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Extramural Research

1999 Progress Report: Endocrine Disruptors and Host Resistance in Lake Apopka Alligators

EPA Grant Number: R826127
Title: Endocrine Disruptors and Host Resistance in Lake Apopka Alligators
Investigators: Schoeb, Trenton R. , Brown, Mary B. , Gross, Timothy S. , Klein, Paul A.
Institution: University of Florida , U.S. Geological Survey
EPA Project Officer: Reese, David H.
Project Period: January 3, 1998 through January 2, 2001
Project Period Covered by this Report: January 3, 1998 through January 2, 1999
Project Amount: $598,253
RFA: Endocrine Disruptors (1997)
Research Category: Endocrine Disruptors

Description:

Objective:

In the early 1980s, numbers of juvenile alligators in Lake Apopka, Florida, declined by 90 percent in association with decreased egg viability, increased neonatal mortality, and organochlorine pesticide contamination. We observed hypoplastic lymphoid organs and bone marrow and weakened antibody responses in young Lake Apopka alligators, suggesting that weakened host defenses could be a contributing factor. The objective of this project is to study effects on neonatal alligators of endocrine disruptors on measures of specific and nonspecific immunity and lymphoid organ and bone marrow development. The approach is to study alligators hatched from eggs from Lake Apopka, from reference lake eggs treated with endocrine disrupting contaminants found in tissues and eggs of Lake Apopka alligators, and from untreated reference lake eggs (controls) for ability to generate T dependent humoral immune responses and for hematologic parameters, peripheral blood antibacterial activity, lymphoid organ and bone marrow morphology, and susceptibility to a pathogenic Mycoplasma sp. of alligators.

Progress Summary:

For studies begun in Year 1 and completed during this reporting period, egg treatments were DDE, dieldrin, endosulfan, methoxychlor, toxaphene, chlordane, low and high dose combinations of these, and dimethylsulfoxide (DMSO) vehicle control, applied topically. Target egg concentrations of each substance (based on available analytical results and on average egg weight) were 2 ppm for individual treatments, 0.33 ppm of each substance for the low dose combination, and 2 ppm of each substance for the high dose combination. Analysis of liver samples verified absorption and presence in tissues of DDE, chlordane, dieldrin, and toxaphene, but not endosulfan or methoxychlor, and concentrations were somewhat variable. Thus, egg treatments for Year 2 were done by injection. These studies were focused on an environmentally relevant range of doses of DDE, by far the predominant contaminant found in tissues and eggs of Lake Apopka alligators, and one mixture based on recent data. Treatments were up to 25 ppm p,p'-DDE; a mixture of DDE, chlordane, dieldrin, methoxychlor, and toxaphene; or DMSO vehicle control. Eggs also were collected from Lake Orange (reference site), Lake Griffin, Emeralda Marsh (adjacent to Lake Griffin), and both north and south shores of Lake Apopka, sites having a range of contaminant levels.

T-dependent antibody responses were assessed by immunization with keyhole limpet hemocyanin (KLH) esterified with dinitrophenol (DNP) (DNP-KLH) and measurement of DNP hapten-specific responses by enzyme-linked immunosorbent assay (ELISA). In Year 1 experiments, no significant differences were found among pesticide treatment or lake study sites. Assessment of antibody responses for Year 2 studies will be completed in April of this year. In results to date, there also were no statistically significant differences among the pesticide treatments in white or red blood cell counts, packed cell volumes (hematocrits), plasma protein, or hemoglobin. We also have found no statistically significant effects of pesticide treatments on peripheral blood antibacterial activity against Staphylococcus aureus.

In Year 1 studies, both experimentally and naturally exposed animals had changes in spleen, thymus, and bone marrow similar to those previously observed for Lake Apopka hatchlings. However, there was no detectable relationship to pesticide treatment or source lake. Recently published findings indicated that similar changes can be caused by chronic corticosteroid treatment and thus, could be related to stress. In Year 2, major efforts were made to optimize housing and care of hatchlings. Histologic findings in each group were similar and were considered to be within normal limits.

In Year 1, we determined the dose and route of inoculation of alligator Mycoplasma sp. experimental infections. In Year 2, alligators from control and DDE treatment groups were inoculated intravenously. Blood, brain and pericardium were cultured, and all limb joints, brain, and any tissues with gross lesions were prepared for histologic examination. There were no statistically significant differences in quantitative culture results among experimental treatments or lake sites. Histopathological examination of tissues is still in progress.

Future Activities:

During the next reporting period, we will complete the studies currently in progress. We also will continue efforts to develop monoclonal antibodies for use in immunoglobulin class specific ELISAs and immunohistochemical enumeration of B and T cells in lymphoid organs. We will work to further optimize culture techniques for assays of proliferative responses of alligator lymphocytes to mitogens and mixed lymphocyte culture. Because we now expect that we may find that in ovo exposure to the test substances does not have demonstrable effects, and because we have observed clutches in which the young developed opportunistic fungal and bacterial infections in ovo, studies will be conducted in the final months of the project to test the hypothesis that in ovo antimicrobial defenses are altered in eggs from contaminated study sites.

Journal Articles:

No journal articles submitted with this report: View all 5 publications for this project

Supplemental Keywords:

health effects, ecological effects, organics, pathogens, bacteria, aquatic, biology, pathology, Florida, FL, agriculture., RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Toxics, Geographic Area, Ecosystem Protection/Environmental Exposure & Risk, exploratory research environmental biology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Health Risk Assessment, pesticides, State, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Risk Assessments, Southeast, Biochemistry, Physical Processes, Biology, Endocrine Disruptors - Human Health, adverse outcomes, ecological effects, aquatic ecosystem, ecological exposure, wildlife, exposure, reptile, host resistance, 2, 3, 7, 8-Tetrachloro-dibenzo-p-dioxin (TCDD), chemical mixtures, Chlordane, animal models, developmental processes, immune system response, pathogen, fetal development, Lake Apopka, FL, biological effects, DDE, Florida, Lake Apopka Alligators

Relevant Websites:

http://www.flmnh.ufl.edu/natsci/herpetology/lakes/lakes.htm

Progress and Final Reports:
Original Abstract
Final Report

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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