Environ. Sci. Technol. 1998, 32, 975-980
Uptake and Transformation of TNT by Hybrid Poplar Trees PHILLIP L. THOMPSON,† LIZ A. RAMER, AND JERALD L. SCHNOOR* Department of Civil and Environmental Engineering, University of Iowa, Iowa City, Iowa 52242
This paper examines the potential for using hybrid poplar trees to remediate sites contaminated with the high explosive 2,4,6-trinitrotoluene (TNT). Laboratory experiments assessed the uptake of [U-14C]TNT from both hydroponic and soil systems. TNT is strongly bound and transformed by root tissues, and it only translocates slightly to the leaves of poplar cuttings. TNT was more bioavailable in the hydroponic system, but this did not affect the distribution of radiolabel among root, stem, or leaf tissues. The translocation of TNT was found to be similar to that reported for other plant species with up to 75% of the explosive uptaken remaining in root tissues and up to 10% eventually being translocated to the leaves. The majority of TNT was not extractable from plant tissues, and less than 10 percent of the applied label was identifiable by HPLC/ radiochromatograph. TNT was transformed by the tree to 4-amino-2,6-dinitrotoluene (4-ADNT), 2-amino-4,6-dinitrotoluene (2-ADNT), and to a number of unidentified compounds which are more polar than TNT. Phytoremediation efforts must consider the fate and toxicity of these metabolites.
77% of the TNT-related label accumulated in root tissues with less than 20% of that fraction being recovered as TNT. The remainder consisted of 4-ADNT, 2-ADNT, a highly polar unknown, and non-extractable residues. About 15% of the applied radiolabel was recovered in the stem. The accumulation of the applied label ranged from 6 to 13% in leaf tissues, and about 1% of the radiolabel was recovered in the pod and seed of the plant. Overall, greater than 80% of the TNT-related radiolabel was determined to be conjugated or transformed in the tissues of all three plant species. Neither the mineralization to 14CO2 nor the production of volatile organic transformation products was observed. Recent interest in phytoremediation has led to the research of TNT uptake by aquatic plants (5). Hughes et al. (5) used mass balances to assess the fate of [U-14C]TNT in both aquatic plants (Myriophyllum aquiticum and Myriophyllum spicaticum) and plant tissue cultures (Catharanthus roseus). They, too, found TNT to be quickly uptaken by each of these plant species. Their average 14C recoveries ranged from 93 ( 4.8 to 99 ( 9.5% with no significant mineralization to 14CO2 over 7 day periods. They were able to extract an average of 27% of the radiolabel from plant tissues and were only able to identify about 6% of the radiolabel uptaken by the plants as TNT, 4-ADNT, or 2-ADNT. Hence, about 94% of the 14C uptaken was unidentified with about 48% of that being nonextractable. Since TNT was detected in plant tissues on only one occasion, they hypothesized that TNT may be transformed during transport into root tissues. In addition to aquatic plants, terrestrial species such as poplar trees are being considered for remediating sites contaminated with organic chemicals as exemplified by fieldscale demonstrations initiated by the U. S. Environmental Protection Agency (6). The objectives of this research were to (1) study the translocation and fate of TNT in a poplar tree hybrid (Populus sp. deltoidesXnigra, DN34) and (2) identify transformation products that must be considered prior to the remediation of TNT contamination.
