Environ. Sci. Technol. 2001, 35, 286-291
Factors Controlling the Rate of DDE Dechlorination to DDMU in Palos Verdes Margin Sediments under Anaerobic Conditions J O H N F . Q U E N S E N , I I I , * ,†,‡ JAMES M. TIEDJE,§ MAHENDRA K. JAIN,† AND S H E R R Y A . M U E L L E R †,| MBI International, P.O. Box 27609, 3900 Collins Road, Lansing, Michigan 48909-0609, and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824
Marine sediments off the coast of the Palos Verdes Peninsula in California have been designated a Superfund site primarily because of the presence of DDE [1,1-dichloro2,2-bis(p-chlorophenyl)ethene]. For decades, it was believed that DDE was not microbially transformed, but anaerobic bacteria in the Palos Verdes sediments reductively dechlorinate DDE to DDMU [1-chloro-2,2-bis(p-chlorophenyl)ethene], which is also found in the sediments. The effects of electron donor to sulfate ratio, available carbon, sampling sites, sediment depth, and temperature on the rate and extent of DDE dechlorination in anaerobic Palos Verdes sediment microcosms were investigated. Dechlorination rates varied, depending on the site and depth from which the sediments were collected, but DDE dechlorination occurred with sediments from all locations studied. Sulfate and low temperatures slowed dechlorination, but in the presence of sulfate and at in situ temperature, the dechlorination rates observed in the microcosms agree well with the observed rate of DDE disappearance from the Palos Verdes margin sediments.
Introduction Following the discovery of its insecticidal properties in the late 1930s, DDT [1,1,1-trichoro-2,2-bis(p-chlorophenyl)ethane] was widely used to control agricultural pests and vectors of malaria and other insect-borne diseases. As manufactured, DDT contained the byproducts DDD [1,1dichloro-2,2-bis(p-chlorophenyl)ethane] and DDE [1,1dichloro-2,2-bis(p-chlorophenyl)ethene], both of which can also be formed by chemical and biological degradation of DDT. Collectively these three compounds are sometimes referred to as DDx or total DDTs. Beginning in the 1960s, the environmental persistence and effects of these compounds became highly scrutinized, resulting in the banning of DDT in the United States and many other industrialized countries in the early 1970s. * Corresponding author telephone: (517)355-9284; fax: (517)3532917; e-mail:
[email protected]. † MBI International. ‡ Present address: Department of Crop & Soil Sciences, Michigan State University, East Lansing, MI 48824. § Michigan State University. | Present address: Ford Motor Company, Chemistry Department, SRL Building, MD 3083, P.O. Box 2053, Dearborn, MI 48121. 286
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Despite the ban, DDx compounds are still present in the environment, occurring at 3422 of 22 000 sites that the U.S. Environmental Protection Agency identified in the agency’s National Sediment Quality Survey. One such site is the continental shelf off the Palos Verdes Peninsula in southern California. DDx containing sediments were discharged through an offshore outfall operated by the Los Angeles County Sanitation District, primarily between 1950 and 1971, and now cover a large area of the ocean floor to a depth of 70 cm (1, 2). Research on the biodegradation of DDx declined drastically after it was banned in the 1970s. Two notable discoveries, however, have been made in recent years. Under aerobic conditions, the ring cleavage of DDT by Alcaligenes eutrophus A5 (3, 4) and of DDE by biphenyl grown cells of Pseudomonas acidovorans M3GY (5) occur via pathways similar to those for biphenyl degradation. Under anaerobic conditions, DDE was reductively dechlorinated to DDMU [1-chloro-2,2-bis(p-chlorophenyl)ethene] in marine sediment slurries (6). Prior to these reports, there was no convincing evidence for the ring cleavage of DDT or for any microbial transformations of DDE. Thus, these findings have important implications regarding the environmental fate of DDx for the bioremediation of DDx-contaminated soils and sediments by both intrinsic and proactive means and should be considered in decisions regarding DDx-contaminated sites. Toward those ends, it is important to understand the factors controlling the rates and extents of these transformations. While our previous work (6) clearly demonstrated the microbially mediated reductive dechlorination of DDE to DDMU in Palos Verdes sediment microcosms, it did not address environmental factors that control the rates of DDE dechlorination. In this paper, we describe the effects of electron donor to sulfate ratio, available carbon, sampling site, sediment depth, and temperature on the rate and extent of DDE dechlorination in Palos Verdes sediment microcosms.
