Phytoremediation of trichloroethylene with hybrid poplars.

Axenic tumor cultures of poplar cells, clone H11-11, were grown in the presence of [14C]-trichloroethylene (TCE) (uniformly labeled). The cells were capable of metabolizing TCE to produce trichloroethanol, di- and trichloroacetic acid. Some of the carbon from TCE was found in insoluble, nonextractable cell residue, and small amounts were mineralized to [14C]CO2. Poplar cuttings grown in soil and exposed to TCE produced the same metabolites. In field trials, trees were planted in soil in test cells and exposed to TCE via underground water injection during the growing season. During the growing season, at least 95% of the TCE was removed from the influent water stream in cells containing trees. Mass balance studies conducted in the laboratory indicated that 70 to 90% of the TCE was transpired; however, greenhouse and field study results showed that less than 5% of the total TCE taken up by the plants is transpired. These results show that significant TCE uptake and degradation occur in poplars. Poplars appear to be useful for in situ remediation of TCE-contaminated sites under proper conditions. ImagesFigure 2

Trichloroethylene (TCE) is one of the most widespread contaminants in the environment of the United States (1). TCE is stable in groundwater and can persist for decades as dense nonaqueous-phase liquids. Difficulty in pinpointing the location of these pools of TCE can greatly hinder attempts at remediation. The fact that TCE is a suspected carcinogen (2) has lent urgency to cleanup efforts. The principal methods for remediating groundwater contaminated with TCE are pumping water from the aquifer and stripping the TCE by aeration or by charcoal absorption. These procedures can take years or decades and can be very expensive (3). Other techniques use bacteria to degrade TCE, although this often requires inducers such as toluene or phenol for degradation to occur (4). Recent research investigating the use of plants to remediate environmental pollution is yielding promising results. With some species of plants the major site of TCE degradation appears to be in the rhizosphere, and the actual role of the plant in remediation may be to supply nutrients to the rhizospheric organisms (5,6).
We have investigated two clones of poplar (Populus trichocarpa x P. deltoides, clones H1-11 and 50-189) as a means to remediate TCE pollution. Poplar was chosen for a variety of reasons. Poplar has a very wide geographical distribution and can be grown from southern Alaska into Central America. Members of the species can be easily crossed sexually. Propagation by cuttings is simple, yielding clones of a given individual, and poplars can be grown axenically in culture. Exogenous genes can be incorporated into the poplar genome, thus there is the potential of incorporating genes that will more completely mineralize TCE or other pollutants. The College of Forest resources at the University of Washington and Washington State University at Puyallup have worked with poplars for over 20 years, and has accumulated an enormous amount of background concerning the physiology and genetics of this genus. The absorption surface of roots in a stand of poplars is enormous and can approach 300,000 km/ha (7). The water usage under the warm, arid conditions of eastern Washington State is about 140 cm per year in a stand of 5-yearold trees at a density of 1750 trees/ha (7).
The investigations discussed in this chapter first utilized axenic cultures of Populus trichocarpa x P. deltoides H 1 1-1 1 tumor cells, then small plants treated with water containing TCE, and, finally, a number of experimental cells that mimicked field trials. We also developed a bioreactor that enabled us to account for the distribution of most of the TCE metabolized by small H 11 -1 1 rooted cuttings.

Axenic Tumor Cell Experiments
Transforming shoots of HI 1 -1 1 with Agrobacterium tumefaciens A281 produced the axenic H 11-1 1 tumor cells. Tumor cells are used because they can be easily grown on simple minimal medium (Murashige and Skoog basal salt medium) (8). The cells were grown on the above medium at various degrees of saturation of TCE with shaking and illumination for 3 or 5 days at 22°C. The vital stain, Trypan blue ( Figure 1 determined the toxicity of TCE, towards the HI 1-11 cells. To study the metabolites of TCE, the poplar cells were incubated with 80 mg TCE/1. The suspension was then centrifuged and a sample was extracted at 20°C with 1 N H2SO4/10% NaCI, then 3 times with methanol, and then 3 times with methyl tertiary butyl ether (MTBE). In order to test for the presence of diand trichloroacetic acid, a second batch of cells was extracted with 1 N sodium hydroxide (NaOH), the extracts acidified, backextracted with MTBE, and the organic acids in the ether extract esterified with diazomethane. The metabolites were then analyzed by gas chromatography with an electron capture detector.
The cell cultures were also tested for the formation of '4C-CO2 from TCE. The gases evolved during 4or 10-day incubations with 14C-TCE were trapped in 1 N NaOH and confirmed to be CO2. The cells and media were separated by centrifugation and the amount of 14C remaining in the media was determined. Insoluble extracted cell residue was combusted and the resulting CO2 analyzed for radioactivity. The results of these analyses are given in the accompanying table (Table 1). The products from the TCE metabolism, trichloroethanol, di-and trichloroacetic acid, and C02, are also among the products produced by the activity of rat and mouse hepatic cytochrome P450 (9). These findings suggest that the oxidative metabolism ofTCE in poplars is similar to the processes in mammals ("green liver" hypothesis).

Whole Plant Experiments
Cuttings of the two hybrid clones noted above were rooted in PVC (polyvinyl chloride) pipes, 20.5 cm diameter, containing a 30-cm bottom layer of sand with 60 cm soil (Sultan silty clay loam) overlay. The plants were dosed with water alone, or with 50 mg TCE/l added directly to the sand layer through an inner watering tube. A total of 8 g TCE was added over a period of 8 months. During the period of growth, transpiration was assayed by enclosing the leaves in plastic bags and pulling the air through a charcoal filter for 0.5 hr. The results were highly variable and indicated transpiration values from undetectable to about 1.6 pg TCE/leaf/hr, with an average of approximately 1.0 pg TCE/leaf/hr. After 8 months, the plants in PVC pipes were harvested and various morphologic measurements were taken ( Table 2). The major difference noted was that the density of the fine roots in the sand layer of TCEexposed plants was less than that of the controls. The plant tissues were analyzed for metabolites of TCE (Table 3) using the same methods that were used for cell analyses. The nature and levels of the chlorinated metabolites suggest that the TCE is oxidized as it moves from bottom roots to the aerial sections of the plant, especially the leaves. These results, together with the products derived from the axenically cultured poplar cells, strongly argue for a role of the plant in the metabolism of TCE, in addition to the well-known degradation of TCE by soil microorganisms.

