A Controlled Field Study

13, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2257. Published .... 2258 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 33, NO. ...... Orlando,. Florida...
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Remediation of Trichloroethylene in an Artificial Aquifer with Trees: A Controlled Field Study L E E A . N E W M A N , * ,† X I A O P I N G W A N G , † INDULIS A. MUIZNIEKS,‡ GORDON EKUAN,§ MARTIN RUSZAJ,| REMI CORTELLUCCI,| DAVID DOMROES,| GEORGE KARSCIG,⊥ TIM NEWMAN,⊥ ROBERT S. CRAMPTON,# RAM A. HASHMONAY,§ MICHAEL G. YOST,# PAUL E. HEILMAN,3 JAMES DUFFY,| MILTON P. GORDON,‡ AND STUART E. STRAND† University of Washington, College of Forest Resources, Department of Biochemistry, Department of Environmental Health, Seattle, Washington, Washington State University, Research and Extension Center, Puyallup, Washington, Occidental Chemical Corporation, Grand Island, New York, Pioneer Chlor Alkali Company, Tacoma, Washington, and Verdant Technologies, Seattle, Washington

Poplar trees have been evaluated in the field for the control of contaminated groundwater movement, but to date, the fate of the contaminants has not been demonstrated. In the present study, we tested a hybrid poplar for the uptake and degradation of trichloroethylene (TCE). Plants were exposed to TCE-contaminated groundwater under field conditions in lined cells for three years. During the growing seasons, the trees were able to remove over 99% of the added TCE. Less than 9% of the TCE was transpired to the atmosphere during the second and third years, and examination of the tissue showed expected metabolites, but at low levels. Chloride did not significantly accumulate in the plant tissues, but chloride ion increased in the soil in amounts that approximately corresponded to TCE loss. These results demonstrate that treatment of TCEcontaminated groundwater with this poplar clone can result in efficient destruction of TCE.

Introduction Trichloroethylene (TCE) is one of the most prevalent groundwater contaminants (1). Conventional remediation technologies for TCE can be both expensive and time consuming. Thus, the use of plants for the uptake and/or dechlorination of TCE (phytoremediation) has been suggested as a more cost effective remediation solution (2). Phytoremediation offers other advantages including low maintenance, high levels of public acceptance, and compatibility with ecological restoration. * To whom correspondence should be addressed. Phone: (206) 616-2388; fax: 685-8279; E-mail: [email protected]. † College of Forest Resources. ‡ Department of Biochemistry. § Research and Extension Center. | Occidental Chemical Corporation. ⊥ Pioneer Chlor Alkali Company. # Department of Environmental Health. 3 Verdant Technologies. 10.1021/es981217k CCC: $18.00 Published on Web 05/21/1999

 1999 American Chemical Society

The application of plants to the remediation of pollutants in the environment has been the subject of several recent reviews (2-4). Anderson and Walton (5) have reported enhanced degradation of TCE in the rhizosphere of various plants. Narayanan et al. (6) described the use of grasses to extract trichloroethylene from shallow contaminated soils, and Schnabel et al. (7) described the uptake of TCE by crop plants. Previous work from our laboratory (8) has shown that hybrid poplars have the ability to degrade TCE, but the extent of this degradation under field conditions has not been documented. Deep-rooted plants such as poplar have been proposed for the remediation of TCE in shallow aquifers (2, 9). Trees are able to take up water from both soil and shallow aquifers, potentially remediating both matrixes. Trees such as poplar have been proposed to prevent movement of polluted groundwater. Where aquifers are too deep for the roots to reach the capillary fringe or where it is not feasible to plant trees directly over the area of contamination, the contaminated water could be pumped from the aquifer and applied to the trees in irrigation water (10). To date, remedial projects in the field have not provided information about the fate of TCE after interaction with the plant occurs. The distribution between uptake, degradation, and transpiration of TCE in plants has not been studied, nor has the final distribution of metabolites been determined. The purpose of this field study was to determine the fate of TCE after interaction with a hybrid poplar clone. This objective was accomplished by monitoring the distribution of TCE due to the following mechanisms: (1) the removal of applied TCE in the artificial aquifers due to the introduction of trees to the system, (2) the transpiration of TCE by leaves using “leaf bags” and open path Fourier transform infrared spectroscopy (OP-FTIR), (3) the accumulation of TCE and other organochlorines in the plant tissue by total organic halide (TOX) analysis and gas chromatography-mass spectroscopy (GC-MS), (4) mineralization of TCE as shown by the accumulation of chloride ion in soil and plant tissue, and (5) degradation of TCE in the soil as affected by the presence of plants and determined using laboratory microcosms of field soils.

