Screening of environmental samples for an estrogenic pollutant: ddt

SCREENING OF ENVIRONMENTAL
SAMPLES FOR AN ESTROGENIC
POLLUTANT: DDT
L. Graham and M. CampbellCooperative Research Programs, 107 Foster Hall, Lincoln University, Jefferson City, MO65109; Phone (573) 681-5312. ABSTRACT
Soil samples collected from three farm communities in southeast Missouri were analyzed for the presence of dichlorodiphenyltrichloroethane (DDT) and its metabolites, using commercially available DDT in a soil test kit. The soil test kit is based on the use of polyclonal antibodies that bind either DDT or a DDT-enzyme conjugate. The same numbers of antibodies are immobilized to the walls of the test tubes. When DDT is present in samples, it competes with the DDT-enzyme conjugate for a limited number of antibody-binding sites. The presence of DDT is determined by a colorimetric reaction in the test tubes that results in the formation of a blue solution. Based on the binding of the DDT molecules, a low concentration of DDT produces a dark blue solution, and conversely, a high concentration of DDT allows fewer DDT-enzyme conjugate molecules to be bound to the antibodies, resulting in a lighter blue solution. Methanol extracts of 11 soil samples were tested.
Nine of the samples showed a level of 0.2 ppm or greater of p’-DDT. Only two samples had levels below 0.2 ppm.
Key words: dichlorodiphenyltrichloroethane, estrogens, polyclonal antibodies,
biomagnification
INTRODUCTION AND BACKGROUND
bald eagles declined, research revealed that degradation products of DDT accumulating in nated with many synthetic chemicals that are the bodies of the affected birds were the cause.
believed to affect the activity of sex hormones Subsequently, DDT usage in the United States such as estrogen. At least 45 environmental was banned in 1972 (Enger et al., 1998). The contaminants, including DDT (Figure 1), that complex structure of DDT makes it very persis- have chemical structures similar to estrogen, tent, and small quantities can remain in the have been reported to cause changes in the environment for a long time. The chemical can reproductive systems of animals. It is postulated enter the food chain through crops and fish and that estrogenic pollutants can replace natural may be ultimately transferred to humans. Studies estrogen (Figure 2) on cell receptors, which may result in a variety of abnormal responses revealed the presence of DDT metabolites in such as the feminizing of male alligators and body fat, urine, semen, breast milk, and blood (Lu,1985). DDT has a half-life in the body between 2-15 years. Additionally, research data pesticides in the United States prior to the 1970’s. It was widely used on crops and in breast cancer. DDT is listed in the National communities around the world for controlling Toxicology Program’s Fifth Annual Report on disease-carrying insects such as mosquitos.
Carcinogens as a “substance which may When populations of ospreys, cormorants, and reasonably be anticipated to be carcinogenic” Proceedings of the 2001 Conference on Environmental Research
Figure 1. Chemical structure of DDT.
Figure 2. Chemical structure of estrogen.
and the Environmental Protection Agency catego- MATERIALS AND METHODS
rizes DDT and its metabolites as probable human Soil collection
carcinogens (Environmental Health Center, 2000).
homes sites located in New Madrid County, that DDT contamination is a global problem Missouri. A 12-inch stainless steel corer was that requires a global solution. Global attention used to collect each sample, which was taken from the A-horizon of the soil profile. The 12- developing nations continues. Studies that inch core of soil was sliced into three, 4- inch sections. Each section was placed in a labeled plastic bag and stored on ice until transported relevant and significant in light of known back to the laboratory. The weight of each adverse environmental and health impacts.
collected sample was recorded before analysis.
For this study, methanol extracts of soil The samples that were analyzed for DDT were were analyzed according to procedures of the taken from the top portion of the core to a Enviro-Gard TM DDT in Soil Test Kit. This semi-quantitative enzyme immunoassay allowed Chemicals
rapid and reliable screening for DDT at concen- trations as low as 0.2 parts per million. The procedures in the kit are based on EPA method 4042. With this kit, samples can be screened EnviroGardTM DDT in Soil Test Kit was
with a 95% confidence of no false negatives at purchased from Strategic Diagnostics.
Extraction of DDT
tion of procedures recommended in the Strate-gic Diagnostics Inc., EnviroGardTM DDT in Soil Proceedings of the 2001 Conference on Environmental Research
Test Kit. A 1:4 ratio (5 g of soil with 20 ml of conjugate is added to the antibody-coated test tubes, it competes with DDT in the sample for capped 50-mL Erlenmeyer flasks. The flasks antibody-binding sites. The color reaction that containing the soil-solvent mixture were al- occurs when the substrate molecules are added lowed to shake overnight (12 hours) on an to the test tubes allows for visual comparisons.
