Environ. Sci. Technol. 2000, 34, 709-713
Bioavailability to Earthworms of Aged DDT, DDE, DDD, and Dieldrin in Soil DOUGLAS E. MORRISON, BOAKAI K. ROBERTSON,† AND MARTIN ALEXANDER* Institute for Comparative and Environmental Toxicology and Department of Soil, Crop, and Atmospheric Sciences, Cornell University, Ithaca, New York 14853
A study was conducted to determine the bioavailability of several pesticides that have persisted for various periods in soils in the field and the laboratory. Based on the concentrations or the percentages of the compound in soil samples that were found in the earthworm Eisenia foetida, ca. 30, 12, 34, and 20% of DDT, DDE, DDD, and a total of the three compounds were bioavailable in a soil treated in the field with DDT 49 years earlier. Only 28 or 43% of dieldrin aged for 49 years was bioavailable based on concentrations in E. foetida or percentages of the compound assimilated by the worms, respectively. Comparably low percentages of DDT, DDE, and DDD but not dieldrin were assimilated by the worms from samples of soil from a waste-disposal site receiving the insecticide ca. 30 years earlier. Aging for 190 days in Kendaia loam in the laboratory markedly reduced the availability to E. foetida of DDT and DDE but not DDD. The amounts of aged or unaged DDT, DDE, and DDD but not dieldrin that were removed from the soils by solid-phase extraction with Tenax TA beads were generally greater with increasing amounts assimilated by the earthworms. The results show that aging markedly reduces the bioavailability of these compounds. Considerable evidence exists that organic compounds may undergo a time-dependent sequestration in soil that results in a decline in bioavailability without a parallel decline in the concentration of the compounds determined by vigorous extraction with organic solvents. For example, the toxicity of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and dieldrin to three species of insects (1), the inhibition of Drosophila melanogaster by lindane (2), the assimilation of phenanthrene by earthworms (3), the toxicity of atrazine to plants (4), and the biodegradability of phenanthrene by bacteria (5) decline with time in soil with either little or no diminution in the concentrations determined by vigorous extraction or decreases far less than are evident by biological tests. Such data suggest that current procedures for analyzing organic pollutants that have persisted in soil do not accurately predict the availability of those toxicants to living organisms. DDT and dieldrin are highly persistent insecticides. For example, more than 50% of the initially applied DDT was present in some soils after more than 15 years (6), and DDT, * Corresponding author phone: (607)255-1717; fax: (607)255-2644; e-mail:
[email protected]. † Present address: Department of Biological Sciences, Alabama State University, Montgomery, AL 36101. 10.1021/es9909879 CCC: $19.00 Published on Web 01/07/2000
2000 American Chemical Society
DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene] were still in the soil 24 years after addition of DDT (7). Similarly, dieldrin was still detected in soil after 15 years (6). A reexamination of the data from long-term monitoring of the persistence of these pesticides and a number of other organic compounds suggested that this aging led to a decline in their bioavailability to indigenous soil microorganisms (8). Although other studies have shown that the bioavailability of organic compounds to animals is reduced as result of their persistence in soil (9-11), few data are available that show the quantitative reduction in availability. An investigation was therefore conducted to assess the decline in bioavailability as a result of aging of DDT, DDE, DDD, and dieldrin in soils in the laboratory and taken from the field. Because the availability to animals of chemicals in solvents is less, often appreciably so, than the same compounds added to soil even without aging (8), the assimilation of the unaged insecticides was measured using soil treated with the test chemicals shortly before the bioassays were conducted. This was not generally done in previous studies. The existence of experimental plots treated in 1949 with known concentrations of individual insecticies (6) provided a unique opportunity to measure the effect of aging in the field. In addition, data are presented to assess the feasibility of using a solid-phase extractant to determine bioavailability by a chemical assay.