Materials and Methods Cell Design and Construction. The hybrid poplar clone H1111 (Populous trichocarpa x P. deltoides) was chosen as our experimental plant based on its demonstrated ability to take up and degrade TCE under laboratory and greenhouse conditions (8). Rooted cuttings were planted on 26 May 1995. Prior to planting, the plants were root and top pruned to 45 cm. Fifteen trees were planted in each cell, with a spacing of approximately 1 m between trees to obtain rapid production of a dense biomass. A site outside Fife, WA, was selected for the test site. The cells used in this experiment were a series of constructed, artificial aquifers. The cell walls were composed of doublewalled 60 mil polyethylene liners, with approximate dimensions of 1.5 m deep by 3.0 m wide by 5.7 m long (Figure 1). The cells contained 0.3 m of coarse sand overlaid with 1.1 m of Sultan silty clay loam topsoil from the site. Each cell had an influent well on one end with a T-shaped, perforated distribution pipe to allow the addition of controlled amounts of either water or TCE-containing water to the sand layer. The bottoms of the cells were sloped 1:40 to the effluent ends where extraction wells were located. Water pumped from the effluent end of the cells was treated on site. Water from both dosed and undosed control VOL. 33, NO. 13, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Diagram of phytoremediation test cells. Top view of cell shows direction of water flow from T-bar distribution port. Side view shows position of influent and effluent wells. cells was mixed, held for 2 days in a settling tank, and passed through a particulate filter and then through two sequential 220 L activated carbon absorption systems for removal of chlorinated organics. Treated water was stored on site prior to reuse. Treated water was analyzed on a monthly basis to ensure that chlorinated organics from the cells were not recirculated. Dosing the Trees and Water Management. Four cells were used for the initial part of this study. One cell was planted with trees but not dosed with TCE (vegetated control); two cells were planted with trees and dosed with TCE; and the fourth cell was left unplanted, but received the same dosage of TCE as the planted cells (unplanted control). The unplanted control cell was kept free of vegetation for the first two years of the test. Dosing of the cells began on 25 July 1995, when the trees had grown 1 m from time of planting. In 1996 and 1997, TCE dosing began in the spring when the trees had leafed out and began to take up water from the aquifer. Either water or water containing TCE was injected into the artificial aquifer through the influent wells from 25 July until 3 November of the first season (1995) with a total dose of 0.93 mol of TCE. Water was obtained from municipal sources and from recirculated water on the site after filtration through the carbon systems. During the first season, TCE concentrations in the influent were increased from 0.038 mM to a maximum of 0.76 mM in order to produce breakthrough of TCE in the effluent of the control cell. The average concentration of TCE added to the cells during this period was approximately 0.38 mM. This level is within the range of TCE concentrations in polluted groundwaters, from less than 40 nM to saturation (8.4 mM). During the second year, the cells were dosed with a target influent TCE concentration of 0.11 mM, a concentration within the range of TCE levels typical of heavily polluted sites (1) and less than TCE levels that exert harmful effects on the trees (8). Each cell was dosed from May to October, producing a total TCE dose of 5.43 mol per cell during 1995 and 1996. All four cells received the same volume of water or TCE-containing water. During the third year, the experi2258

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ment was continued with only the planted, undosed cell and one planted dosed cell, with an additional 6.26 mol of TCE added to the dosed cell prior to bud set (the cessation of terminal growth in autumn and the formation of buds that will lead to the following year’s growth). The goal of water management was to maintain water levels in the bottom of the cells within the range of 15-25 cm. Over the winter months and prior to dosing in the spring, the excess water that accumulated was pumped out of the cells. During the growing, season water levels in the cells were determined by inserting a graduated rod into the effluent well of the cell and directly observing the level of water in the sand layer. Excess water was pumped out of a cell as needed, and in cells with water depths less than 15 cm, water was added to the cell, although high rates of water uptake by the trees sometimes precluded water level maintenance by this method. The application rate of water varied over the growing season depending on the transpiration rate of the plants. Supplemental surface irrigation was necessary in 1996 and 1997 because water consumption by the plants exceeded the amount of water that could be injected into aquifer. To prevent the upper soil in the cells from becoming completely dry during the summer, the cells received periodic surface irrigation starting in 1996. Water applied to the surface did not contain TCE. When necessary during the course of the experiment, water was removed from the effluent wells to provide samples or to maintain target water levels. TCE/water solutions for injection into the cells were mixed in 100 L polyethylene drums that were set up over, and connected to, the injection wells in the cells. One hundred liters of water was added to the drum, and the TCE stock solution was added via a separatory funnel through Teflon tubing to the bottom of the drum. The solution was mixed with gentle stirring, a sample was removed from the drum for analysis of influent TCE levels, and the solution was gravity-fed into the T-bar distribution system in the underground aquifer. During the first year of operation, TCE was added from a stock solution containing 0.7 M TCE and 40% ethanol. The stock solution was diluted to the target concentration in 100 L of water immediately prior to injection into the cells, resulting in concentrations of ethanol in the injected water that ranged 0.4-9 mM. This procedure was adopted because of the low solubility of TCE in water and to ensure sufficient mixing prior to injection into the cells. In the second and third year, saturated aqueous stock solutions of TCE containing about 8.4 mM TCE were used. The period of dosing with TCE in the second season was from 3 May through 17 October 1996. The concentration of TCE in the influent of the cells during the second year averaged 0.12 ( 0.03 mM. Water was added daily to the cells as it was needed by the trees, hence water volumes varied from day to day. However, all of the cells, whether planted or not, received the same volume of water on any given day. At the latter part of the third year water, the rate of water addition was increased above that needed to maintain a constant level of 15-25 cm in the effluent well. The cell receiving TCE was dosed with 600 L day-1 from 10 October to 21 November, with the TCE concentration in the water remaining as constant as possible given the temperature fluctuations (and thus solubility changes) in the field. This additional dosing resulted in an additional 1.91 mol of TCE being added to the cell in 1997. Water Analysis. Water samples from the influent were collected by filling a 50 mL sample bottle to overflowing and immediately capping with a Teflon-lined septum to exclude air. Effluent water samples were collected by using a vacuum pump to pull water from the effluent extraction well through a copper tube leading to the bottom of a 50 mL sample bottle