Introduction
Experimental Section
Improper handling of the high explosive 2,4,6-trinitrotoluene (TNT) has led to the contamination of soil and groundwater in many countries (1). Uptake of TNT by a number of terrestrial plants has been studied over the past decade (24). These studies were prompted by a concern over the effects on the food chain of plants exposed to TNT contamination. In 1986, Leggett and Palazzo published their study of [U-14C]TNT uptake by yellow nutsedge (Cyperus esculentus.) (2). TNT and its transformation products 4-amino-2,6dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2ADNT) were detected in all plant tissues (leaves, roots, rhizomes, tubers) with 4-ADNT appearing as the most prevalent compound. It was proposed that 4-ADNT and 2-ADNT were formed in the plant, because care was taken to ensure no metabolites were in the hydroponic nutrient solution. Other researchers have studied the fate of [U-14C]TNT in bush beans, wheat, and blando brome (3) using mass balances in both hydroponic and soil systems. Research concluded that all three species of plants had a high capacity to absorb TNT with bush bean showing the highest rate of uptake (3). After 60 days of exposure, bush bean had up to
Analytical grade (>99% purity) TNT was obtained from Chemservice (West Chester, PA). Deionized water (Nanopure) was used, and all other chemicals were of reagent grade or better. TNT solutions were prepared by diluting the appropriate volumes from a 100 mg/L stock solution. Such stock solutions of TNT were the sources for all experiments (i.e. No solvents were used to facilitate TNT solubility.) External standards for TNT were obtained from Accustandard (New Haven, CT) as were standards for the major transformation products of TNT: 1,3,5-trinitrobenzene (TNB), 2,4-dinitrotoluene, 4-amino-2,6-dinitrotoluene (4ADNT), 2-amino-4,6-dinitrotoluene (2-ADNT), and 2,4diamino-6-nitrotoluene (2,4-DANT) (7). The measurement of TNT and its transformation products was performed on hydroponic solutions before and after treatment using a Gilson 308 HPLC isocratic pump, a Spectra 100 UV detector set at 254 nm and an injection loop volume of 100 µL. A Packard Series A-500 radiochromatography detector with Packard Optima-Flo M scintillation cocktail (3 mL/min) was also used. A guard column protected a 25 cm Hypersil CPS LC-CN (Supelco; Bellefonte, PA) separation column, and the mobile phase consisted of a water-methanol-tetrahydrofuran (60.5: 25:14.5, v/v/v) mixture fed at a flow rate of 1.5 mL min-1. TNT and its transformation products were also quantified by HPLC using a 2-proponal:water (20:80, v/v) mobile phase and a Supelco LC-8 separation column (5).
* Corresponding author e-mail:
[email protected]; phone: (319) 335-5649; fax: (319) 335-5585. † Present address: Department of Civil and Environmental Engineering, Seattle University, Seattle, WA 98117. S0013-936X(97)00799-2 CCC: $15.00 Published on Web 02/20/1998
1998 American Chemical Society
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Experimental Design. Hybrid poplar cuttings were collected in the winter from a nursery stand at Amana, Iowa. The cuttings were trimmed to 8 in. in length and grown hydroponically in a greenhouse for 30 days. All hydroponic media were half-strength Hoagland nutrient solutions (8) adjusted to pH 7.0 with 1.0 N NaOH. Prerooted cuttings were transferred to foil-wrapped Erlenmeyer flasks (250 mL) that had been acid washed with 1.0 M HNO3. The initial volume of hydroponic solution in each flask was 150 mL. Evaporation from the flasks was prevented by filling the opening with glass wool. After monitoring the daily transpiration for a period of several days, similarly transpiring cuttings were selected for use. Plants were randomly positioned under fluorescent lighting having an average light intensity of 250 µmol/m2 s in the photosynthetically active region (PAR) as measured by a calibrated sensor (LI-COR). The photoperiod for all experiments was typically 16 h of light and 8 h of darkness unless otherwise noted. Transpiration and biomass were measured by weighing individual flasks and cuttings with a digital balance. All samples were prepared by centrifugation at 12 000 rpm for 10 min followed by 1:1 dilution with methanol. Evaluation of TNT Uptake. The uptake of TNT was studied with a series of experiments that were designed to evaluate the loss of TNT from hydroponic solution over time. Four treatments (performed in triplicate) were live trees, heatinactivated trees, fresh roots only, and a glassware control. Heat-inactivated trees were prepared by drying the trees in a drying oven at 103 °C for 1 h. All reactors were maintained