Environ. Sci. Technol. 2000, 34, 1663-1670
Phytodegradation of p,p′-DDT and the Enantiomers of o,p′-DDT A R T H U R W . G A R R I S O N , * ,† VALENTINE A. NZENGUNG,‡ JIMMY K. AVANTS,† J. JACKSON ELLINGTON,† WILLIAM J. JONES,† DARRELL RENNELS,† AND N. LEE WOLFE† Ecosystems Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Athens, Georgia 30605, and Department of Geology, University of Georgia, Athens, Georgia 30602
The reductive dechlorination of p,p′- and o,p′-DDT at the µg/mL level in the presence of the aquatic plant Elodea (Elodea canadensis) and the terrestrial plant kudzu (Pueraria thunbergiana) is described; studies included analysis of the enantiomers of chiral o,p′-DDT and its chiral degradation product, o,p′-DDD, to determine whether the reaction was enantioselective. The degradation process was followed by GC using a γ-cyclodextrin-based chiral phase. The halflives for degradation of both o,p′- and p,p′-DDT by Elodea and kudzu ranged from 1 to 3 days, apparently depending on growth conditions of the plants. The only products identified were o,p′- and p,p′-DDD; no DDE or DDA were detected. Phytodegradation experiments using Elodea and carbon-14 labeled p,p′-DDT indicated that up to 22% of DDT analogues were covalently bound within the plant. DDT degradation by Elodea was only about 40% slower after γ irradiation at 300 krads, indicating the major process not to be dependent upon live microbes. Dead Elodea was shown to maintain reductive activity at about the same rate as fresh plants. The reactions with Elodea and kudzu were not enantioselective in the formation of o,p′DDD from o,p′-DDT. Reductive dehalogenation of o,p′DDT by a partially purified extract of Elodea, by the porphyrin hematin, and by human hemoglobin was also shown to be nonenantioselective, with reaction rates similar to that for the whole plant. This evidence suggests that the phytodegradation process may be catalyzed by an achiral enzyme cofactor or other achiral biomolecule.
Introduction The uptake and degradation of pesticides and other toxic pollutants by plants may be a major environmental sink as well as a potential phytoremediation process (1, 2). Several plants and plant enzyme systems can degrade organic pollutants; examples include reduction of nitro groups in munitions and explosive chemicals to amines by aquatic plants and axenic plant tissue cultures (3), phytotransformation of organophosphorus pesticides by axenic plant tissue and plant extracts (4), and reductive dehalogenation of chlorinated aliphatics by plants and plant extracts (5, 6). Some * Corresponding author phone: (706)355-8219; fax: (706)355-8202; e-mail:
[email protected]. † U.S. Environmental Protection Agency. ‡ University of Georgia. 10.1021/es990265h CCC: $19.00 Published on Web 03/14/2000
2000 American Chemical Society
suspect enzymes have been extracted from the plants and partially characterized. The enantiomers of a chiral compound generally have different biological properties, including microbial degradation rates. Enantiomeric degradation profiles of persistent pesticides may relate to the time frame of environmental fate and transport processes and allow better source definition (7-10). Also, whether a particular environmental reaction is enantioselective or not could help to better define environmental processes (11). o,p′-DDT [1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane] (Figure 1) comprises up to 25% of technical grade DDT (12) and, as opposed to p,p′-DDT [1,1,1-trichloro2,2-bis-(p-chlorophenyl)ethane], contains an unsymmetrically substituted carbon atom. Thus, o,p′-DDT is chiral. The o,p′-isomer is an endocrine disrupter, being estrogenically active in both avian and mammalian systems (13), and is suspected to have human estrogenic effects because of its strong estrogenic activity in rats (14, 15). In fact, the (-)enantiomer of o,p′-DDT has been shown to be a considerably more active estrogen mimic than the (+)-enantiomer (14). o,p′-DDT is found in the environment wherever p,p′-DDT is found, for example, in