Materials and Methods Sediment Collection and Characteristics. Marine sediments from three sites along the 60 m depth contour of the Palos Verdes (California) margin were collected as previously described (6). Site nomenclature and locations (3C: 33°43.85′ N, 118°24.13′ W; 6C: 22°42.51′ N, 118°21.20′ W; 8C: 33°42.00′ N, 118°20.05′ W) were as in the Los Angeles County Sanitation District Survey of 1989 (2, 7). Sediment characteristics (sand, silt, clay, total carbon, and pH) were similar to those of sediments collected previously from sites 3C, 8C, and 5C (33°42.913′ N, 118°21.893′ W) (6). Sulfate concentrations in the pore water were 2400-2600 µg/mL near the sediment surface and were gradually depleted with depth; minimum concentrations of 1700-1800 µg/mL occurred 45-50 cm below the sediment surface. These data indicate that sulfate reduction is occurring in the sediment column and that conditions are sulfidogenic at all depths sampled. The mean annual temperature of the sediments in situ is 12 °C. Microcosms. Anaerobic microcosms consisting of 7 mL of anaerobic sediment slurries in tightly sealed 20-mL glass vials were prepared within 3 months of sediment collection by previously described methods (6). The slurries consisted of equal volumes of wet sediment and autoclaved anaerobic artificial seawater medium (6) and averaged 3.1 g of sediment (dry weight) per vial. When the experimental design required a sulfate concentration less than the 27 mM found in seawater, the ionic strength was maintained by increasing the amount of NaCl in the medium. When the experimental design required the addition of carbon substrates, an equal molar 10.1021/es0012873 CCC: $20.00
2001 American Chemical Society Published on Web 12/09/2000
mixture of pyruvate, lactate, and glycerol was added to the medium prior to autoclaving. Uniformly ring-labeled [14C]DDE (13.0 mCi/mmol, >98% pure by HPLC, Sigma Chemical Co., St. Louis, MO) and nonlabeled DDE (Supelco, Inc., Bellefonte, PA) were dissolved in acetone to give expected concentrations of 29 µg/µL and 147 321 dpm/µL. Sevenmicroliter portions of the mixture were added to each microcosm, giving 638 nmol of DDE/microcosm or 206 nmol of DDE/g of sediment dry weight. Autoclaved slurries served as controls for nonbiological effects. All microcosms were incubated in the dark at the appropriate temperature. At designated sampling times, the entire contents of four replicate vials were extracted and analyzed by thin-layer chromatography combined with liquid scintillation counting (6). DDMU accumulation was standardized on the dry weight of sediment in each microcosm. First-order rate constants (b) for the dechlorination of DDE to DDMU were calculated by fitting data for the (initial DDE concentration - DDMU formed) in nmol/g of sediment dry weight versus time in weeks to an exponential decay regression model beginning with the sampling time immediately preceding the first detection of DDMU. Dechlorination rates after the lag period are more predictive of dechlorination rates in the field because lag times are characteristic of microcosms and derive from the disturbances in setting up microcosms. Half-lives were calculated from the first-order rate coefficients (λ1/2 ) ln 2/b). Analysis of Methane and Sulfate. Prior to extraction, the methane in the headspace of each microcosm was determined by gas chromatography using a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector and 8 ft × 1/8 in. diameter stainless steel Supelco column (SP-1000 liquid phase on 60/80 Carbopack B). Nitrogen was the carrier gas, and temperatures were as follows: inlet, 200 °C; detector, 220 °C; and oven, 40 °C. Data were collected with a Hewlett-Packard 3393A integrator. For quantitation, a four-point external standard calibration curve was generated from 1%, 5%, 10%, and 25% methane in air; calculations were performed in an Excel spreadsheet. Sulfate concentrations in the pore water of collected sediments and in microcosms were determined by capillary electrophoresis as previously described (6). Data collection and quantitation were performed using Turbochrome software and a five-point external standard calibration curve generated from standards containing from 2.91 to 116 µg/ mL (ppm) SO42- in seawater medium. Experimental Designs. Effects of Electron Donor to Sulfate Ratio. Anaerobic sediment slurries were prepared from site 3C sediments and were incubated with [14C]DDE in a complete factorial design with three sulfate levels, two carbon levels, and four replicates. Autoclaved slurries served as biological controls. Live treatments were sampled at 4-week intervals for 32 weeks while autoclaved control treatments were sampled at 8-week intervals for 32 weeks. The three levels of sulfate were designated high, medium, and low. The low sulfate treatments were prepared using sulfate-free seawater medium, and the resulting slurries initially contained approximately 900 µg/mL of sulfate from the sediment pore water. For the medium and high sulfate treatments, sulfate was added to achieve initial sulfate concentrations of 1400 and 2064 µg/mL, respectively, as determined by capillary electrophoresis. At the time the microcosms were prepared, the pore water sulfate content of the sediment, homogenized over depth, was approximately 2200 µg/mL. (Full strength seawater is 27 mM (8) or approximately 2600 µg/mL). The two levels of carbon consisted of no additional carbon or addition of a mixture of pyruvate, lactate, and glycerol (5.55 mM each in the anaerobic medium used to prepare the slurries) estimated to provide sulfate-reducing microorgan-