at 25 °C and under continuous light. Erlenmeyer flasks (250 mL) containing 150 mL hydroponic solutions were sampled at intervals ranging from 1 to 8 h. These solutions were spiked with TNT from a 100 mg/L stock solution and 1-6 µCi of uniformly ring labeled [U-14C]TNT (>99% purity; DuPont, Boston, MA) which had a specific activity of 17.9 mCi/mmol. Purity of the radiolabel was verified by radiochromatography. Determination of [U-14C] Translocation. After the completion of an experiment, each plant was separated into leaves, stems, and roots. The fresh weights of each tissue type were immediately measured. Then, each tissue sample was split for extraction and dry weight analyses, respectively. Tissues used for dry weight analysis were dried in an oven at 35 °C for 72 h and analyzed for 14C by using an RJ Harvey Bio-oxidizer (BO). Sample blanks were performed using 0.05 g of mannitol or unspiked plant tissues. Standards were prepared by applying approximately 60 000 dpm of radiolabeled TNT to 0.05 g of mannitol which was then combusted. The combusted TNT standard was compared to a scintillation vial containing 10 mL of cocktail that had also been spiked with the same TNT standard. Bio-oxidizer efficiency was checked in this way at the beginning and at the end of each run. These combustion efficiency values ranged from 90 to 100 percent. Samples no greater than 1.0 g in size were oxidized for 4 min. CO2 from the BO was trapped in 15 mL of RJ Harvey 14Carbon Liquid Scintillation (LS) cocktail. Ten milliliters of the sample cocktail was then transferred for analysis using a Beckman LS 6000IC scintillation counter. Plant Tissue Extraction. A plant tissue extraction procedure utilizing methods from Anderson (9) and Briggs (10) was used. Tissue samples used for extraction were finely chopped with pruning shears and then homogenized in a high-speed blender for 5 min. The weighed sample was then inserted into a 50 mL polypropylene centrifuge tube to which 40 mL of pure acetone was then added. The samples were allowed to equilibrate for 72 h on a heated shaker table adjusted to 100 rpm and 40 °C. The samples were then filtered through 7.0 cm diameter, 0.2 µm mesh glass fiber filters (Fisher Scientific) using a ceramic filtration funnel. The 976
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TABLE 1. Characteristics of Iowa Army Ammunition Plant (IAAP) Soil Used for Experiments soil % organic KP for TNT extractable extractable Mn S pH matter (L/kg) Ca (ppm) Mg (ppm) (ppm) (ppm) 7.7
3.8
5.3
2300
260
4.8
19
K (ppm) P (ppm) Na (ppm) Fe (ppm) B (ppm) Zn (ppm) Cu (ppm) 180
24
19
23.8
0.7
1.2
1.2
filtrate (100 µL) was analyzed by LS while the filter and filter residue were reserved for bio-oxidizer analysis. The filtrate was mixed with 10 mL of deionized water, and the acetone was evaporated using a Bu ¨ chi rotary evaporator set to 50 °C. After distillation, part of the sample was transferred to two 1.5 mL microfuge tubes while the remainder was stored in a glass vial at 4 °C. No radioactivity distilled over with the acetone. After centrifugation at 12 000 rpm for 10 min, the plant extracts were analyzed using HPLC equipped with radiochemical detection. Volatilization Studies. The transformation of TNT to CO2 or volatile organic compounds was studied in a plant growth chamber. The chamber had one inlet connected to an air tank with a flow rate of 1.68 L/min and five outlets with combined flow rates of 1.68 L/min. One end of three of the outlets was connected to the glass sidearm of a Erlenmeyer flask while the other end led to the chamber exterior where it was connected to a 27 gauge needle that bubbled through a test tube containing 10 mL of 3 N NaOH. The other two chamber outlets were exits for air contained within the chamber itself, and they were bubbled through NaOH traps as well. The exhaust from all five NaOH traps was evacuated to the atmosphere with an air pump. Air flow rates were metered with gages from Gilmont (Barrington, IL). Since evaporation from the NaOH traps could occur, the initial and final mass of each trap was recorded. Volatile organics were trapped in activated carbon-containing traps (Orbo tube 32 large, Supelco) placed inline before the NaOH traps. Transpired water was condensed in the chamber by a copper coil that was continuously cooled with 4 °C water from a circulating water bath, maintaining the relative humidity between 50 and 60% at 25 °C. Plant lighting was about 200 µmol/m2 s in the PAR, and the photoperiod was 16 h. Plant Uptake from Soil. Prior to the addition of soil, glass beakers (800 mL) were acid washed, filled with 250 g of 5 mm diameter glass beads then with 250 g of 3 