO2 production from the radiolabeled chlorinated hydrocarbons: CO2 counts ) (A - B)3. The amount of 14C radioactivity in both the NaOH solution and the supernatant was determined by mixing 0.2 mL solution with 7 mL liquid scintillation cocktail (Utima Gold) and counting in a Beckman LS 7000 liquid scintillation counter.
Results and Discussion CT and Water Removal from the Test Bed Planted with Poplar. The mass of CT in the effluent water from the test bed was significantly lower than that in the influent water (Figure 2). The total amount of CT added to the test bed during the mass balance season of 2002 was 4.2 mol, while the total amount of CT recovered in the effluent was only 0.01 mol, which represented a 99.8% removal of CT added (Table 1). There was a small amount of CF present in the influent water during the beginning and end of the season, but the total chloroform was only about 10% of the total CT 5746
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FIGURE 4. Concentration of carbon tetrachloride and chloroform in poplar trunk tissue with respect to height above soil surface. added to the bed (Table 1 and Figure 2). By the end of the growing season, 98.7% of the total CF in the influent water was removed (Table 1). The percent consumption of water during the mass balance studying period was 99%, which was nearly the same as the percent removal of CT (Table 1). Compared to the removal of CT mass, the concentration of CT in the test bed effluent did not decrease to the same extent (Figure 3). The average CT concentrations in the influent and effluent were 12.3 ( 1.5 and 4.6 ( 3.1 mg L-1, respectively, corresponding to a concentration decrease of 62%. Similar results were observed in previous experiments with TCE, in which 99% of the TCE mass was removed in poplar-planted beds, while the concentration of TCE fell by only 58% (13). Interestingly, CF concentrations in the effluent tended to increase as compared to the influent, probably due to exudation of CF from biological reduction in the root tissues (15) or microbial reduction of CT to CF in anaerobic sites in the soil. Transpiration of CT by Poplar. No CT was detected in any leaf bag assays. When CT was injected into the bags, an average of 77% of the influent CT mass was recovered. The result suggests that transpiration was not a significant pathway of CT removal. CT Concentrations in Trunk Tissue and Diffusion from Poplar Trunks, Soils, and Exposed Surface Roots. No significant trends of CT and CF concentrations with height above soil surface were observed in trunk samples up to 1.8 m where the first branching occurred (Figure 4). The CT concentration at the highest sampling point was significantly greater than lower points. These observations are inconsistent with significant decreases in tree trunk concentration of TCE
TABLE 2. Flux of CT from the Trunks of Poplar as a Function of Position in the Test Bed and Height above Ground Level flux of CT from trunk, nmol CT h-1 cm-2 sample height position of tree in the test bed
ground level
0.25 m
0.4 m
0.57 cm
near the effluent end near the influent end
1.25 ( 0.21 1.87 ( 0.32
0.57 ( 0.07 1.56 ( 0.05
0.86 ( 0.11 2.63 ( 1.21
1.00 ( 0.26 1.33 ( 0.58
TABLE 3. Inorganic and Organic Chloride Concentration in Plant Tissues after CT Exposure chloride ion (mmol TOX (mmol
kg-1)
kg-1)
treatments
roots
branches
leaves
control, unexposed trees CT-exposed trees control, unexposed trees CT-exposed trees
7.2 ( 1.5 6.1 0.46 ( 0.06 0.59
7.2 6.8 0.45 0.62
64 ( 27 50 ( 12 2.7 ( 1.2 2.6 ( 0.78
TABLE 4. Mass Balance of Chlorine in Poplar Test Beds Exposed to CT from May 16 to October 17, 2002 source of chlorine
Chlorine Removal, mol influent water effluent water removal
CT chlorine chloroform chlorine Cl- ion in makeup water Cl- ion in top water Cl- in rainfall total chlorine removed
16.67 1.28 5.44 0.18 0.03
0.04 0.01 0.04
Estimated Chlorine Recovery, mol Cl- ion recovered from soil bed chlorine recovered from leaf bags chlorine recovered from trunk diffusion chlorine recovered from soil surface chlorine recovered from surface root diffusion total chlorine recovered recovery efficiency (total Cl recovered/total Cl removed) a
16.63 1.27 5.40 0.18 0.03 23.5
32.1 NDa 0.41 0.001 ND 32.5 138%
ND: none detected.