Materials and Methods Chemicals. DDT (75% p,p′-isomer, 18% o,p′-isomer), DDE (99.5% pure), DDD (99.5% pure), and dieldrin (99.5% pure) were obtained from ChemService (West Chester, PA). Hexane (HPLC grade), acetone (ACS grade), diethyl ether (reagent grade), and Florisil (60/100 mesh) were obtained from VWR Scientific Products (Bridgeport, NJ). Tenax TA 20/35 mesh, a porous polymer based on 2,6-diphenyl-p-phenylene oxide, was purchased from Alltech Associates (Deerfield, IL). Soils. Samples of Chester loam (pH 5.5, 6.5% organic matter) and Sassafras silt loam (pH 5.2, 4.4% organic matter) that had been treated with DDT and dieldrin in 1949 or that did not receive the insecticides were obtained at depths of 0-25 cm from experimental plots at the Beltsville Agricultural Research Center (U.S. Department of Agriculture, Beltsville, MD). DDT and dieldrin initially had been mixed in separate plots at rates of 448 and 11.2 kg/ha of soil, respectively (6). Samples of Kendaia loam (pH 6.6, 12.5% organic matter) were obtained from Aurora, NY. Samples of a sandy loam (pH 6.6, 6.0% organic matter) that had been contaminated with DDT and dieldrin approximately 30 years earlier and an adjacent uncontaminated silt loam (pH 6.4, 5.4% organic matter) from a remediation site at the U.S. Navy Surface Weapons Testing Center in Dahlgren, VA, were provided by Remediation Technologies. Earthworms. Mature redworms (Eisenia foetida) from Carolina Biological Supply (Burlington, NC) were maintained in an aerated Styrofoam box containing a commercial worm bedding (Magic Products, Amherst Junction, WI), which was kept moist with Cl2-free deionized water. The worms were fed a mixture of crude protein and carbohydrate (Magic Worm Food) and were active when introduced into the soils. Determination of Pesticide Residues. Worms were frozen at -10 °C and ground with a mortar and pestle. The ground tissue (ca. 2 g) or 10 g of soil was subject to Soxhlet extraction by EPA Method 3540 (12) except that 150 mL of a 1:1 hexane: acetone mixture in a 25-mL round-bottom flask was used for the extraction. The tissues or soil samples were mixed with VOL. 34, NO. 4, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
709
10 g of anhydrous Na2SO4 in paper extraction thimbles and then extracted for approximately 16 h. The extracts were concentrated to 10 mL in a vacuum evaporator and then cleaned on a Florisil column by EPA Method 3620 (12). The fractions containing DDT congeners or dieldrin were concentrated under vacuum to 2 mL and then diluted to 10 mL in hexanes. The chemicals were analyzed with a gas chromatograph (model 5880, Hewlett-Packard) equipped with an electroncapture detector according to EPA Method 8080 (12) except that an HP-608 column (30 m, 0.53-mm i.d., 0.5-µm film thickness; Hewlett-Packard) was used, the N2 (99.99% pure) flow rate was 10 mL/min, and the temperatures of the column, injector, and detector were 225, 275, and 275 °C, respectively. The retention times of DDT, DDE, DDD, and dieldrin were 4.7, 2.8, 4.0, and 3.0 min, respectively. The concentrations found in Chester loam treated in 1949 with DDT were 10.0 mg of DDT, 5.38 mg of DDE, and 4.10 mg of DDD/kg. Chester loam treated in 1949 with dieldrin contained 9.56 mg of the insecticide per kilogram. The soil from the Dahlgren remediation site contained 81.6 mg of DDT, 9.64 mg of DDE, 33.2 mg of DDD, and 9.35 mg of dieldrin per kg. Aging of Chemicals in Laboratory. DDT, DDE, and DDD in hexanes were added to 2 kg of Chester loam that had not previously been treated and contained no pesticides. The final concentrations were 13.6, 5.28, and 3.26 mg/kg of soil, respectively. The soil was thoroughly mixed with a Tefloncoated spatula and stored in an EPA-certified ultraclean 1-L glass jar with a Teflon-lined screwcap. The height of the headspace above the soil was 5 cm. The soil was stored in the dark for 90 days at approximately 22 °C, and then earthworm uptake of the compounds was determined. Prior to aging, 3.0-3.2 kg of Kendaia loam (in a 4-L glass jar with a Teflon-lined cap), which had been sterilized by 2.5 Mrad of γ-irradiation, was amended with DDT, DDD, DDE, or dieldrin under aseptic conditions. The first three compounds dissolved in hexane or dieldrin dissolved in acetone were added to soil, which was then mixed thoroughly with