Doctoral Dissertations

Date of Award

5-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Lee R. Shugart

Committee Members

Walter Farkas, Richard Strange, S. Marshall Adams, Mark S. Greeley, Jr.

Abstract

While the embryos of many North American fish species are commonly used in toxicity testing, a non-indigenous, but equally useful fish species has been largely overlooked by organizations preparing protocols for such use in research and environmental regulation. The Japanese medaka (Oryzias latipes) has been used in basic and applied research in the United States and Japan for many years. All life-history stages have been used and studied, including numerous different types of embryo and embryo-larval assays. However, a medaka embryo assay has yet to be recommended by the American Society for Testing and Material, the United States Environmental Protection Agency or the American Public Health Association for use as a test species in water quality control.

We have developed and refined methods for evaluating toxicity and abnormalities in embryos exposed to contaminants in a variety of media. Individual embryos exposed to benzo[a]pyrene showed both increased mortalities and abnormalities. In the two field applications, individual embryos were exposed to: 1) soil extracts from two hydrocarbon-contaminated sites on U. S. Army Kwajalein Atoll, in the General Republic of the Marshall Islands, or 2) water samples from ten sites on East Fork Poplar Creek, in Oak Ridge, TN, a stream that originates inside the DOE Y-12 Plant.

The two soil extracts were from diesel fuel contaminated soil; treated embryos showed significant responses ranging from increased mortalities to abnormalities and developmental delays. The stream has a history of industrial contamination, including polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and heavy metals, (particularly mercury); other typical industrial discharges (i.e. chlorine, ammonia, and nitrates) are also present at some sites. Treatment groups showed high mortality and dose-dependent mortality in diluted samples compared to control and reference water samples. Medaka fry exposed to the water samples (96 h) did not have significant mortalities. This study demonstrates the flexibility of this medaka embryo assay for assessing the toxicity of complex environmental sites.

The incubation of individual embryos provided several advantages over group incubations. These advantages included (1) lower exposure potential for technicians using individual vials rather than Petri dishes, (2) increased ability to observe and track developmental abnormalities in developing fry during individual incubations, (3) greatly reduced problems with fungal, bacterial and parasitic contamination in individual vials, and (4) smaller samples sizes could be used with individually incubated embryos compared to either type of group incubation. Overall, the incubation of single embryos appears to be more applicable to toxicity testing, with both laboratory-generated solutions and in solutions obtained from soil extracts and stream effluents.

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