Mediterranean surficial sediments (16); in the bioavailable (dissolved) fraction of water from the Missouri River (17); and in 150 fish samples from seven countries in tropical Asia and Oceania (18), where o,p′-DDT averaged 17% of the sum of o,p′- and p,p′-DDT. The main analogues of DDT found in the environment, as shown in Figure 1, are o,p′- and p,p′-DDT; o,p′- DDD [1,1dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane] and p,p′-DDD [1,1-dichloro-2-bis-(p-chlorophenyl)ethane]; and o,p′-DDE [1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethylene] and p,p′-DDE [1,1-dichloro-2,2-bis-(p-chlorophenyl)ethylene]. o,p′- and p,p′-DDT are presumed to degrade in the environment by similar pathways. The principal products of dechlorination of these two isomers in reducing environments are o,p′-DDD and p,p′-DDD, respectively (19, 20). Reductive dechlorination of o,p′- and p,p′DDD can ultimately produce small yields of o,p′-DDA [o-chlorophenyl p-chlorophenyl acetic acid] and p,p′-DDA [bis (p-chlorophenyl) acetic acid] as well as several other minor products. o,p′-DDE and p,p′-DDE are produced by dehydrochlorination of the respective DDT under aerobic conditions; they are the principal DDT analogues found in soils after weathering and also are important sediment pollutants. Microbial degradation of DDT, DDD, and DDE is generally slow, resulting in environmental persistence of these compounds. Under certain conditions, however, DDT may degrade to DDD with a half-life of a few days (as described later in this paper). Regardless, because o,p′-DDT and o,p′-DDD are chiral molecules, their degradation by biological processes and their transport across biological membranes are expected to be enantioselective, that is, one enantiomer will degrade faster than the other or be preferentially transported. Only a few reports on the chirality of o,p′-DDT and -DDD in the environment have been found in the literature. These include observations of the occurrence of unequal concentrations of the enantiomers of o,p′-DDT in soil extracts (21, 22) and in cod liver oil (23) and of o,p′-DDD in fish tissue (24). o,p′-DDT enantiomers have been separated by chiral GC (12, 25) and HPLC (12). This report describes results of our investigation into the reductive dehalogenation of o,p′- and p,p′-DDT in the presence of Elodea (Elodea canadensis), an aquatic plant, and the terrestrial plant kudzu (Pueraria thunbergiana). New VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Structures of the main analogues of DDT found in the environment. chiral GC separation techniques were used to determine the enantioselectivity of o,p′-DDT reduction; the presence or absence of enantioselectivity should help in deduction of the reaction mechanism.
Experimental Section Plant Studies. Twenty grams of Elodea canadensis, collected from Lake Herrick on the University of Georgia campus, was rinsed with distilled water and placed in each of a series of 15 serum bottles containing 100 mL of the boiled and centrifuged aqueous extract of a sample of the lake sediment. The water in the bottles was dosed by syringe with 1 µg/mL each of o,p′- and p,p′-DDT (100 µg of each in 100 µL of methanol) or, for the racemization studies, the pure (+)- or (-)-enantiomer of o,p′-DDT. The bottles were capped with Teflon-lined aluminum crimp caps. A bottle was used for analysis at time zero and at predetermined times thereafter. Control samples included the following: (1) a blank of Elodea and the aqueous soil extract, not dosed with DDT, (2) Elodea and distilled water (instead of soil extract) dosed with the two DDT isomers, (3) soil extract without Elodea dosed with the DDT isomers, and (4) distilled water dosed with the DDT isomers. These samples and controls composed one kinetic experiment; this experiment was repeated seven times over the course of 18 months with Elodea collected under various climatic conditions (e.g., daily average temperatures ranging from 2 to 30 °C). Extraction and Cleanup. The combination of Elodea and water in the bottle was extracted twice by blending for 3 min with 60 mL of a 1:1 mixture of hexane and acetonitrile. The extract was separated from the aqueous-plant phase by centrifugation, passed through 2 g of sodium sulfate, and then evaporated just to dryness at 35 °C with a stream of nitrogen. The residue was taken up in 5 mL of methanol, which was diluted with 20 mL of distilled water and passed through a previously prepared 3 mL C-18 solid-phase 1664
