Exposure of three generations of the estuarine sheepshead minnow (Cyprinodon variegatus) to the androgen, 17beta-trenbolone: effects on survival, development, and reproduction.

Estimating long-term effects of endocrine-disrupting chemicals on a species is important to assessing the overall risk to the populations. The present study reports the results of a 42-week exposure of estuarine sheepshead minnows (Cyprinodon variegatus) to the androgen, 17beta-trenbolone (Tb) conducted to determine if partial-(F0) or single-generation (F1) fish exposures identify multigenerational (F0-F3) effects of androgens on fish. Adult F0 fish were exposed to 0.007, 0.027, 0.13, 0.87,and 4.1 microg Tb/L, the F1 generation to < or =0.87 microg Tb/L, the F2 fish to < or =0.13 microg Tb/L, and the F3 fish to < or =0.027 microg Tb/L. The highest concentrations with reproducing populations at the end of the F0, F1, and F2 generations were 4.1, 0.87, and 0.027 microg Tb/L, respectively. Reproduction in the F0, F1, and F2 generations was significantly reduced at 0.87, 0.027, and 0.027 microg Tb/L, respectively. Fish were significantly masculinized in the F1 generation exposed to 0.13 microg Tb/L or greater. Female plasma vitellogenin was significantly reduced in F0 fish exposed to > or =0.87 microg Tb/L. Gonadosomatic indices of the F0 and F1 generations were significantly increased at 0.87 and 0.13 microg Tb/L in the F0 and F1 generation, respectively, and were accompanied by ovarian histological changes. Reproduction was the most consistently sensitive measure of androgen effects and, after a life-cycle exposure, the daily reproductive rate predicted concentrations affecting successive generations. The present study provides evidence that a multiple generation exposure of fish to some endocrine-disrupting chemicals can result in developmental and reproductive changes that have a much greater impact on the success of a species than was indicated from shorter term exposures.

Multigenerational exposure of the estuarine sheepshead minnow (Cyprinodon variegatus) to 17 ß-estradiol. I. organism-level effects over three generations.

A 280-d study examined the effects of 17ß-estradiol (E2) on reproduction and development of the sheepshead minnow (Cyprinodon variegatus) exposed from the parental (F0) through three subsequent (F1, F2, and F3) generations and evaluated the need for multigenerational assessments of the risks of endocrine-disrupting chemicals. This first three-generation study exposed adult F0 and F1 fish to measured concentrations of 0.01, 0.04, 0.08, 0.2, and 0.3 µg E2/L; the F2 and F3 generations were exposed to 0.2 µg E2/L or less. The cumulative 21-d production of normal embryos was significantly reduced in the F0 generation at 0.3 µg E2/L and in the F1 and F2 generations at 0.08 µg E2/L or more. The daily reproductive rate was significantly reduced in all three generations at 0.08 µg E2/L or more during spawning days 8 to 14 and 15 to 21. The proportion of infertile eggs from F1 fish was significantly increased above that of the solvent controls at 0.04 and 0.2 µg E2/L and from F2 fish at 0.04 µg E2/L or more. Changes in liver, kidney, and gonadal tissues were seen in the F0 and F1 generations exposed to 0.2 µg E2/L or more. The female gonadosomatic index was significantly decreased at 0.3 µg E2/L in the F0 and F1 generations. Estradiol affected the hepatosomatic index only in female F1 fish, but not in a dose-dependent manner. All F1 fish in 0.3 µg E2/L appeared to be phenotypically female. Our results indicate that life-cycle exposure to E2 significantly decreased embryo production by F1 and F2 fish at concentrations lower than those affecting the F0 generation, and they emphasize the importance of evaluating the impact of an estrogenic chemical on reproduction through a minimum of two (F0 and F1) generations.

Multigenerational exposure of the estuarine sheepshead minnow (Cyprinodon variegatus) to 17ß-estradiol. II. Population-level effects through two life cycles.