mm diameter glass beads to aid drainage. Clean (uncontaminated) soil from the Iowa Army Ammunition Plant (IAAP; Middletown, Iowa) was used. Soil characteristics can be found in Table 1. Soil was added (425 g) to each beaker after it was sieved to particle sizes less than or equal to 2 mm. Field capacity was evaluated by saturating the bead layers with water, adding the soil and then saturating the soil, and it was estimated at approximately 96 mL of water, a sufficient volume for at least 1 day of transpiration. Beakers were dosed with solutions containing 4.4 mg of TNT and 8.1 µCi of [U-14C]TNT 3 days after the initial planting and tree survival had been established. This equilibration period was used to approximate the initial transpiration. This volume of water (40 mL) was then used to dose the plants with the solution containing the [U-14C]TNT. Each beaker was then covered with two staggered layers of aluminum foil. Daily transpiration for each tree was measured gravimetrically. Evaporative losses were small and were accounted for by measuring the losses from unplanted controls that had glass pipets in place of the live cutting. At the end of each experiment, trees were removed from the beakers with the roots predominantly intact. The majority
TABLE 2. Mass Balance Results for [U-14C]TNT Uptake Experiments from Hydroponic Solutionsa Percent of Applied [U-14C] time (days)
hydroponic medium
plant oxidation
total recovered
2 4 7 20 42
13.5 ( 12.2 16.1 ( 14.3 6.0 ( 7.6 1.5 ( 0.6 3.67 ( 2.04
85.1 ( 19.6 83.8 ( 5.7 82.3 ( 10.1 86.1 ( 1.9 73.4 ( 13.1
98.6 ( 8.0 99.9 ( 9.6 95.0 ( 5.3 87.6 ( 2.3 74.4 ( 12.5
a n ) 3 for each group and values are averages ( 1 Standard Deviation.
FIGURE 1. Loss of [U-14C]TNT from hydroponic solution (n ) 3 for each group, pH 7.0 and 25 °C). Where [TNT] is the concentration of TNT as measured by UV detection. [Radiolabel] is the concentration of 14C as measured by liquid scintillation counting and [4-ADNT] is the concentration of 4-amino-2,6-nitrotoluene as measured by UV detection.
FIGURE 2. Distribution of [U-14C]-label into roots, stems, and leaves of poplar cuttings over time. (n ) 3 for each group, error bars represent (1 standard deviation, pH 7.0 and 25 °C). of the beads were left in the reactor and the soil was separated and dried at 100 °C for 24 h. Plant roots were washed with deionized water to remove as much soil as possible. This wash water was combined with rinse water from cleaning the glass beads. The combined rinse waters were evaporated and the residual soil was mixed with the other soils for biooxidizer analysis.
FIGURE 3. Uptake and partitioning of applied [U-14C]TNT by poplar cuttings from hydroponics, fresh soil, and aged (270 days) soil systems after 20 days. (n ) 6 for fresh soil, n ) 2 for aged soil and n ) 3 for hydroponics. Error bars represent (1 standard deviation.)
FIGURE 4. Relative distribution of [U-14C]-label in plant tissues were similar for poplar cuttings grown in hydroponics, fresh soil and aged (270 days) soil systems after 20 days. (n ) 6 for fresh soil, n ) 2 for aged soil and n ) 3 for hydroponics. Error bars represent (1 standard deviation.) An experiment with aged-TNT soils was also performed by preparing three beakers with soil as detailed above, covering them with foil and allowing them to age for 280 days at 20 °C. After aging, the soils were air-dried at 30 °C, combined and ground to pass a 2 mm sieve. Four 600-mL glass beakers were used for one dead cutting control and VOL. 32, NO. 7, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 6. Radiochromatogram of root extract (1) compared to standards (2) using an LC-8 separation column. The only detectable peaks were four unknown polar compounds, [U-14C]TNT (B), 2-amino4,6-dinitrotoluene (D), and 4-amino-2,6-dinitrotoluene (E). A clear signal for 2,4-diamino-6-nitrotoluene (A) was not discernible.
FIGURE 5. Radiochromatogram of poplar root extract (1) compared to the UV chromatograms of the same root extract (2) and to 10 mg/L standards (3). Identified peaks were 2,4-diamino-6-nitrotoluene (A), [U-14C]TNT (C), 4-amino-2,6-dinitrotoluene (D), and 2-amino-2,4dinitrotoluene (E). Trinitrobenzene (B) was not detected. At least two unidentified peaks were detected before, and possibly two others may have been eluted simultaneously with 2,4-diamino-6nitrotoluene. three live cuttings. Glass beads were again added to each beaker to aid drainage of the soil. In all, 187.5 g of 5 mm diameter beads followed by an equal amount of 3 mm diameter glass beads was added to each beaker. Then, 274 g of aged soil (which was homogenized with a blender) was added to each beaker along with a prerooted cutting.