mounted in a vacuum jar. The effluent was allowed to overflow the bottle with at least 3 vol before the bottle was capped with a Teflon-lined septum, excluding air. Samples were analyzed for the presence of TCE and its chlorinated metabolites according to EPA Analytical Method 8260A. For analysis, a Fisons GC 8000 was used in conjunction with a Fisons MD 800 MSD. The GC-MS was linked to a Tekmar Precept II Vial Autosampler and a Tekmar LSC 2000 Purge and Trap Concentrator. The column for the GC, with open split injection, was a J&W 0.53 mm × 75 m DB624 with a film thickness of 3 µm. GC temperature program was 35 °C for 5 min, ramp at 8 °C min-1, and held at 160 °C for 3 min. Samples were analyzed for TCE and the products of its reductive dechlorination (TCE/R): all three isomers of dichloroethylene, 1,1,2-trichloroethane, and vinyl chloride. Detection limits for undiluted samples were maintained at 38 nM, but a majority of the samples were diluted with a corresponding detection limit of 3.8 µM. Dissolved oxygen (DO) in effluent water was measured with a portable DO electrode and meter (Yellow Springs Instruments) by lowering the electrode into the water accumulated in the effluent well. Plant Tissue Analysis, October 1995 and June 1996. Leaf, branch, and root samples were collected and analyzed for the presence of TCE and oxidative metabolites. Tissue samples were removed from the trees and flash frozen in liquid nitrogen in the field. Tissues were then sealed in bottles with aluminum-lined closures and stored at -20 °C until analysis. Samples were analyzed for TCE and oxidative metabolites as described in Newman et al. (8). During the fall of 1995, samples were taken from representative plants throughout the cells and mixed for a composite sample from each cell. During June of the second year, leaf and soil samples were collected from trees at both the influent and effluent ends of the cells and held separately. Soil samples were collected from the surface and from 30 cm deep, placed in glass jars with aluminum lined closures and stored at -20 °C. Plant Tissue and Soil Analysis, Fall and Winter 1996. In September 1996, one tree was harvested from the influent end of each cell. Measurements were taken to determine the masses of the trunk and branches and total leaf mass and surface area. Samples were collected and stored as described above. Soil and root samples were taken from multiple horizons as the trees were excavated. Leaf litter was collected during the winter of 1996 to determine the magnitude of the decrease in TCE and metabolites that would be present in the leaves after weathering. The amounts of TCE and its oxidative metabolites, trichloroethanol, trichloroacetic acid, and dichloroacetic acid, were determined by extracting milled plant tissue samples with methyl-tert-butyl ether (MTBE) according to the protocol in Newman et al. (8). Reductive metabolites (dichloroethylene isomers) were analyzed by placing a weighed portion (0.3-0.5 g) of milled plant tissue into a 25 mL screw cap vial along with 20 mL of aqueous 1 N H2SO4/10% NaCl and 20 µL of internal standard (200 µg mL-1 bromochloromethane in methanol). The vial was capped and shaken for 10 min. The vial headspace was then sampled for 10 min with a 100 µm poly(dimethyl siloxane) solid-phase microextraction (SPME) fiber. The SPME fiber was immediately thermally desorbed for 20 min in a Fisons MD-800 GC-MS injection port maintained at 300 °C. The capillary GC column was a J&W DB-VRX 60 m × 0.32 mm i.d. with a 1.8 µm film thickness operated with a helium flow of 1.7 mL min-1. A column temperature program of 45 °C for 4 min, 5 °C min-1 to 80 °C with a 4 min hold, then 15 °C min-1 to 250 °C with a 4 min hold was used. The mass spectrometer was operated in the electron impact mode with a scan range of 35-200 m/z.

Plant Analysis, Summer 1997. Leaf samples were collected throughout the crowns of standing trees, mixed and stored as above. Branch samples were also collected and stored as previously described. Tissue samples were analyzed for both oxidative and reductive metabolites as described above. Plant tissue metabolite concentrations for all three years were derived from the average of one to four analyses, with nondetects counted as a zero reading. Concentrations measured in control, nondosed plant tissues (less than 250 ng g-1 of oxidative metabolites and less than 1.1 µg g-1 DCEs) were subtracted from the averages. Transpiration of TCE. Two methods were used to determine the amount of TCE transpired by the plants during the second and third years of the experiment. The first method (leaf bag) involved measuring the amount of TCE that was transpired from a singe leaf. A leaf was enclosed in a Teflon bag (Norton Performance Plastics) loosely fitted around the petiole to allow passage of air into the bag. Brass wires inside the bag prevented the bag from collapsing against the leaf surface and permitted passage of air over the leaf. The bag was fitted with a metal exit port, which was attached to an activated carbon tube (SKC Inc.) by heat shrink Teflon tubing. The carbon tube was heated to 35 °C to prevent condensed water from interfering with the adsorption of TCE to the carbon. Air was pulled through the leaf bag and carbon tube for 2 h at a flow rate of 1 L min-1. The front and rear portions of carbon from the tube were placed in two septum-capped vials, 1.0 mL of carbon disulfide was added, and the vials were vortexed for 30 min. Methylisobutyl ketone (MIBK) was added as an internal standard (10 µL). The sample was analyzed on a Hewlett-Packard 5890 GC with a J&W 0.53 mm × 30 m DB-1 column and flame ionization detector. The GC temperature program was 35 °C for 4 min, ramped at 10 °C min-1, and held at 100 °C for 4 min. Two types of negative controls for the leaf bag assays were performed: one in which the leaf tested was on a tree in the nondosed cell, and one in which there was no leaf in the bag. No TCE was detected in the carbon tubes of these control tests. To determine the recovery efficiency, two types of spiked, positive controls were also performed. In the first test, an empty bag was dosed with a known amount of TCE and air was pulled through the bag for 1 h. In the second test, a bag placed around a leaf was dosed with a known amount of TCE and air was again pulled through the bag for 1 h. Recovery efficiencies in the spiked controls ranged 70-80% of the added TCE. Since the detection limit for the GC measurement of TCE in the extracts from the activated carbon trap was about 1 × 10-4 µmol of TCE mL-1, the detection limit of the leaf bag method was about 0.8 nmol of TCE m-3 air. In the second season (1996), leaf bag tests were always performed on leaves in full sun growing on the current year’s wood. During the third season (1997), the following tests were run: leaves in full sun growing on the current year’s wood, leaves in full sun growing on a previous year’s wood, and leaves in full shade growing on the previous year’s wood. A day-long study was also done in which a set of leaves from a single tree were analyzed at 2 h intervals from sunrise to sunset to determine if transpiration of TCE varied diurnally. Total leaf area was calculated by measuring the mass of the total leaves of a tree that was harvested at the end of the 1996 season. The area/mass ratio of individual leaves was used to determine the leaf area of the entire tree. The second method for transpiration determination used open-path Fourier transform infrared (OP-FTIR) spectroscopy to measure ambient TCE concentration in the vicinity of the trees (11). Two experiments were conducted. In the first experiment, a retroreflector was placed at one side of VOL. 33, NO. 13, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Water Balancea 1996 planted TCE-exposed injection top water rainfallb total added recovered through 10/10/97 recovered 10/10-11/21/97 total recovered/year