The evaluation of multigeneration, population-level impacts is particularly important in the risk assessment of endocrine-disrupting compounds, because adverse effects may not be evident during the first generation of exposure. Population models were developed for the sheepshead minnow (Cyprinodon variegatus) exposed to 17ß-estradiol (E2) for two complete generations (F1 and F2) to determine population-level effects of multigenerational exposure to a model estrogen. Stage-structured matrix models were used to determine interactions between treatment and the number of generations exposed. Reproduction was significantly reduced in both the 0.08 and 0.2 µg E2/L treatments in both generations, and embryo and larval stages experienced reduced survival at 0.2 µg/L in the second generation only. However, increased female to male sex ratio in these treatments compensated for the loss in reproductive output, and significant population-level effects only occurred in the 0.2 µg E2/L treatment of the F2 population. The F2 population in the 0.2 µg E2/L treatment also had an altered, stable stage distribution relative to the control population of both generations and the Fl population in the 0.2 µg E2/L treatment, resulting in additional population-level effects. These results demonstrate that continued exposure to E2 had compounding effects on sheepshead minnow populations and that long-term exposures may be necessary to understand the risk that exposures to environmental estrogens pose to native populations. Although population-level effects did not occur in the Fl generation, a risk decision based on Fl organism-level effects would be protective of the population exposed for two generations.

Comparison of estrogen-responsive plasma protein biomarkers and reproductive endpoints in sheepshead minnows exposed to 17beta-trenbolone.

Protein profiling can be used for detection of biomarkers that can be applied diagnostically to screen chemicals for endocrine modifying activity. In previous studies, mass spectral analysis revealed four peptides (2950.5, 2972.5, 3003.4, 3025.5m/z) in the plasma of estrogen agonist-treated male and gravid female sheepshead minnows (Cyprinodon variegatus, SHM), which served as distinct estrogenic biomarkers. In this study, a 21-day reproductive assay with adult SHM was conducted to investigate possible dose-related effects of the synthetic androgen, 17beta-trenbolone, on expression of these four estrogen-responsive peptides. In addition, the response of the peptide biomarkers were compared to traditional reproductive endpoints of fecundity, histopathology, secondary sex characteristics, length, weight, hepatosomatic index, female gonadosomatic index and plasma vitellogenin (VTG) levels. Fish were continuously exposed to 0.005, 0.05, and 5.0 microg/l, a solvent control (triethylene glycol, TEG), and a seawater control (SW) using an intermittent flow-through dosing system. Plasma was analyzed for the presence of the four peptide biomarkers by MALDI-TOF MS and VTG protein by quantitative ELISA. Male fish from the trenbolone treatments and controls showed no expression of the four peptide biomarkers or measurable levels of VTG. The estrogen-responsive biomarkers and plasma VTG were constitutively expressed in females from the SW, TEG, 0.005 and 0.05 microg/l exposures. All four peptide biomarkers were significantly reduced (p<0.0002 to p<0.005) at the 5.0 microg/l treatment level which corresponded with significant reductions in fecundity and changes in ovarian morphology. A distinct but non-significant reduction in VTG was also observed in female fish from the 5.0 microg/l treatment. Results of this study suggest application of these estrogen-responsive protein biomarkers may be a cost effective alternative to fecundity measures which are labor intensive and expensive to conduct.

Contaminated sediment testing with the bivalve Mulinia lateralis: culture refinement for organism availability.

Availability of test species for estuarine benthic assessment is limited; therefore, a method was developed by the U.S. Environmental Protection Agency for using the dwarf surf clam (Mulinia lateralis) to identify adverse biological effects of bulk estuarine sediments. A multilaboratory evaluation of the draft method resulted in favorable responses from the participants with respect to general testing and handling of the clam. However, availability of good-quality test organisms was limited. An investigation of culture techniques determined that culture vessel topography dramatically influenced survival, because larvae accumulated in bottom depressions. Conditioning of brood stock was enhanced by algae naturally containing both 20:5n-3 and 22:6n-3 fatty acids. Survival of larvae to metamorphosis at 14 d postspawn was greatly increased by maintenance at a lighting of 734 lux with addition of gradually increasing amounts of Isochyris galbana and Chaetoceros calcitrans. Greater than 50% survival of 600 juveniles to testing size by 14 d postmetamorphosis was accomplished by intermittent delivery of algae 12 times per day in a total of 13 L of seawater. By 21 d postmetamorphosis, an additional 27% achieved testable size. As a result of identification of these culture parameters, test bivalves can be readily available to improve predictions of adverse biological effects on benthic communities beyond those presently determined through amphipod exposures.