Results and Discussion Uptake and Translocation of [U-14C]TNT. An experiment evaluating the removal of [U-14C]TNT from hydroponic solution was performed. TNT losses were primarily a result of plant uptake, and microbial transformation was negligible during the 24-h experiment (11). Figure 1 shows that the loss of TNT from solution by live poplars was faster than that of the radiolabel (the pseudo-first-order rate constant for TNT loss from solution was twice that of the radiolabel, 0.02 versus 0.01/h). The mass balance of label could not be accounted for solely by adding the TNT and 4-ADNT concentrations. Thus, the presence of an unknown product(s) was implied. An unknown product was detected using radiochromatography, and the unknown had a retention time of about 2 min. This retention time indicated that the product was more polar than TNT or any of its known transformation products. The concentrations of both radiolabel and TNT remained constant for the nonsterile controls. The translocation of [U-14C]TNT was evaluated by monitoring the accumulation of [U-14C] in root, stem, and leaf tissues for 2-, 4-, 7-, 20- and 42-day periods (Figure 2). The mass balances for these experiments are presented in Table 978
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FIGURE 7. Tissue extracts of poplar cuttings exposed to a spike input of [U-14C]TNT for 7 days were analyzed by HPLC-radiochemical detection (n ) 3 cuttings and error bars represent (1 standard deviation.) The small percentage of identified [14C]TNT-related compounds consist of TNT, 4-ADNT, 2-ADNT, and 2,4-DANT in both root and stem tissues. In addition, there are unknown polar products formed. 2. Closure on mass balances averaged 91%. The apparent decrease in recovery over time was not statistically significant (p < 0.05). After 2 days, about 78% of the radiolabel uptaken by poplars remained in root tissues while about 13 and 9% were translocated to the stem and leaves, respectively. After 7 days, there appeared to be slightly more radiolabel in the leaves and stems. Similarly, after 20 days, the amount of radiolabel increased in the stems and leaves while the levels in the roots decreased. The data through 20 days suggested that the radiolabel may eventually leave the root tissues, ultimately being
FIGURE 8. Possible aerobic biodegradation pathways for TNT adapted from Rieger and Knackmuss (7) and modified. The products shown in boldface were detected in poplar tree tissues. translocated to the leaves. This hypothesis was tested by exposing cuttings in a longer term experiment to a [U-14C]TNT spike for 42 days. The results from the 42-day exposure once again showed that about 74% of the uptaken radiolabel was immobilized in the root tissues of the cuttings. Similar results with respect to the partioning of [U-14C]TNT in plant tissues have been found for other terrestrial plant species (3, 4). Uptake of [U-14C]TNT from Soil. Translocation by poplars growing in [U-14C]TNT spiked IAAP soil was also evaluated after 20 days of uptake. The average percent recovery of radiolabel for this experiment was 83.6 ( 2.6%. Roughly 75% of the applied radiolabel remained in the soil by the end of the 20-day uptake period. These mass balances were comparable to the 20 day hydroponic experiment which had an average recovery of 87.6 ( 2.3% (Table 2). The two experiments differed however in the amount of label taken up by the trees. Figure 3 demonstrates that the label was more bioavailable in the hydroponic experiments as might be expected. In soils, TNT binds to humic material (12) and clays (13) and is more difficult for plants to uptake. It should be noted that the trees in the hydroponic experiment were able to transpire roughly two times the water as the trees in soil even though they were comparable in size. Hence, the lower amount of uptake was related to water availability as well. As with hydroponics, a large majority of the radiolabel remained in root tissues after 20 days, and only 8.4 ( 2.2% of the absorbed label was detected in leaf tissues. In comparing the soil and hydroponic systems (Figure 4), it was apparent that the absorbed label was distributed in the plant in a similar manner regardless of the exposure medium. TNT Uptake from Aged Soil. An experiment with agedTNT soils was also performed to observe the effect of time on the bioavailability of TNT (and its transformation products). It was anticipated that there would be a significant decrease in bioavailability due to the irreversible binding (hysteresis) that TNT is known to undergo in soils (14). The mass balance recovery for the aged soil experiment was 88.0 ( 6.0%. Figure 3 indicates that the amount of radiolabel uptaken by the poplar cuttings was significantly lower from the aged soils than freshly spiked soils. Hence,