1997

+ + 32 100 5640 9170 46 900

+ + 32 100 5640 7590 45 300

+ 31 700 5640 9170 46 500

+ 32 100 3270 7590 43 000

1190

1430

2720

35 000

+ + 81 800 8730 7590 98 100 530 19 730 20 300

+ 64 300 8340 7590 80 200 744

a

Water was added to cells either by injection into the sand/gravel layer at the bottom of the cell, surface watering, or by rainfall. Recovered water was taken from the effluent wells. Water loss occurred either through evaporation from the soil or plant uptake. Volumes in liters. b Due to heavy winter rains in the Pacific Northwest, the cells were covered during the winter months to prevent flooding of the cells while the trees were dormant. The differences in the amount of rainfall reflect the different dates when the cells were covered.

the TCE-exposed trees on scaffolding approximately 3.6 m above the ground level. The unistatic (source and detector) OP-FTIR device was set up on a second scaffolding at the same height such that the beam path of 6 m would go near and partially through the canopy of the trees. In the second experiment, a row of trees was pulled over such that the beam path would go directly over the bent trunks of the trees. For both experiments, the optical path length was 12 m, which yielded a detection limit of 50 ppb (v/v, 0.3 µmol m-3) for TCE at 1 cm-1 spectral resolution. The OP-FTIR limit of detection for TCE was determined from measurements of the instrument absorbance signal-to-noise ratio under the same temporal and path length conditions as the field data. Noise was measured from an absorbance spectrum generated with 2 successive single-beam spectra collected in the field over a 12 m optical path (11). Five times the peak-to-peak noise was used as the minimum detectable absorbance peak in the TCE absorption spectral range between 750 and 970 cm-1 (12). A TCE absorbance spectrum from a reference library (Infrared Analysis Inc.) was used to establish the height of an absorbance peak for a known concentration. Background spectra were taken over an open field, upwind from the site to ensure that any detected contamination was from the trees or other on-site sources. Instrument calibration, spectra collection, and data analysis to retrieve concentration were performed following the procedures in the draft US-EPA standard method TO-16 (13). These measurements were taken in September 1996. Rhizosphere Activity. The degradation of TCE in the rhizosphere soil was assayed in the second year. Approximately 2 L of soil from both planted and unplanted cells was collected from 30 cm below the surface and placed in glass metabolic chambers (14) in triplicate. The soil samples were field dry and mixed during sampling. Rooted cuttings of H11-11 stock were planted in the soil from the planted cell and held for approximately 1 week until roots started to penetrate into the soil. The soil chambers were sealed with glass plates and both sets of soil were dosed with TCEsaturated water to a final concentration of 38 µM TCE and 1 × 106 dpm uniformly labeled [14C]TCE (Sigma, St. Louis). After 2 weeks, gases were stripped from the soil by an air stream and CO2 was trapped in 1 N NaOH. The amount of 14C in the CO was measured by scintillation counting. 2 Chloride Analysis. The first method, a modified turbidimetric method using a Hitachi U2000 visible wavelength spectrophotometer, was used only for aqueous samples. The procedure was calibrated between 0 and 0.3 mM Cl-1 using a 10 mm path-length cell. Samples were prepared for analysis by treating a 40 mL sample with 0.2 mL of 0.1 N HNO3, and then 0.2 mL of 0.1 N AgNO3 was added to precipitate the inorganic chloride as a AgCl colloid. After 10 min in the absence of light, the absorbance was measured at 550 nm and concentrations reported in millimolar chloride ion. 2260

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A modification of the mercury(II) thiocyanate method (15) was used to determine chloride ion in additional aqueous samples and in soil and plant tissue samples. Plant tissues were extracted with water, decolorized with activated carbon. Reagent Fe(NO3)3 was added to the extract followed by HNO3 and Hg(SCN)2. The chloride recovery using this method was 90-110% efficient as determined from soil and plant samples spiked with chloride. On 15 Aug 1997, a set of soil samples was obtained for chloride analysis. Soil cores (5 cm diameter) were obtained from the cells at 1.4 and 4.8 m from the influent end of the cells. The cores were separated into subsamples at 0.2 m intervals, representing depths from the surface to 1.0 m deep. Plant samples were harvested by the method used for collecting samples for metabolite analysis. Fixed organic halides in the plant tissues were analyzed by Analytical Resources, Inc., Seattle, WA, using a modification of the total organic halide method (TOX) (16). Inorganic chloride ion was removed from plant tissue samples prior to TOX analysis by washing ground tissue with deionized water (50 mL g-1 tissue). Washed samples were dried at 70 °C and weighed directly into the combustion boats for analysis.