a Teflon-coated spatula. The final concentrations of DDT, DDD, DDE, and dieldrin were 46.6, 11.2, 19.8, and 13.6 mg/ kg, respectively. The soils were placed in a hood for 2-3 days with the caps of the jars slightly loosened to allow the solvent to evaporate. The moisture content was then adjusted to 80% of field capacity. The bottles were stored at approximately 22 °C in the dark for aging. No microbial growth was observed in 7 days on nutrient agar to which 0.1-g samples of soil were added prior to and after the aging period. Bioavailability. Six redworms were placed in 60 g (dry wt) of pesticide-amended soil contained in 250-mL glass jars. The soil had been adjusted to 90% of field capacity with Cl2-free deionized water before the worms were added, and the jars were covered with Saran wrap bearing holes for air entry. The soils were kept under constant room lighting, and after 8 days, the worms were carefully removed, rinsed, and allowed to purge their gut contents for 24 h on moistened filter paper. All worms were active after the 8-day period. The worms in each soil sample were then weighed, the mass varying from 1.5 to 2.5 g of fresh weight per replicate. The worms were sealed in glass Petri dishes, frozen at -10 °C for 25-48 h, and ground with approximately 10 g of anhydrous Na2SO4 with a mortar and pestle. The tissue was transferred to paper thimbles and subject to Soxhlet extraction, and the extracts were cleaned with a Florisil column and analyzed. To compare the bioavailabilities of compounds that had aged in Chester loam for 49 years in the field or 90 days in the laboratory relative to the bioavailabilities of these pesticides when freshly added to soil, DDT, DDE, and DDD were thoroughly mixed into pesticide-free soil to give 13.6, 5.28, and 3.26 mg/kg of soil, respectively. These were the 710
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 4, 2000
concentrations found after 49 years in the soil. Each treatment was replicated four times. In a study of the relative bioavailabilities of compounds aged in Kendaia loam in the laboratory for 0 and 190 days, triplicate soil samples were amended with DDT, DDE, or DDD to give 46.6, 19.8, and 11.2 mg/kg. A determination was also made of the uptake by worms of dieldrin that had aged for 49 years in the field and that had been freshly added. The labels in the experimental plots that gave the original soil types had deteriorated, and sometimes were illegible. Consequently, although the aged dieldrin was in Chester loam, Sassafras silt loam was inadvertently used for the unaged dieldrin. The soil in which the dieldrin was not aged was amended to give 9.56 mg/kg, which was the concentration of aged dieldrin that was found in the field. Four replicate samples were used. Measurements were also made of the bioavailabilities of DDT, DDE, DDD, and dieldrin present for approximately 30 years in soil from the remediation site in Dahlgren and in an adjacent soil to which the compounds were freshly added. DDT, DDE, DDD, and dieldrin were added to the uncontaminated soil to give 45.9, 11.0, 15.6, and 13.6 mg/kg, respectively. The data for earthworm uptake are expressed on the basis of fresh weight of tissue. Solid-Phase Extraction. Tenax TA was used by the method of Cornelissen et al. (13) with slight modification. A single extraction rather than consecutive extractions was performed. Soil (0.2 or 0.5 g) was placed in a 30-mL glass separatory funnel equipped with a Teflon stopcock and stopper. Sterile inorganic salts solution (25 mL) containing 10 mg of Na azide to prevent biodegradation and 0.1 or 0.2 g of Tenax TA beads were then added to the funnels. The salts solution contained 0.8 g each of K2HPO4 and NH4NO3, 0.1 g each of MgSO4‚7H2O and CaCl2‚2H2O, and 0.1 g of FeCl3‚6H2O/L. The beads had been initially conditioned by washing with acetone (1 g in 10 mL solvent) followed by rinsing three times in hexane (1 g in 10 mL solvent) and then drying. The funnel was placed inside a rotary extractor designed in accordance with EPA specifications (12). The sample was shaken end-over-end at 20 rpm for 14-16 h. Tenax TA beads were found to be an adequate sink for organic pollutants in sediments (14). Subsequent extractions did not show a detectable level of the compounds. The single extractions appeared to be complete in