aging decreased bioavailability. The distribution of radiolabel was virtually identical between plant tissues for the hydroponic, fresh soil and aged systems, indicating that the aging process did not transform the TNT-related radiolabel to products that were more easily translocated in plant tissues (Figure 4). Transformation of [U-14C]TNT. To understand the transformation of TNT by the hybrid poplar, plant tissues were extracted. About half of the absorbed TNT-related label remained unextractable with either methanol or acetone, which is comparable to the findings of others (5). Radiochromatograms indicated that no transformation of [U-14C]TNT had occurred in the glassware and heatinactivated controls. Analysis of the hydroponic solution containing live cuttings indicated the presence of three radioactive compounds: TNT, 4-ADNT, and an unknown [U-14C] peak that eluted at about 2 min. HPLC analyses of root extracts are presented in Figure 5. The radiochromatogram (top) shows the presence of TNT and its tranformation products 4-ADNT, 2-ADNT, and possibly 2,4-DANT. At least two unidentified products were also present. The double-shouldering in the 2,4-DANT region indicated the presence of two other unknown compounds. Figure 5 (middle) is an example of the simultaneous quantification of cold compound after the elution of the strongly UV-absorbing plant material. Extracts were also analyzed by HPLC-radiochemical detection with separation utilizing an LC-8 column. Figure 6 (top) illustrates the fate of TNT in the roots and compares the retention time of these peaks to those of standards (Figure 6, bottom). 4-ADNT and 2-ADNT had longer retention times with this column and the results once again indicated their presence in root extracts. The previous method had measured the presence of up to four unknown compounds of which two were coeluted with 2,4-DANT. This alternate method was able to separate these compounds more readily, and it detected four of these polar unknowns. Figure 7 summarizes the fate of [U-14C]TNT in hybrid poplar tissues. The low values on the ordinate and the large error bars reveal the large variability of small numbers. TNT was transformed by poplar cuttings to 4-ADNT and 2-ADNT as shown by dual column/dual detector HPLC analysis. Since VOL. 32, NO. 7, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Comparison of Calculated and Experimental Root Concentration Factors (RCF) and Transpiration Stream Concentration Factors (TSCF) for TNT (n ) 18 for TNT, Errors Are Equal to ( 1 Standard Deviation) Briggs, 1982 (calculated) chemical
RCF (mL/g)
TNT
1.70
Burken, 1996 (calculated)
TSCF
RCF (mL/g)
0.78
3.50
4-ADNT and 2-ADNT were produced, it is possible that the hydroxylamino intermediates and their azoxy condensation products were formed even though these compounds were not detected. The results also suggested that 2,4-DANT was produced. In addition to the formation of bound residues, the poplars transformed TNT to a number of unidentified compounds which were more polar than TNT as indicated by their retention times on the HPLC. One of these unknowns may be the only TNT-related product that is translocated to leaf tissues. It is possible that these unknowns represent heterocyclic conjugates of 4-ADNT or 2-ADNT with plant products or imine-like transformation products as suggested by Thorne (15). It is unlikely that the unidentified products are TNB, 2,4 dinitrotoluene, 2,6-dinitrotoluene, or 2-nitrotoluene, since these compounds all have retention times that are much longer than these more polar unknowns. No plant respiration to 14CO2 was detected, and microbially generated 14CO was within the range of radiolabel purity for exposure 2 periods of up to 4 days. Figure 8 summarizes possible aerobic biodegradation pathways for TNT. The results presented here support studies using other plant species with respect to the transformation of TNT to 4-ADNT and 2-ADNT (2-5) and build upon a previous report on the detection of a polar TNT transformation product in root tissues (3) by showing that TNT may be transformed to a number of polar products in root and leaf tissues. It is the first report of these products in poplar tissues that represent conditions at a hazardous waste site where phytoremediation will be used. Further research is needed for identifying the unknown transformation products and assessing their bioavailabililty under field conditions. RCF and TSCF Estimates for TNT. Root concentration factors (RCF) and transpiration stream concentration factors (TSCF) were calculated from results using whole plants, an initial input of chemical, and eqs 1 and 2.