Results and Discussion Effect of TCE on Tree Growth. The trees in the experiment grew rapidly, attaining mean heights of 3 m by the end of the first season, 7 m by the end of the second season, and 11 m after three growing seasons. Growth was not significantly affected by TCE treatment. Mean parameters of the trees at 2 years were 7.0 m height, 18.7 cm trunk diameter at breast height, 18 m2 leaf area per tree, 6.2 g dry weight leaves per tree, and 71 branches per tree. Water Uptake by Poplars. Table 1 shows the amount of water added to the planted and unplanted cells, and the amount of water removed from each of the cells during the second and third growing seasons. The high transpiration of water by these trees suggests that they have the potential, under appropriate circumstances, to lower the rate of flow of water from a contaminated site, thus reducing the spreading of a water-soluble pollutant. TCE Removal from the Artificial Aquifer. TCE/Rs were detected in the effluent of the unplanted cell nine weeks after the start of injection into the aquifer (Figure 2). Five weeks later (9 November), TCE/R was detected in water samples from the effluent of the planted cells. TCE/R levels in the effluents of the planted cells increased substantially following leaf drop (9 November). The cumulative data for 1995 (Table 2) show that 0.37 mol of TCE/R was recovered in the unplanted control cell, compared to 0.02 and 0.06 mol of TCE/R recovered from the planted cells. Recoveries of TCE/R in the second year were 3.30 mol from the unplanted cell, and 0.04-0.06 mol from the two

FIGURE 2. Daily influent and effluent masses of TCE and reductive dechlorination products (TCE/R). Daily masses of TCE and TCE/R were determined from measured concentrations and water volumes from September 1995 through December 1997. TCE influent concentrations averaged 0.11 mM. Fluctuations in data reflect variations in water volumes added due to climatic conditions and growth of trees, and temperature influenced changes in saturation levels of stock solutions.

TABLE 2. Influent and Effluent Mass of TCE and TCE/R, Cummulative Totals, 1995-1997a mass of TCE and TCE/R 1995 TCE in the influent of all cells recovered in the effluent cell with no trees cell with trees cell with trees

1996

1997

total mass % recovered recovered

0.93 4.50 6.26

11.70

0.37 3.30 0.02 0.04 0.02 0.06 0.06

3.66 0.08 0.12

67 1 2

a Shown is the mass of TCE added to each cell over the three year period, the mass or TCE/R recovered from each cell, and the percent recoveries of TCE/R in the effluent relative to the influent. Data for 1997 are given prior to bud set. Values given are in moles.

planted cells. Recovery of TCE/R during the third growing season followed the same trend with only 0.38% of the added TCE recovered in the cell with trees (prior to bud set). Overall, the mass of TCE/R recovered in the effluent from the unplanted cell was 67% of the mass of TCE added. In contrast, the mass of TCE/R recovered from the planted cells was only 1-2% of the TCE applied. The recovery of TCE/R from the planted cells was low when the transpiration of water was high (Figure 2) and higher when transpiration was low (during the period between bud set and the end of the season). The apparent linkage between transpiration and low recovery of TCE/R suggests that the majority of TCE loss from the aquifer was associated with plant uptake of water. Reductive dechlorination products from each cell during the first two years of experiments included all three dichloroethylene (DCE) isomers, with cis-1,2-DCE predominating (Table 3). Trace amounts of other compounds, including vinyl chloride, 1,1-dichloroethane, and 1,1,2-trichloroethane were detected three times during 1997 (data not shown) and

totaled less than 0.006% of the TCE added. It is likely that these compounds were produced from microbial and abiotic reduction of TCE in anaerobic zones of the soil. In the first growing season, anaerobic conditions may have been enhanced by the addition of the ethanol that was used as a solvent for TCE. This hypothesis is supported by low levels of DO observed in the effluent wells (averaging 2.9 mg L-1, ranging 1.2-5.3) and by the steady decrease in levels of chlorinated compounds other than TCE during the course of the second growing season, when ethanol was no longer used as a solvent for TCE. DO levels in the cell effluent wells in August of the second season averaged 4.5 mg L-1. TCE/R mass recovered from the planted, exposed cell that was used for the entire 3 year period was less than 1% of the TCE mass added (Table 2), leaving 99% of the TCE mass unaccounted for. However, TCE/R concentrations in the effluent water from the planted cells were not decreased to the same extent. The combined concentrations of TCE/R from the planted cells in 1996 averaged 0.05 ( 0.03 mM in the effluent water versus 0.12 ( 0.02 mM TCE in the influent water. In contrast, the average combined concentrations of TCE/R in the effluent of the unplanted control cell were 0.09 ( 0.04 mM for the same year. Decreased TCE/R concentrations may indicate an ability of the trees and the rhizosphere to reduce TCE concentration of the water. TCE and Metabolites in Tissue. Table 4 shows the distribution of metabolites in the plant tissue over the 3 year period of TCE exposure. Di- and trichloroacetic acids were the major metabolites in the leaves during the first 2 years. The pattern in these years was seen previously in the tissues of TCE-exposed trees grown in the greenhouse (8). The pattern changed in the third summer with the chloroacetic acids decreasing both in concentration and in proportion to TCE in the tissue. TCE was the major chlorinated compound detected in the branches or trunks of plants with the exception of the fall of 1996. This behavior might be expected considering VOL. 33, NO. 13, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Recovery of TCE and TCE/R from the Effluent Wells in 1995 and 1996a 1995

planted cell

TCE cDCE tDCE TCE cDCE tDCE TCE cDCE tDCE

planted cell unplanted cell

1996

Oct

Nov

Dec

May

June

July

0.00 0.00 0.00 0.06 0.13 0.00 30.9 21.2 0.00

1.12 9.29 0.00 2.59 29.7 0.02 24.0 183 0.14

0.50 9.52 0.00 0.98 27.8 0.04 2.39 104 0.22

0.14 8.14 0.07 0.19 7.35 0.03 1.42 29.6 0.33

0.03 2.10 0.01 0.03 1.34 0.01 137 40.5 0.53

0.63 2.04 0.03 1.90 0.00 890 22.8 0.05

Aug

Sept

4.03 2.56 0.02 4.80 6.59 0.04 999 72.2

13.0 4.26 0.05 25.6 12.4 0.08 875 20.0

Oct 1.03 0.23 1.02 0.38 195 11.6

a Monthly recoveries of TCE and TCE/R (mM) from effluent wells, with amounts for each product given. Amounts given are in millimoles. TCE, trichloroethylene; cDCE; cis-1,2-dichloroethylene; tDCE, trans-1,2-dichloroethylene.