with height observed previously (16, 17), and they do not support the hypothesis that CT was lost to the atmosphere by diffusion from the trunk. Diffusion of CT from the trunks of two poplars in the test bed ranged from 0.57 to 2.63 nmol of CT h-1 cm-2, averaging 1.4 nmol of CT h-1 cm-2 (Table 2). There was no apparent trend of CT flux with height above the soil surface. CF flux was not determined due to interference from the solvent peak. No CT was recovered from diffusion chambers placed on surface roots. The measurements of CT diffusion from soils were performed at five locations in the test bed. CT flux from soils was highly variable, averaging 4 ( 6.5 nmol of CT h-1 cm-2. During the gradient and diffusion data acquisition periods (September 2-22, 2002), the trees were fully leafed out and actively transpiring with an average water removal rate of 383 L/day (as determined by the difference in influent and effluent water volumes), and CT and CF were being removed at the average rates of 35.7 and 1.9 mmol/day, respectively. Chloride in Poplar Trees. There was no significant difference in the levels of chloride ion between CT-exposed and -unexposed trees (Table 4). The concentrations of TOX in the plant tissues were lower than the concentrations of chloride ion. TOX levels were the same in the leaves of CTexposed and -unexposed trees, while root and branch TOX levels were slightly elevated in the CT-exposed trees as compared to the unexposed trees. Nonetheless, these results indicate that the accumulation of CT and its metabolites in
FIGURE 5. Concentration of chloride ion in the soil of the test bed at the beginning and ending of mass balance period in 2002. plant tissues was negligible as compared to the total amount of CT removed in the test bed. Chloride Accumulation in the Soil of the Test Bed. Figure 5 shows the distribution of chloride concentration in the soil of the test bed at the beginning of the mass balance period (May 16, 2002) and at its end (October 17, 2002). Soil chloride levels increased significantly toward the effluent end of the test bed, a trend that was also observed previously for TCE (13). Chloride concentrations in the soil samples collected from the two sampling positions closest to the effluent of the test bed increased significantly (p ) 0.05) during the mass balance period, while concentrations of chloride in the first two sampling positions closest to the influent did not increase significantly during the mass balance period. The high standard deviations of the means suggest that the chloride distribution was highly variable, possibly due to preferential flow channels in the heterogeneous soil, although there were no significant trends of chloride concentration with soil depth. The total accumulation of soil chloride was the difference between the total mass of soil chloride in the test bed on October 17, 2002 and the total mass of soil chloride on May 16, 2002. The total mass of soil chloride was calculated from the average chloride concentrations in the soil cores at the positions 3 and 4 multiplied by the average soil density (1.01.1 g/cm3) and one-half of the test bed volume (8.3 m3). Since the chloride concentrations of the soil samples collected at the positions 1 and 2 were not significantly different between the beginning and the end of the mass balance period, they were not used to estimate the total chloride accumulation in the bed. The estimated total mass of chloride ion was 20 ( 9.2 mol at the beginning of the mass balance period and VOL. 38, NO. 21, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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52 ( 12 mol at the end. Therefore, the difference in total mass of chloride between the beginning and the end of the mass balance period was 32 ( 15 mol, which represents the total accumulation of chloride ion in the soil. The large error of the estimate of chloride accumulation resulted from heterogeneity of the soil samples, but the difference between the chloride mass in the test bed soil at the beginning and end of the mass balance period was significantly different from zero (p ) 0.05). Chlorine Mass Balance. The sources of chlorine added to and removed from the test bed during the mass balance period from May 16 to October 17, 2002 are summarized in Table 4. The total amounts of chlorine in the CT and CF added to the test bed during this period were 16.6 and 1.3 mol, respectively, and the total CT-chlorine and CF-chlorine recovered from the effluent of the bed were 0.04 and 0.01 mol, respectively. City water used to prepare the influent water had a chloride concentration of approximately 113 µM L-1, which contributed a significant amount of chlorine (5.4 mol) to the soil after transpiration loss of water (Table 4). The total chlorine removed from all sources during the 2002 mass balance period was 23.5 mol. CT chlorine lost to the atmosphere due to transpiration from leaves was negligible since no CT transpiration was detected. CT chlorine lost to the atmosphere by diffusion from the trunk was 0.41 mol of Cl, as estimated from the average flux observed (1.4 nmol of CT/h/cm2), multiplied by four mol of Cl per mole of CT, the area of the trunks to the first branch (about 3.1 m2), and by the total hours of daylight during the mass balance period (about 2400 h). Chlorine in CT lost to the atmosphere by diffusion from the soil surface was approximately 1.0 mmol of Cl, as calculated from the average flux (4 nmol of CT h-1 cm-2) multiplied by 4 mol of Cl per mole of CT, the soil surface area of the test bed minus the area occupied by tree trunks and exposed roots (16.6 m2), and by the number of hours in the mass balance period (24 h × 151 days). The amount of chloride accumulated in the soil of the test bed exceeded the total chlorine removed during the growing season, 32 ( 15 mol as compared to 23.5 mol, respectively. Because of experimental error, closure on the mass balance was not complete (recovered chloride was 138% of the applied chlorine). Nonetheless, these results suggest that dechlorination was