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extraction tube (Supelclean LC-18 SPE from Supelco, Bellefonte, PA). The analytes remaining on the C-18 tube were eluted twice with 5 mL of methanol; the combined eluates were evaporated to 1 mL and analyzed for the DDT congeners by chiral GC. The analytical procedures and degradation reactions for the kudzu experiments were similar to those for Elodea. Twenty grams of Kudzu stems and leaves were washed with distilled water and cut into about 10 cm lengths and 2 cm2 pieces, respectively, before loading into the serum bottles. For the chlordane degradation experiments with Elodea and Kudzu, 1 µg/mL of cis- and trans-chlordane were dosed separately into sets of serum bottles containing the plant material. Otherwise, the degradation reaction conditions and the extraction and cleanup techniques were the same as for DDT. Some of the Elodea-water samples were also analyzed for acidic degradation products, such as DDA, by chiral GC. The extraction and cleanup procedures were similar to those described above for DDT, except that the sample was made to pH 1 with dilute HCl before blending, and the extract was washed by shaking in a separatory funnel with 100 mL of water of pH 1. One milliliter of the final methanol eluant (10 mL total) from the C-18 column was derivatized with diazomethane to form the methyl ester for GC analysis. Standards of p,p′-DDA were also derivatized with diazomethane; we could find no commercial source of o,p′-DDA to serve as a standard. Gas Chromatography. For chiral GC of the DDT compounds, a 12 m × 0.25 mm i.d. Chiraldex G-PT chiral column (chiral phase: permethyl-trifluoroacetoxypropyl γ-cyclodextrin) (Astec, Whippany, NJ) was connected in tandem with a 15 m × 0.25 mm i.d. × 0.50 µm film thickness XTI-5 column (5% diphenyl-95% dimethyl polysiloxane phase) (Restek Corp., Bellefonte, PA). The DDT congeners/isomers and enantiomers were separated using the following conditions: column gas flow: 3 cm3/min; temperature program: 120 °C for 2 min, ramp to 180 °C @ 3 °C/min, hold at 180 °C for 60 min. The injector was operated in the split mode at 210 °C, with split ratios ranging from 1/60 to 1/100; 1 µL of sample was injected. Detection was by electron capture at 350 °C. The methylated p,p′-DDT acid analogue, p,p′-DDA, was analyzed on the same chiral column under the same conditions. cis- and trans-Chlordane enantiomers were separated to the baseline on a Chiraldex dimethyl-β-cyclodextrin column (Astec, catalog no. 77022), 20 m × 0.25 mm i.d., operated at 145 °C for 1 min and then at 0.25 °C/min to 170°, and then held for 20 min. Preparative Separation of o,p′-DDT and -DDD Enantiomers. Chiral Technologies Inc. (Exton, PA) separated the enantiomers of o,p′-DDT and -DDD and provided enough for identification of the enantiomers separated by GC during our experiments as well as for future endocrine disrupter studies. Between 200 and 300 mg of each enantiomer of each compound was successfully separated (enantiomer purity of greater than 99% for each of the four enantiomers) by HPLC using a 2 cm × 25 cm CHIRACEL OJ cellulose ester based column. For the o,p′-DDT separation, the eluent was ethanol at a flow rate of 0.61 mL/min and a temperature of 40 °C, while o,p′-DDD was separated using hexane/ethanol in a ratio of 98/2 at a flow rate of 1.5 mL/min at room temperature. Mass Balance Experiments with Carbon-14 Labeled p,p′DDT. A stock solution of 12C-p,p′-DDT (1 mg/mL) and 14Cp,p′-DDT (0.2 mCi/ml) isomers in methanol was used in all radiolabel studies. Two sets of serum bottles, seven of samples and seven of controls, were prepared; the sample bottles contained Elodea and lake sediment extract as described above under Plant studies. The control bottles contained