TABLE 4. TCE and Metabolites in Plant Tissues Fall 1995 TCE TCAA DCAA TCEtOH

leaves

brach

roots surface

ND 0.20 0.25 ND

0.10 0.15 ND ND

0.21 ND ND ND

Spring 1996 leaves branch

influent old growth

influent new growth

effluent old growth

effluent new growth

influent

effluent

0.06 40 19 0.11

ND 16 1.5 ND

ND 0.42 0.11 0.03

ND 1.6 1.6 ND

3.2 ND ND ND

1.8 ND ND ND

TCE TCAA DCAA TCEtOH

Fall 1996 leaves

TCE TCAA DCAA TCEtOH c DCE t DCE 1,1-DCE CH

old growth

new growth

yellowed

weathered

branch

trunk

surface

roots 30 cm

60 cm

ND 21 10 0.31 NS ND NS ND

ND 22 6.1 0.33 NS ND NS ND

ND 20 12 0.21 NS ND NS ND

ND 1.9 0.6 0.25 0.47 0.1 11 ND

0.68 0.74 0.08 0.17 0.29 0.13 17 ND

1.3 0.03 ND ND ND ND NS 0.09

0.14 ND 0.08 ND ND ND NS ND

0.25 ND ND ND ND ND ND ND

0.33 ND 0.29 ND ND ND ND ND

Summer 1997 leaves TCE TCAA DCAA TCEtOH c DCE t DCE 1,1-DCE CF

that these tissues were less metabolically active than leaves or roots. TCE concentrations were lower in the branch samples collected in the fall of 1996, perhaps because leaf petioles were included in the branch samples, and petioles are more metabolically active than branches. These samples also contained relative high amounts of 1,1-DCE. That compound was not detected in other samples, but significant levels of cis-1,2-DCE appeared in the branch samples collected in 1997. Transpiration of TCE. In 1996, the average rate of TCE transpiration as detected in the leaf bags was 2 × 10-8 mol 2262

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2.5 2.3 0.47 0.56 0.52 ND ND 0.49

branch 41 0.08 ND 0.56 7.2 ND ND ND

of TCE h-1 leaf-1. With leaf area of the tested leaves averaging 400 cm2, transpiration of TCE per cm2 leaf area averaged 5 × 10-11 mol of TCE h-1. Transpiration of TCE was not detected in any of the OP-FTIR spectra. Although it had a higher detection limit than the leaf bag methods, the OP-FTIR method sampled the entire canopy atmosphere with minimal disturbance of the trees. The failure to detect any airborne TCE by OP-FTIR is in agreement the results of the more sensitive leaf bag method. Total leaf area on all trees in a cell was approximately 2.3 × 106 cm2, giving an estimated transpiration of TCE per cell

TABLE 5. Inorganic and Organic Chloride Concentrations in Plant Tissuesa chloride ion (mmol kg-1)

control, unexposed trees TCE-exposed trees

tox (mmol kg-1)

roots

stems

leaves

roots

stems

leaves

6.4 ( 1.4 14 ( 8

7.4 8.3 ( 0.7

64 ( 28 75 ( 34

0.46 ( 0.06 0.96 ( 0.81

0.45 1.0 ( 0.7

1.4 ( 1.5 1.8 ( 1.1

a Plant tissues were extracted and chloride ion determined. Total organic halide (TOX) was determined on tissue samples after inorganic halide was removed.

FIGURE 3. Distribution of chloride ion in soil of cells with trees. Soils were sampled on August 15, 1997, at various depths at two locations in each cell: near the influent and near the effluent. The horizontal dotted line indicates the background level of chloride in soils that were not exposed to either TCE or to chloride in the water recirculated between cells. of 1.2 × 10-4 mol of TCE h-1. Assuming there were 210 days with active transpiration of TCE in a growing season and that the trees were transpired 12 h d-1, we estimate that the total amount of TCE transpired per cell in a season was about 0.30 mol. The 0.30 mol transpired represents approximately 9% of the 3.2 mol of TCE lost from the planted cells in 1996. TCE was not detected in 1997 by the leaf bag method, although measurements were taken on leaves of various sizes, on different positions on the trees, or taken during different times of the day. Degradation of TCE in the Rhizosphere. The 2 week laboratory study in 1996 showed no enhancement of degradation of TCE associated with the roots. The unplanted soil mineralized 7% of the added TCE to CO2, whereas the rhizosphere soil from the planted cell mineralized 5%. This study indicated no rhizosphere enhancement of soil organisms that metabolized TCE. Chloride Concentrations in Water, Tissue, and Soil. Chloride ion concentrations in the water leaving the TCEexposed cells were less than the levels in the influent water (70 ( 45 versus 113 ( 42 µM, respectively), presumably due to chloride adsorption and precipitation in the soil. The total mass of chloride leaving the TCE-exposed cells (3 mmol Cl-) was less than the chloride mass in the influent water (210 mmol Cl-) during a 13 day period in 1996, due to the reduction in water in the system through tree uptake.