the dominant fate of CT applied to the test bed planted with poplar. CT Mineralization in Poplar-Vegetated Soil. There was no enhancement of 14CO2 production in vegetated soil as compared with unvegetated control soil under either aerobic or anaerobic conditions. Radiolabeled CO2 production was generally higher in unvegetated soils than in vegetated soils, although the differences were not significant. For most treatments, the amount of CO2 production from the 5 day laboratory incubations was less than 1% of the total CT dosed. To estimate the contribution of CT mineralization by root zone soil to the total removal of CT, the average rate of 14CO2 production in the vegetated soil (0.0036 µmol (g soil)-1 day-1) was applied to the entire volume of test bed soil. About 0.047 mol of CT could have been oxidized in the test bed during the mass balance period, a negligible amount as compared to the total removal of CT (4.16 mol) during the season (Table 1). Evaluation of CT Removal Mechanisms. Nondestructive removal processes suggested for phytoremediation of chlorinated hydrocarbons, such as CT and TCE, include transpiration from the leaves and diffusion from the trunk and roots (16). Our results show that CT losses to the atmosphere by either of these routes was insignificant as compared to the total amount of CT removed in the test beds (Table 4). Mechanisms that have been proposed for destructive phytoremediation of chlorinated hydrocarbons are enhanced rhizosphere microbial degradation (11, 18) and plant uptake 5748
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and metabolism (phytodegradation) (12, 14). We found no enhancement of CT mineralization in vegetated soils as compared to unvegetated soils, and the estimated amount of CT mineralized by root zone soil was insignificant as compared with the total removal of CT. Poplar cells are capable of oxidizing CT to carbon dioxide (14). Therefore, the removal of CT in the test bed was likely due to uptake and mineralization of CT by poplar trees. Several considerations suggest that plant-mediated oxidation of CT probably occurred in root tissues of poplar. CT transpiration from the leaves and CT diffusion from the trunks and exposed surface roots were minimal; there was no significant accumulation of either inorganic or organic chlorine in poplar tissues. These results suggest that CT was mineralized before it reached the above-ground portion of the trees. Chloride ions produced from CT oxidation in the roots could have been excreted by the roots to maintain a balance of charge and ion concentration. Chloride ion is an essential nutrient and an important co-ion for plants, which therefore regulate chloride ion concentrations in plant tissues. Channels for transport of chloride ion into and out of plant cells and vacuoles are well-known (19), and chloride effluxes from plants have been observed (20). Implications for Phytoremediation of TCE. Ma and Burken (16) found that TCE diffusion from poplar trunks was a potentially important process for TCE volatilization. However, the relative importance of diffusive removal of TCE to total TCE removal has not been quantitatively determined in a mass balance experiment under field conditions. The only mass balance field experiment with TCE to date found significant chloride ion accumulation in the soil, suggesting that destruction of TCE was an important removal mechanism (13), although trunk diffusive loss of TCE to the atmosphere was not measured. Our trunk diffusive fluxes (1.4 nmol of CT h-1 cm-2) were similar to the fluxes reported by Ma and Burken for leafed-out poplar cuttings exposed to TCE: 1.8 nmol of TCE h-1 cm-2 at 50 mg/L TCE in soil or 0.62 nmol of TCE h-1 cm-2 at 20 mg/L TCE in hydroponics.16 Importantly, if the average of these fluxes, 1.2 nmol of TCE h-1 cm-2, is applied to the trunk surface area of the 15 trees used in the test bed study reported by Newman et al. (13), trunk diffusion of TCE could have accounted for only 0.22 mol of TCE during the growing season, which is less than 7% of the observed average removal of TCE (3.2 mol of TCE per growing season) (1996 and 1997). Since measured trunk diffusion rates cannot account for either CT or TCE removal observed in mass balance field trials, trunk diffusion is apparently not an important removal pathway for phytoremediation of VCHCs. The inability of poplar to reduce the concentration of VCHCs in treated water has important implications for phytoremediation plantations treating pumped groundwater by irrigation. If VCHC concentration cannot be reduced to regulatory levels in the root zone, rainfall on such plantations is likely to push polluted water past the depth of roots, resulting in contamination of vadose zones. In summary, our mass balance study of CT phytoremediation suggests that poplar plantations can effectively break down CT into harmless components. Poplar trees removed more than 99% of dosed CT with minimal transpiration and diffusion of CT into the air. Accumulation of CT and its metabolites in the tissues of poplars exposed to CT was negligible. The amount of chlorine applied as CT did not exceed the amount of chloride ion that accumulated in the test bed soil during the growing season. Root zone soils did not mineralize CT at a significant rate, and the rate in vegetated soils was not faster than in nonvegetated soil. These results support the hypothesis that during phytoremediation by poplar trees, the dominant fate of CT is degradation in plant tissues.
Acknowledgments We thank Indulis A. Muiznieks for technical assistance on the test bed site and Angela Wilson for analysis of VCHCs. This work was supported by funding from U.S. NIEHS Grant 5 P42 ES04696-07, the U.S. EPA Grant R822329-01-3, and the U.S. DOE EMSP program through Grant DE-FG07-96ER20256, and material support from Occidental Chemical Corporation.
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Received for review January 16, 2004. Revised manuscript received July 16, 2004. Accepted July 26, 2004. ES0499187
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