only lake sediment extract (100 mL). These samples and controls were dosed with 100 µL of the radioactive stock solution to give a concentration of 1 µg/mL of p,p′-DDT (12Cplus 14C-p,p′-DDT). The specific activity in each bottle was 11 800 DPM/mL. Controls of Elodea and lake sediment extract without DDT were also prepared to account for natural background 14C activity in the plants. Samples and controls were analyzed at predetermined time intervals and at the same time as a set of nonlabeled samples (containing 12Cp,p′-DDT); the nonlabeled samples were analyzed using the analytical procedure described above. The modified method of Kriegman-King and Reinhard (26) was used to quantify the distribution of the labeled parent compound and metabolites in the solution phase. Three 10 mL aliquots of the reaction solution were taken from sacrificed sample bottles with a gastight syringe and treated in three different ways. Aliquot 1 was acidified with 2 mL of 1 N H2SO4, purged with nitrogen for 10-15 min, and then counted using a Beckman LS 6000L liquid scintillation counter. This procedure strips the volatiles and CO2 from the solution, leaving the water soluble nonvolatiles behind. Aliquot 2 was treated with 2 mL of 1 N NaOH, purged for 10-15 min, and then counted. This treatment strips the nonCO2 volatiles. Aliquot 3 was added to 10 mL scintillation cocktail containing 2 mL of 1 N NaOH to determine the total activity. The 14CO2 activity was estimated by subtracting the counts in aliquot 1 from aliquot 2. The nonvolatile fraction in solution was calculated by subtracting the counts in aliquot 2 from aliquot 3. The method was verified and found efficient in stripping 14CO2 under acidic conditions and retaining 14CO under basic conditions (5). 2 The excess solution phase was removed from the bottles with a gastight syringe and the residual Elodea extracted by sonication three times with 25 mL of a binary solvent (1:1 methanol:acetonitrile). This fraction, containing extractable plant components, was then counted. The nonextractable or covalently bound fraction was directly quantified by combusting the oven dried plant residue in a Packard Model 307 sample oxidizer (Packard Instrument Co., Downers Grove, IL). The evolved 14CO2 was trapped and quantified using the liquid scintillation counter. The controls containing the radiolabel but no Elodea were used to monitor losses due to sorption on container walls and artifacts; recoveries of the labeled compound were better than 95%. Irradiation with Co-60. Ten serum bottles containing Elodea plants and the aqueous sediment extract were irradiated at the Center for Applied Isotope Studies at the University of Georgia, Athens, GA. Irradiation was from a cobalt-60 source at 300 krads for 4.9 h. The Elodea did not lose its green coloration following γ irradiation. The irradiated samples were dosed with o,p′- and p,p′-DDT on the day after irradiation as described above (under Plant studies), and the kinetics of degradation were followed as with the nonirradiated samples. Total bacterial colony plate counts of the aqueous phase of the Elodea-water samples were determined for both anaerobic and aerobic bacteria at 24 and 48 h after the beginning and at 24 and 48 h after the end of the kinetic experiment involving the irradiated samples. This work was performed by the Diagnostic Assistance Laboratory of the College of Veterinary Mecicine at the University of Georgia, Athens, GA. The three media used were basic blood agar, cooked meat broth, and thioglycollate broth with indicator. Microbial Transformation. Anaerobic biotransformation experiments were performed in 28 mL Bellco culture tubes initially containing sterilized freshwater sediment (1.0 g dry weight), 1.5 mL RAMM (reduced anaerobic mineral medium) (27), and 10 mg/L of o,p′-DDT. The experimental culture tubes were prepared inside an anaerobic glovebag (N2