Chloride and fixed TOX concentrations, although very low, were higher in TCE-treated poplar tissue than in the unexposed trees, but the differences were not statistically significant (Table 5). The low level of TOX is in agreement with the low levels of TCE metabolites detected in plant tissue by GC-MS (Table 4). The chloride ion levels (up to 3.9 mmol kg-1) in soil from the planted, TCE-exposed cell were 10 times higher than in soil that was not exposed to TCE or to recirculated water (0.39 mmol kg-1). Figure 3 shows the distribution of chloride ion in the soils of one of the TCE-exposed cells planted with trees and in the nondosed, vegetated control cell. Chloride ion was greatest at the 0.4-0.6 m depth in both cells and levels tended to be highest at the effluent end of the cells. Because of high water uptake by the trees, soil above the capillary fringe was usually dry and thus chloride likely accumulated in that layer. With only one exception, chloride levels were greater in the TCE-exposed soil samples than in the samples from the control cell, which did not receive TCE, but did received recirculated water. Our interpretation of these results is that TCE was taken up by the plants, dechlorinated in the plant tissue, and that the chloride was excreted by the roots and accumulated in the soil. The expulsion of chloride from intact roots has not been reported in the plant literature, but we have observed leakage of chloride ion from roots when poplar plants grown in the presence of chloride are transplanted to chloride-free VOL. 33, NO. 13, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 6. Mass Balance for Chlorine in TCE-Exposed Cell with Hybrid Poplara 1995 TCE-chlorine lost from water in systemc TCE-chlorine recovered from transpirationd TCE-chlorine recovered from oxidative metabolites in plant tissued leaves branches trunk rootsg excess chloride ion in soilb chloride balance recovery efficiency

mol of chlorine or chloride ion 1996

2.75

13.4

0.28e

0.87e

0.03 × 10-3 0.03 × 10-3 0.05 × 10-3 0.03 × 10-3 ND

0.006 0.005 0.01 0.006 ND

1997b

3-year total loss

11.32

27.47

0f

3-year total recovered

1.15

0.002 0.002 0.003 0.002 18

0.008 0.007 0.013 0.008 18 27.5

19.2 70%

a Chlorine added in the form of TCE-chlorine was balanced against the amount of TCE-chlorine, metabolite-chlorine and free chloride ion recovered from the system. Masses given cover the three years that theexperiment ran. ND, not determined. b Prior to 15 August 1997. c Corrected for presence of reductive dechlorination products. d Leaf areas and mass of tree tissues per cell were adjusted by the ratio of tree heights at the end of the respective growing season to that measured at the end of 1996. e Calculated using average of leaf bag assays in 1996, 1.5 × 10-11 mol h-1 cm-2 leaf. f No TCE was recovered from the leaf bag assay in 1997. g Mass of roots was calculated as 0.40 times the mass of the above ground woody parts of the plants (20).

hydroponic solutions (data not shown). Furthermore, chloride ion is known to be transported into and out of plant root cells in vitro (17-19), chloride and nitrate are thought to be transported by the same carrier system (20), uptake of chloride and nitrate by many plants is competitive (21), and excess nitrate can be excreted from roots of some plants (22). Thus, it is reasonable to expect that chloride ions may be expelled from plant tissue that is producing excess chloride by dechlorination. This hypothesis will be tested with further experimentation. While it is certainly possible that the TCE loss from the planted cells was due to enhancement of microbial degradation in the rhizosphere, the magnitude of TCE mass that was lost plus the results from the laboratory study, though short term, indicated otherwise. At the rate that TCE mineralization was measured in the laboratory with rhizosphere soil [5 × 10-8 mol of TCE (kg of soil) -1 day-1], the mass of TCE removal in a cell containing about 19 400 kg soil over a 210 day season would be 5 × 10-8 × 19 400 × 210 ) 0.20 mol of TCE. With observed losses of 4.45 and 6.24 mol of TCE in the planted cells in the years 1996 and 1997, respectively, rhizosphere degradation would account for less than 5% of the observed losses of TCE. However, it is possible that different results would be obtained if different soils were used. Due to the short duration and limited sampling of the laboratory tests of soil activity, we cannot say conclusively that microbial degradation did not contribute to the observed TCE loss. The excess of chloride in the soil of the TCE-exposed cell shown in Figure 3 suggests that dechlorination was a significant fate of TCE. The total mass of chloride in the cells was calculated from the average soil Cl- concentrations in the soil cores multiplied by the average soil density and the cell volumes. The estimated accumulated mass of chloride was 44 mol of Cl- for the soil in the cell with TCE-exposed trees and 26 mol of Cl- for the soil in the control cell with unexposed trees. Since both cells received the same amounts of chloride ion in the influent water (either from municipal water or from recirculated water), the difference in accumulated chloride, 18 mol of Cl-, was due to degradation of the TCE added to the cell from the start of the experiment in 1995 to the sampling date, 15 Aug 1997. The total chlorine in the TCE applied to the exposed cell during this period was 27.64 mol. The total TCE chlorine in the effluent from this 2264

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cell was 0.17 mol, leaving approximately 27.5 mol of Clunaccounted for. Thus, the chloride recovered in the soil in the TCE-exposed cell represented approximately 65% of the chlorine in the lost TCE. The distribution of chlorine from TCE in one of the TCEexposed cells is shown in Table 6, which presents a summary of the masses of chlorine in the TCE which was lost in the cell from 1995 to 1997 (i.e., influent TCE chlorine minus effluent TCE chlorine), the estimated TCE chlorine lost due to transpiration, the estimated chlorine in oxidative metabolites in the plant tissue, and the chloride present in the soil of the TCE-exposed cell compared to that in the unexposed, planted control cell. The total recovered chlorine accounts for 70% of the lost TCE chlorine. One explanation for the remaining missing chloride is that some chloride ion was probably washed from the TCE-exposed cell during winter rains. More than 93% of the recovered chlorine was accounted for as chloride ion in the soil, and less than 6% as transpired TCE chlorine. Plant metabolites accounted for an insignificant fraction of the total recovered chlorine. Thus, chloride ion accumulation in the soils of the planted cells exposed to TCE was enough to account for a substantial portion of the chloride that would be produced from complete degradation of the TCE lost in the cell. Further work is needed in order to determine the range of volatile chlorinated compounds that can be treated by phytoremediation and the level of such activities in or associated with various tree species. For phytoremediation to be successful, we must also determine the maximum loading of TCE and other chlorinated compounds that can be treated without giving rise to plant toxicity, accumulation of toxic metabolic intermediates, or atmospheric emissions. Knowledge of other factors such as the depths at which roots can intercept and halt the movement of a contaminated groundwater plume and the land area required to be planted for a given hydrologic condition are also necessary for the engineered application of phytoremediation. Further experimentation is also needed to define the relative roles of microbial rhizosphere degradation versus degradation of TCE by the plant tissue itself under different environmental conditions. Taking into consideration these caveats, the results of this investigation show that hybrid poplar trees tolerate