Eberbach shaker. After overnight shaking, the Samples that contain very low concentrations of soil slurry was centrifuged at 8,000 rpm for 15 DDT bind to very few antibodies in the test minutes at 4°C. The solvent supernatant was tube, so large numbers of the prepared DDT- carefully decanted into sterile, labeled test tubes enzyme conjugate molecules bind with the antibodies and cause the development of a dark The EnviroGardTM DDT in soil test kit
blue solution. Samples with high concentrations (EPA Method 44042)
of DDT bind with more antibodies so fewer Before use, all components were stored at molecules of the DDT-enzyme conjugate bind 4°C. On the day of analysis, the test kit com- to the antibodies and the solution has a lighter ponents were allowed to come to an ambient blue color. After the stop solution is added, temperature of 26°C before proceeding with the test. The test components consist of the follow- spectrophotometer. The calibration curve, plotted ing: 20 12 x 75mm antibody-coated test tubes; from the prepared DDT calibrators, is used to one vial of assay diluent; one vial as a negative extrapolate the concentrations of the samples.
control (methanol); one calibrator vial containing Occurrence of residues
in methanol calibrator vial; one of 10.0 ppm of DDE for each sample are recorded in Table DDT in methanol calibrator vial; one DDT- 1. Data indicates the presence of DDT in all enzyme conjugate vial; one vial of substrate; and soil samples collected from the three southeast a 20-place test tube rack. The solvent extracts Missouri communities. Three out of the eleven were analyzed according to test kit procedures.
samples collected contained concentrations of RESULTS AND DISCUSSION
DDT greater than 10 ppm. Two out of elevensamples contained concentrations of DDT lower DDT determination
than 1ppm. The site with the highest concentra- tion was located in North Lilbourn, Missouri, in Soil Test Kit is that it is based on the use of polyclonal antibodies that bind to either DDT orDDT-enzyme conjugate. All coated test tubes Possible health impact
have the same number of antibody-binding sites and receive a matching number of DDT-enzyme the environment is of concern. DDT can enter conjugate molecules. When the DDT-enzyme the body mainly through consumption of con- Proceedings of the 2001 Conference on Environmental Research
taminated foods, and by inhalation of contami- Ecological effects
nated air. Since the body’s natural hormonal balance is necessary to maintain normal body chemical that can be biomagnified. Synthetic activities, estrogenic contaminants that enter the estrogens are insoluble in water, are soluble in body may affect that balance. Estrogen and fat, and are slowly biodegraded by natural other hormones cause their effects by binding to processes. This means that they become more receptor molecules in tissues of the breast, concentrated in the fatty tissue of an organism at uterus, brain, and testis. The action of DDT is higher trophic levels in the food chain. As noted in the introduction, research has shown that triggering or blocking a response to the body’s populations of predatory birds such as falcons, natural hormones. The mimicking effects of hawks, and eagles have declined because DDT DDT disrupt normal estrogen metabolism in the accumulated in their bodies and altered normal breast development in young girls; infertility in CONCLUSION
Since the 1940’s, four billion pounds of DDT have been used worldwide and 80% ofthat has been used in agriculture (Fransis and Table 1. Concentrations of DDT and/or its
metabolites in methanol extracts of soil samples
Magnus, 1994). Our data indicates that DDT collected from three southeast Missouri farm was broadly used in the three southeast Mis- souri farm communities we studied. Because DDT is very persistent in the environment, p
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samples. We can also reasonably conclude that DDT may be present in water and wildlife in thearea. Future studies will be made to confirm this speculation. The data from this study will be shared with the communities in hopes that it will help them develop appropriate strategies to protect human health and improve the quality of ACKNOWLEDGMENT
Missouri State Matching-Mission Enhancement funds. The authors wish to acknowledge the Proceedings of the 2001 Conference on Environmental Research
cooperation and helpfulness of laboratory Environmental Health Center, 2000. Environ- technicians in the Lincoln University Coopera- mental Writer-DDT (C14H9C15) /DDE (C14H8C14)/DDD tive Research Program. Sincere appreciation is expressed to individuals from the communities that allowed the collection of soil samples from Lu, Frank C, 1985. Basic Toxicology. Hemi- REFERENCES
Enger, E.D., J.R. Kormelink, B.F. Smith, and Magnus, B., 1994. Toxic Substances in the mental Science “The Study of Interrela- tionships.” William C. Brown Publish-ers, Dubuque, Iowa.
Proceedings of the 2001 Conference on Environmental Research

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