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Vitellogenin Gene Expression in Fathead Minnows and Pearl Dace from Reference (non-dosed) and Lakes Dosed with EE2 in the Canadian Experimental Lakes Area

Project Purpose:
The purpose was to further develop a vitellogenin gene expression assay as an indicator of estrogen exposure and to investigate the effects of long-term exposure to an endocrine-disrupting pharmaceutical in a whole lake ecosystem.

Project Description(s):
Fisheries and Oceans Canada dosed a lake in the Experimental Lakes Area (ELA) of northwestern Ontario with 17-ethynylestradiol (EE2) for three years beginning in 2001. EE2 was added to the lake at environmentally relevant levels with a target concentration of ~4-6 ng/L. The U.S. EPA collaborated in this study by evaluating vitellogenin gene expression in indigenous fathead minnows (Pimephale promelas) and pearl dace (Margariscus margarita) collected from a reference lake and a continuously dosed lake and in laboratory cultured fathead minnows exposed to water and sediments from the dosed lake and reference lakes. This experiment examined population, organismal, biochemical and cellular-level effects in lake trout, white sucker, fathead minnow, and pearl dace exposed to environmentally-relevant (4-6 ng/L) concentrations of the synthetic estrogen, 17-ethynylestradiol. The USEPA collaborated in this study by evaluating vitellogenin (vtg) gene expression in: 1) indigenous fathead minnows and pearl dace collected from the dosed and reference lakes in 2001 through 2003, before and after dosing; 2) indigenous minnows collected in 2001from the reference lake and deployed for 1, 3, 7 and 13 days in the dosed and reference lakes; and 3) Cincinnati cultured minnows exposed to water collected in 2001 through 2004 from ELA lakes and shipped to Cincinnati. RT-PCR methods were used to measure vtg expression. In addition to water exposures, embryo-larval fish and adult male fathead minnows were exposed to reference and dosed lake sediment elutriates. Indigenous male fathead minnows and pearl dace collected at all time intervals from the dosed lake showed a constant level of elevation in vitellogenin gene expression. Gene expression in the 2001 fathead minnow deployment study was detected within 24 hours after deployment of control fish into the treated lake and stayed elevated for the entire 13-day study. Highly variable gene expression was found in fathead minnow fry exposed to dosed lake sediment elutriates, but no significant gene expression was found in fry exposed to reference lake sediment elutriates. Male adult fathead minnows exposed to elutriates from sediments collected in 2004 in the previously dosed lake showed significant vitellogenin gene expression. Results indicate that RT-PCR analyses of total RNA can be used to provide a rapid and timely estimate of exposure to estrogenic substances.

Project Outcomes:
EPA APM 273 Report - EPA 600/R-04/173, Publication submitted: Collapse of a Fish Population Following Exposure to a Synthetic Estrogen. Kidd, Blanchfield, Mills, Palace, Evans, Lazorchak, Flick

Contact
Jim Lazorchak at lazorchak.jim@epa.gov

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