Grantee Research Project Results
Final Report: Development and Preliminary Validation of an Amphibian Lifecycle Test Method for Monitoring Endocrine Disruption
EPA Contract Number: 68D03024Title: Development and Preliminary Validation of an Amphibian Lifecycle Test Method for Monitoring Endocrine Disruption
Investigators: Fort, Douglas J.
Small Business: Fort Environmental Laboratories Inc.
EPA Contact: Richards, April
Phase: I
Project Period: April 1, 2003 through September 1, 2003
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2003) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)
Description:
Concerns regarding both the presence of endocrine disruptors in food, water, or other environmental media and the potential risk they pose to humans and wildlife have been growing in recent years. Passage in 1996 of the Food Quality Protection Act and Amendments to the Safe Drinking Water Act reflected these concerns and required the U.S. Environmental Protection Agency (EPA) to develop a screening program, using appropriately validated test systems and other scientifically relevant information, to determine whether certain substances may have an endocrine effect in wildlife and humans.
The proposed work resulted in the development of an assay to test substances that have the capacity to disturb reproductive and developmental processes during the lifecycle of a vertebrate animal by interfering with the endocrine system. The primary goal of the proposed research was to standardize, validate, and commercialize an amphibian lifecycle (XLCA) model using Xenopus as a system for evaluation of endocrine-disrupting chemicals (EDCs) found in the workplace or the environment. Specifically, use of X. tropicalis as a model system was standardized and evaluated to determine the effect of EDCs on various aspects of the amphibian lifecycle by conducting studies with a series of known mammalian EDCs and chemicals with unknown activity. 17ß-Estradiol (E2), androstene dione, and methoxychlor were used to develop and standardize the model. Because none of the currently developed amphibian toxicological test methods are capable of specifically addressing lifecycle effects in amphibians, and the Endocrine Disruptor Screening Program (EDSP) developed and implemented by EPA includes the use of amphibians as a vertebrate model, the successful completion of this amphibian lifecycle model will provide the scientific community with a cost-effective, rapid, reliable method of testing EDCs with amphibians. Results from these studies were used to generate a final study protocol from which a Guidance Document ultimately will be prepared following Phase II interlaboratory validation studies.
Because the life histories of most amphibians incorporate both an aquatic and terrestrial phase, the following exposure protocol was evaluated. This protocol was designed for use together as an assay suite or individually, depending on the objective of the study. The approach involved exposure of embryonic test organisms through advanced development, metamorphosis, and sexual maturity. Specimens surviving through sexual maturity then were bred in a crossover design (exposed female with unexposed male and exposed male with unexposed female), and the progeny were cultured under standard conditions to evaluate transgenerational effects. Sexual maturity in X. tropicalis was achieved in 4-5 months, and the progeny were evaluated for an additional week. An evaluation of growth kinetics, normalcy of development, metamorphosis, sexual maturation (primary and secondary sexual development), reproductive fitness, and developmental fitness in the resulting progeny (F1 generation) was performed.
Summary/Accomplishments (Outputs/Outcomes):
Results from the present study strongly indicated that chronic exposure to E2 slightly delayed development, had a marginal affect on the normalcy of early larval development, dramatically skewed sex ratios toward the female gender, induced gonadal malformations that included some intersexual development at low test concentrations, and induced liver abnormalities. Furthermore, chronic E2 exposure impaired both female and male reproductive fitness. In females, a concentration-dependent decrease in the total number of oocytes present in the ovaries and increase in oocyte necrosis were observed. In males, a concentration-dependent reduction in testis weight and sperm cell count, as well as an increase in sperm cell dysmorphology was noted. Chronic exposure to E2 decreased breeding success in both sexes, decreased fertilization in exposed females, and decreased embryo-larval viability in the F1 progeny.
Results from these studies also suggested that chronic exposure to androstene dione marginally inhibited development similar to E2, had a marginal affect on the normalcy of early larval development, dramatically skewed sex ratios toward the male gender, induced gonadal malformations that included some intersexual development at low test concentrations, and caused liver abnormalities. However, as opposed to chronic exposure of E2, androstene dione reduced reproductive fitness in female specimens, but not male specimens. In females, a concentration-dependent decrease in the total number of oocytes present in the ovaries and increase in oocyte necrosis were observed. Androstene dione appeared to stimulate maturation of the oocytes in vivo. In males, a concentration-dependent increase in testis weight and sperm cell count were noted. Chronic exposure to androstene dione did not markedly alter breeding success in both sexes or decrease embryo-larval viability in the F1 progeny in contrast to the effects of E2.