atmosphere) and inoculated with 3 mL of a methanogenic microbial consortium cultivated from anaerobic sediment amended with DDT. Inoculated tubes were sealed with Teflon-lined butyl rubber stoppers and incubated at 30 °C. Strict anaerobic conditions were utilized throughout the experiments (28). Sterile controls were prepared by autoclaving tubes after addition of the inoculum followed by aseptic addition of DDT. At selected times, the entire contents of individual tubes were extracted by vigorous shaking, once with acetone and twice with hexane-acetone (1:1). Acetone and hexane extracts were combined, and cleanup of the hexane portion was performed on a Florisil column. DDT and transformation products were analyzed by GC/ECD. Degradation Studies with a Plant Extract, Hematin, and Hemoglobin. Elodea, collected from Lake Herrick as mentioned above, was ground in liquid nitrogen and extracted with a tris buffer at pH 8. The extract was boiled (microwave), partially cleaned up by a process of pH adjustment and centrifugation, desalted, passed through a hydrophobic interaction phenyl sepharose column, and then fractionated by size exclusion chromatography (29). A fraction of approximate molecular weight 1200 tested active for the dehalogenation of several halogenated aliphatic compounds (in the presence of ascorbic acid). A portion of this active fraction was kept sealed and refrigerated at 4 °C for protection against microbial contamination until use in these DDT degradation studies. This active fraction (concentration unknown), dissolved in a matrix of 50 mM tris buffer (pH 8.5) and 10% glycerol, was tested for DDT degradation as follows: 10 mL of the active matrix, 100 µL of 1 mM ascorbic acid, and 10 µL of a solution containing 100 µg/mL each of o,p′- and p,p′-DDT were mixed and divided into 10 samples. A blank contained the matrix and ascorbic acid, but no DDT, and a control contained only ascorbic acid and the DDT compounds. Each of the 10 samples was analyzed for DDT and DDD at predetermined times, and the blank and control were analyzed at the end of the experiment. For analysis, each sample (1 mL) was extracted two times by shaking with 1 mL of hexane for 2 min; the hexane extracts were combined, dried with 2 g of sodium sulfate, and then evaporated to 0.5 mL for GC analysis according to the conditions described above. The extracted water layer of each sample was acidified, extracted, derivatized with diazomethane, and analyzed for DDA by GC. Degradation by hematin and hemoglobin generally followed procedures used by Zoro (19) and Miskus (30). One hundred milligrams of hematin from bovine blood (Sigma, St. Louis, MO) was mixed with 300 mg of sodium dithionite in 500 mL of 0.05 M sodium hydroxide, and 5 g human hemoglobin (Sigma) was mixed with 750 mg of sodium dithionite in 500 mL of boiled, distilled water. Each solution was spiked with 1 mg each of o,p′- and p,p′-DDT and divided into 10 samples. Controls consisted of the following: (1) DDT in water or in 0.05 M sodium hydroxide, (2) DDT with dithionite in water or in 0.05 M sodium hydroxide, (3) DDT with hematin in 0.05 M sodium hydroxide, and (4) DDT with hemoglobin in water. Each of the 10 samples was analyzed for DDT and DDD at predetermined times, and the controls were analyzed at occasional times during the experiment, including at the end. For analysis, 50 mL of acetonitrile was added to each sample (50 mL) followed by extraction with 40 mL and again with 25 mL of hexane. The combined extracts were dried by passage through 2 g of sodium sulfate and then evaporated to 1 mL for GC analysis.
Results GC Separations. As shown in Figure 2, the combination of the γ-cyclodextrin-based chiral column with the XTI-5 column, in that order, provided excellent separation of the VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Degradation of o,p′- and p,p′-DDT in Elodea-Water Matrices date of expt spiking
lag time
Jan 97 ∼3 days (for each compd) a 2 Mar 97 ∼3 days (for each compd) a 3 Aug 97 ∼2 days (for each compd) a av k and t1/2 for expts 1-3b 1
4+ Jan 98