exposure to groundwater containing moderate levels of TCE. Under the conditions of this experiment, 99% of the added TCE was removed in the planted cells. Up to age 2 years, the trees transpired about 9% of the TCE added and after age two, no transpiration of TCE was detected. The presence of an excess of chloride in the soil together with no increase in TOX in the plant indicated dechlorination of the applied TCE. Thus, these results are of great importance to the remediation of TCE contaminated soils and groundwaters.

Acknowledgments This work was funded by Occidental Chemical, U.S. NIEHS Grant 5 P42 ES04696-07, the Consortium for Risk Evaluation with Stakeholder Participation (CRESP) through U.S. DOE Cooperative Agreement DE-FC01-95EW55084, and U.S. DOE EMSP program through Grant DE-FG07-96-ER20256. Technical assistance was provided by Curtis Bod, Diane Fogle, B. Brook Shurtleff, and Stephanie Stanley. We would like to acknowledge the contributions of the late Dr. Irving Gordon who participated in the initial formulation of the ideas presented in this paper.

Literature Cited (1) Patrick, R.; Ford, E.; Quarles, J. Groundwater contamination in the United States, 2nd ed.; University of Pennsylvania Press: Philadelphia, PA, 1987. (2) Cunningham, S. D.; Ow, D. W. Plant Physiol. 1996, 110, 715719. (3) Schnoor, J. L.; Licht, L. A.; McCutcheon, S. C.; Wolfe, N. L.; Carreira, L. H. Environ. Sci. Technol. 1995, 29, A318-A323. (4) Anderson, T. A.; Guthrie, E. A.; Walton, B. T. Environ. Sci. Technol. 1993, 27, 2630-2636. (5) Anderson, T.; Walton, B. Comparative plant uptake and microbial degradation of trichloroethylene in the rhizospheres of five plant species - - implications; Oak Ridge Lab., Martin Marietta, 1992. (6) Narayanan, M.; Davis, L. C.; Erickson, L. E. Environ. Sci. Technol. 1995, 29, 2437-2444.

(7) Schnabel, W. E.; Dietz, A. C.; Burken, J. G.; Schnoor, J. L.; Alvarez, P. J. Water Res. 1997, 31, 816-824. (8) Newman, L. A.; Strand, S. E.; Choe, N.; Duffy, J.; Ekuan, G.; Ruszaj, M.; Shurtleff, B. B.; Wilmoth, J.; Heilman, P.; Gordon, M. P. Environ. Sci. Technol. 1997, 31, 1062-1067. (9) Nair, D. R.; Schnoor, J. L. Water Res. 1994, 28, 1199-1205. (10) University Washington; Occidental Chemical Co. Removal of Organic Contaminants from Water or Gas by Phytoremediation. Patent Patent applied for, filed August 20, 1998. (11) Russwurm, G. M.; Childers, J. W. FTIR Open-Path Monitoring Guidance Document; US-EPA, 1995. (12) Strang, C. R.; Levine, S. P. Am. Ind. Hyg. Assoc. J. 1989, 50, 7884. (13) Anonymous. Compendium Method TO-16: Long-Path OpenPath Fourier Transform Infrared Monitoring of Atmospheric Gases, Draft; U.S. Environmental Protection Agency, 1996. (14) Gordon, M. P.; Choe, N.; Duffy, J.; Ekuan, G.; Heilman, P.; Muiznieks, I.; Newman, L. A.; Ruszaj, M.; Shurtleff, B. B.; Strand, S. E.; J., W. In Phytoremedialion of Soil and Water Contaminants; E. Kruger, Ed.; Symposium Series 664; Am. Chem. Soc.: Orlando, Florida, 1996; pp 177-185. (15) Adriano, D. C.; Doner, H. E. In Methods of Soil Analysis, Part 2. Chemical and Microbiological Properties; Page, A. L., Miller, R. H., Keeney, D. R., Eds.; ASA-SSSA: Madison, WI, 1982; pp 449462. (16) Anonymous. Test Methods for Evaluating Solid Waste, SW-84b; U.S. EPA Office of Solid Waste, 1994. (17) Felle, H. H. Plant Physiol. 1994, 106, 1131-1136. (18) Marten, I.; Lohse, G.; Hedrich, R. Nature 1991, 353, 758-762. (19) Cram, W. J. J. Membr. Biol. 1983, 74, 51-58. (20) Pope, A. J.; Leigh, R. A. Planta 1990, 181, 406-413. (21) Marschner, H. Mineral Nutrition of Higher Plants, 2nd ed.; Academic Press: London, 1995. (22) Clarkson, D. In Inherent Variation in Plant Growth: Physiological Mechanisms and Ecological Consequences; Lambers, H., Poorter, H., Van Vuren, M. M. I., Eds.; Backhuys Publ.: Leiden, Netherlands, 1998; pp 221-235.

Received for review November 23, 1998. Revised manuscript received March 24, 1999. Accepted April 12, 1999. ES981217K

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