Root uptake ) RCF (mL/g) × root mass (g) × water concentration (mg/mL) (1) Shoot uptake ) TSCF × transpiration (L) × water concentration (mg/L) (2) The experimentally determined RCF and TSCF values presented in Table 3 are compared to the theoretical values calculated using eqs 3 and 4 and eqs 5 and 6 which are the RCF and TSCF relationships, respectively (10, 16).
log(RCF - 0.82) ) 0.77 log Kow - 1.52
(3)
log(RCF - 3.0) ) 0.65 log Kow - 1.57
(4)
{ {
} }
log TSCF ) 0.784 exp -
[(log Kow - 1.78)2] 2.44
(5)
log TSCF ) 0.75 exp -
[(log Kow - 2.5)2] 2.4
(6)
The empirically determined RCF and TSCF relationships assume that the log Kow of the radiolabeled compound does not change (the metabolites have the same log Kow as the 980
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experimental
TSCF
RCF (mL/g)
TSCF
0.65
49.0 ( 30.0
0.46 ( 0.19
parent compound which is not accurate). Thus the RCF for TNT was underestimated compared to the experimentally determined values and was likely due to the transformation of TNT in the root tissues to compounds that were less mobile. These results illustrate the difficulty with using empirical relationships for predicting plant uptake and translocation of organic compounds.
Acknowledgments The authors thank the U.S. EPA National Network for Environmental Management Studies (NNEMS) Fellowship Program, the U.S. Army Corps of Engineers (Omaha District), the U.S. Army Engineers Waterways Experiment Station, and the Center for Health Effects of Environmental Contamination for funding this research. We especially thank Steve McCutcheon, Rodger Allison, Kevin Howe, Jerry Miller, Ron Spanggord, Jim Jordahl, and Craig Just for their generous cooperation.
Literature Cited (1) Spain, J. C., Ed. Biodegradation of Nitroaromatic Compounds; Plenum Press: New York, 1995. (2) Palazzo, J.; Leggett, D. C. J. Environ. Qual. 1986, 15, 49. (3) Cataldo, D. A.; Harvey, S.; Fellows, R. J.; Bean, R. M.; McVeety B. D. An Evaluation of the Environmental Fate and Behavior of Munitions Materiel (TNT, RDX) in Soil and Plant Systems: TNT; U.S. DOE Contract 90-012748, 1989. (4) Gorge, E.; Brandt, S.; Werner, D. Environ. Sci. Pollut. Res. 1994, 1, 229. (5) Hughes, J. B.; Shanks, J.; Vanderford, M.; Lauritzen, J.; Bhadra, R. Environ. Sci.. Technol. 1997, 31, 266. (6) Kling, J. Environ. Sci. and Technol. 1997, 31, 129A. (7) Rieger, P. G.; Knackmuss, H. J. Biodegradation of 2,4,6trinitrotoluene and related nitroaromatic compounds. In Biodegradation of Nitroaromatic Compounds.; Spain, J. C., Ed.; Plenum Press: New York, 1995. (8) Epstein, E. Mineral nutrition of plants: Principles and perspectives; John Wiley & Sons: New York, 1972. (9) Anderson, J. M. J. Chromatogr. 1985, 330, 347. (10) Briggs, G. G.; Bromilow, R. H.; Evans, A. A. Pestic. Sci. 1982, 13, 495. (11) Thompson, P. L.; Ramer, L. A.; Guffey, A. P.; Schnoor, J. L. Environ. Toxicol. Chem. 1998, in press. (12) Li, A. Z.; K. A. Marx; Walker, J.; Kaplan, D. L. Environ. Sci. Technol. 1997, 31, 584. (13) Haderlein, S. B.; Weissmahr, K. W.; Schwarzenbach, R. P. Environ. Sci. Technol 1996, 30, 612. (14) Comfort, S. D.; Shea, P. J.; Hundal, L. S.; Li, Z.; Woodbury, B. L.; Martin, J. L.; Powers, W. L. J. Environ. Qual. 1995, 24, 1174. (15) Thorn, K. A. Covalent binding of the reductive degradation products of TNT to humic substances examined by N-15 NMR; 213th National Meeting of the American Chemical Society; San Francisco, California, 1997. (16) Burken, J. G. Uptake and Fate of Organic Contaminants by Hybrid Poplar Trees; Ph.D. Thesis, University of Iowa, Iowa City, Iowa, 1996.
Received for review September 8, 1997. Revised manuscript received December 4, 1997. Accepted December 15, 1997. ES970799N