isms with enough electron donors to reduce all of the sulfate at the medium sulfate level. Effects of Added Carbon. To determine if the differences between sediments in their abilities to support DDE dechlorination persisted when readily utilizable carbon was added, separate microcosm experiments were set up with sediments, homogenized over the depths of the cores, from sites 3C, 6C, and 8C using sulfate-free seawater medium. Treatments consisted of no carbon addition, a low carbon addition, a high carbon addition, and autoclaved controls. The carbon substrates were the same as described above, and they were added to the anaerobic media to concentrations of 2.1 and 8.4 mM each in the low and high carbon treatments, respectively. The low carbon addition was enough to deplete about half of the sulfate initially present, and the high carbon addition was enough to deplete twice the initial sulfate present. The microcosms were incubated at room temperature. DDE Dechlorination with Sediment Depth. To determine if DDE dechlorinating microorganisms exist at depth in the sediment, a single 48-cm core from site 6C was cut into twelve 4-cm slices, and separate [14C]DDE-amended microcosms were set up in quadruplicate with sediments from each slice and the anaerobic sulfate-free seawater medium. The microcosms were incubated at room temperature for 24 weeks prior to extraction and analysis. Effects of Temperature on DDE Metabolism. Microcosms were prepared from site 3C sediment slurried with either sulfate-free or sulfate-containing seawater medium. Separate sets of microcosms were incubated at 10 and 15 °C in environmental control chambers and in the laboratory (approximately 22 °C). In each case, chart recorders were used to continuously monitor the actual temperatures, and these results verified that the indicated temperatures were maintained. At each temperature, autoclaved microcosms served as controls.
Results Effects of Sulfate on DDE Dechlorination. Addition of carbon affected the initial rate of sulfate utilization and the time to sulfate depletion (Figures 1 and 2). Without carbon addition, the low and medium levels of sulfate were gradually depleted over the first 12 weeks. Methane was first detectable in the low sulfate treatment at 12 and 16 weeks in the medium sulfate treatment. In the high sulfate treatment, most of the sulfate was gone by 16 weeks, but low levels persisted throughout the experiment in most replicates, and only traces of methane were ever detected. With the addition of carbon, the low concentration of sulfate was depleted within the first 4 weeks, the medium concentration within the first 8 weeks, and the high concentration by 16 weeks. Methane was first detected at 12 weeks with the low concentration of sulfate and at 20 weeks with the medium and high concentrations of sulfate; relatively large amounts of methane were produced in all treatments by 24 weeks. Without carbon addition, the lag time to DDE dechlorination was 16 weeks regardless of the amount of sulfate initially present (Figure 1). For the low and medium sulfate treatments, all DDE dechlorination took place after sulfate depletion (at 12 weeks), and the dechlorination rates were not significantly different from each other (Table 1). Sulfate was always present in the high sulfate treatment, and the dechlorination rate was slower such that only half as much DDMU was formed by 32 weeks. Addition of carbon clearly shortened the lag time until DDE dechlorination from 16 to 8 weeks in the low sulfate treatments and from 16 to 12 weeks in the medium and high sulfate treatments (Figures 1 and 2). For the low sulfate treatments, dechlorination occurred only after sulfate depletion (i.e., only under methanogenic conditions), and the VOL. 35, NO. 2, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. First-Order Rate Coefficients (b) with Standard Errors (SE) for DDE Dechlorination to DDMUa and Corresponding Half-Lives for Different Initial Carbon Substrate and Sulfate Concentrations C amendmentb
S amendmentb
interval (weeks)
b
SE
95% CI
P
half-life (weeks)
+ + + +
L M H L M H H
12-32 12-32 12-32 4-32 8-32 8-16 16-32
0.0214 0.0120 0.0070 0.0189 0.0311 0.0098 0.0378
0.0026 0.0040 0.0022 0.0007 0.0021 0.0009 0.0049
0.0160-0.0268 0.0037-0.0203 0.0024-0.0116 0.0175-0.2030 0.0268-0.0354 0.0078-0.0118 0.0275-0.0481