Mass Balance Studies
The mass balance of TCE in hybrid poplar trees was determined in a bioreactor. The leaves, stem and roots of the plant were enclosed in separate sections of the chamber to allow the study of plant uptake, incorporation, transpiration, and rhizosphere degradation without interference from soil volatilization. The major problems we faced were due to the volatility of TCE and its suspected metabolites, and the absorption of these materials by commonly used laboratory materials such as rubber, tygon, and various sealants. The chamber is shown in Figure 2. It is constructed of glass, aluminum foil, Teflon, and inert inorganic materials. Note that the separate sections for roots, stems, and leaves are independently aspirated to prevent volatile transpirates from leaking into other chambers. Controls without plants show only 0.03% leakage of the TCE into the foliage chamber.
Small rooted poplar cuttings (approximately 20 cm tall) were planted in a peat moss/vermiculite mixture in the root section and TCE was added to the root section to a final concentration of 5 mg/l and spiked with 2 x 106 dpm 14C-TCE. After 7 days approximately 0.8% of the added 14C was detected in the transpirate.
Several conclusions can be drawn from these three laboratory experiments. First, TCE can be taken up by the poplars. Second, the plants transpire some of the material, and some is metabolized to known extractable metabolites, as found in Tables 1 and 2. Finally, some nonextractable material is fixed in tissue; the nature of incorporation in tissues is under investigation. We believe that the contribution of each of these pathways towards the destruction of TCE depends upon variables such as concentration of TCE, size of tree, nature of soil microflora, type of soil, temperature, light intensity, relative humidity, wind velocity, etc.

Controlled Field Trials
A controlled field trial was set up in collaboration with the Occidental Chemical Corporation. Double-walled cells, 3.7 m x 6.1 m X 1.5 m deep, were constructed of 60-gauge high-density polyethylene sheeting. The bottoms had about 0.3 m sand overlaid with 1.1 m Sultan silt loam. In order to ensure a uniform flow of input water, a T-shaped input pipe was used at the bottom of the influent pipe and a 1/40 slope was oriented toward the effluent well. Four cells were each planted with fifteen 30-cm plants of poplar clone H 11-1 1. Two of these cells were dosed with water containing 50 mg/I TCE; the other two cells received only water. The fifth cell, which had no vegetation, also received water containing 50 mg/l TCE. Each of the cells Mininert injection port Figure 2. Diagram of bioreactor. The temperature of the carbon tubes was left at 40°C to prevent condensation of water. received the same volume of liquid; however, variable amounts of liquid were pumped out of the cells on a daily basis to maintain a level of about 20 cm of liquid in the bottom of the cells.
About 9 weeks after injection of TCE into the-cells, breakthrough of TCE and related metabolites, particularly cis-1,2dichloroethylene, occurred in the cells that did not contain trees. The occurrence of this compound is indicative of anaerobic bacterial dehalogenation of TCE, perhaps because of ethanol, which was initially used as a solvent for the TCE in the stock solutions. The use of ethanol was discontinued, and the concentrations of these compounds decreased. In cells with trees, very little TCE or metabolites could be detected in the effluent until after leaf drop ( Figure 3). Throughout the course of this first year of the experiment, a total of 957 mmol TCE was added to each dosed cell. Of this amount a total of 366 mmol was recovered from the unplanted cell (cell 8, 38% recovery), while 21 and 61 mmol of TCE were recovered from cells 3 and 5, which were planted with hybrid poplars (2 and 6%, respectively). The stems, roots, and leaves of the trees exposed to TCE showed the same products of oxidative metabolism which were seen in the greenhouse studies and tissue culture experiments (Tables 1,  2). The 1-year-old trees (approximately 3.5 m tall) were very effective in removing TCE from the input water. These experiments are being continued.

Conclusion
The results of the above experiments show that TCE has multiple fates in poplars. Although the mass balance data would have us believe that most of the TCE taken up by the plant is transpired, the field and greenhouse studies show that very little is transpired under field conditions. The compound also can undergo oxidation to chloromethyl derivatives such as trichloroethanol, dichloroacetic acid, trichloroacetic acid, or complete mineralization to C02, which are oxidative metabolites found in mammalian livers (9). Variable amounts of 14C from TCE are found fixed in insoluble, non-extractable residues. Plant uptake coupled with TCE metabolism serves to remove TCE from groundwater and from the soil environment. The details of these reactions and methods for increasing the mass flow through these pathways are subjects of current investigations. We are also attempting to introduce enzymes into  poplars to increase their tolerance to higher levels of TCE and to increase the fraction of TCE that is mineralized to CO2.
These experiments show that roots of actively metabolizing poplars are able to intercept a plume of TCE-contaminated water and metabolize most of the TCE taken up. The system shows promise where there is sufficient space to plant trees and when the roots can reach the contaminated region. NOTE ADDED IN PROOF. As a result of 2 more years of operation of the experimental field trial, we have found that approximately 99% of the TCE is removed from influent water. There is virtually no detectable transpiration of TCE on the part of the more mature trees, which are now 40 ft tall. An increase in chloride ion, derived from TCE dechlorination, appears in the soil surrounding the roots of the trees.