Chronic exposure to methoxychlor delayed development, including slowing the rate of metamorphosis; had a slight affect on the normalcy of larval development; caused a skewed sex ratio toward the female gender; and induced gonad, liver, and thyroid abnormalities in juvenile specimens. It should be noted, however, that only a few cases of inter-sex gonad development were found in this study. Furthermore, chronic methoxychlor exposure impaired both female and male reproductive fitness. In females, a concentration-dependent decrease in ovary weight, total number of oocytes present in the ovaries, oocyte maturity, and oocyte necrosis were observed. In males, less of a concentration-dependent response was observed. However, in the 100 µg/L methoxychlor exposure treatment, decreased testis weight, sperm cell counts, and normalcy of the sperm cells were noted. Methoxychlor was found to bioaccumulate in the carcasses, but it accumulated more dramatically in the gonads of X. tropicalis.
Conclusions:
Although many pesticides and some industrial chemicals have undergone extensive toxicological testing, this testing may have been inadequate to determine whether they interact with the endocrine system and whether additional testing is needed for EPA to assess and characterize both human health and ecological risk. Notwithstanding recognition that the scientific knowledge related to endocrine disruptors is still evolving, there is appropriate widespread agreement that the development of a screening and testing program is needed. By standardizing and validating the X. tropicalis lifecycle assay system for EDC testing, Fort Environmental Laboratories, Inc., provides the scientific community, chemical and pharmaceutical producing industries, appropriate regulatory agencies, and ultimately the public with a versatile amphibian reproduction, development, and growth assay. Furthermore, this model will enhance understanding of the significance of the effects of EDCs on the reproductive systems of amphibians.
The technical feasibility of the XLCA model to evaluate the effects of EDCs is high. Although more work will be required to validate the model and prepare the guidance document, the XLCA model appears to fit the criteria established for emerging Tier 2 test methods that currently are being sought by the EDSP. The model is relatively straightforward and could be commercialized following method standardization and validation. Results from the Phase I studies have shed light on attributes of the model in that it is capable of rapidly and cost-effectively testing materials with widespread endocrine activity for effects on development, growth, and reproduction, each of which may be affected by EDCs. The ability to rapidly and cost-effectively screen for and evaluate the mechanisms of EDCs is an attractive alternative to the current laborious and expensive testing systems used today. Increasing concerns over the widespread finding of EDCs in the environment have dramatically increased the need for standardized assays, such as the XLCA, because no other assay of this type is currently available.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 3 publications | 3 publications in selected types | All 3 journal articles |
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Type | Citation | ||
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Fort DJ, Guiney PD, Weeks JA, Thomas JH, Rogers RL, Noll AM, Spaulding, CD. Effect of methoxychlor on various life stages of Xenopus laevis. Toxicological Sciences 2004; 81(2):454-466. |
68D03024 (Final) 68D03044 (Final) EPD04054 (Final) |
not available |
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Fort DJ, Rogers RL, Thomas JH. Development of an anuran lifecycle assay using Xenopus tropicalis. Environmental Toxicology and Chemistry. |
68D03024 (Final) |
not available |
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Fort DJ, Rogers RL, Thomas JH, Noll AM, Spaulding CD, Guiney PD, Weeks JA. Evaluation of the developmental and reproductive toxicity of methoxychlor using an anuran (Xenopus tropicalis) chronic exposure model. Toxicological Sciences. |
68D03024 (Final) |
not available |
Supplemental Keywords:
endocrine-disrupting chemicals, EDC, amphibian lifecycle, Endocrine Disruptor Screening Program, EDSP, Xenopus tropicalis, reproduction, development, growth, guidance document, methoxychlor, androstene dione, 17b-estradiol, small business, SBIR., RFA, Health, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, RESEARCH, POLLUTANTS/TOXICS, Environmental Chemistry, Chemicals, Endocrine Disruptors - Environmental Exposure & Risk, Monitoring/Modeling, Monitoring, endocrine disruptors, Environmental Microbiology, Biochemistry, Environmental Monitoring, Endocrine Disruptors - Human Health, Biology, bioindicator, endocrine disruptor screening program, assays, amphibian lifecycle testing, Xenopus lifecycle model, animal model, EDCs, endocrine disrupting chemicals, developmental biology, biomonitoring, amphibian lifecycle test, animal modelsSBIR Phase II:
Validation of an Amphibian Lifecycle Test Method for Monitoring Endocrine Disruption | Final ReportThe 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.