Long-term exposure to environmental concentrations of the pharmaceutical ethynylestradiol causes reproductive failure in fish

Abstract

Heightened concern over endocrine-disrupting chemicals is driven by the hypothesis that they could reduce reproductive success and affect wildlife populations, but there is little evidence for this expectation. The pharmaceutical ethynylestradiol (EE2) is a potent endocrine modulator and is present in the aquatic environment at biologically active concentrations. To investigate impacts on reproductive success and mechanisms of disruption, we exposed breeding populations (n = 12) of zebrafish (Danio rerio) over multiple generations to environmentally relevant concentrations of EE2. Life-long exposure to 5 ng/L EE2 in the F1 generation caused a 56% reduction in fecundity and complete population failure with no fertilization. Conversely, the same level of exposure for up to 40 days in mature adults in the parental F0 generation had no impact on reproductive success. Infertility in the F1 generation after life-long exposure to 5 ng/L EE2 was due to disturbed sexual differentiation, with males having no functional testes and either undifferentiated or intersex gonads. These F1 males also showed a reduced vitellogenic response when compared with F0 males, indicating an acclimation to EE2 exposure. Depuration studies found only a partial recovery in reproductive capacity after 5 months. Significantly, even though the F1 males lacked functional testes, they showed male-pattern reproductive behavior, inducing the spawning act and competing with healthy males to disrupt fertilization. Endocrine disruption is therefore likely to affect breeding dynamics and reproductive success in group-spawning fish. Our findings raise major concerns about the population-level impacts for wildlife of long-term exposure to low concentrations of estrogenic endocrine disruptors.

Figure 1. Between-tank and between-day variations in number of eggs over a 20-day period. (A) Total number of eggs in five random control tanks. (B) Mean (± SEM) number of eggs for the same tanks.

Figure 2. Reproductive success in the F₀ generation of zebrafish exposed to 0.5, 5, and 50 ng/L EE₂, 5 ng/L E₂, and unexposed controls for three consecutive 5-day periods: (A) 1–5 days, (B) 6–10 days, and (C) 11–15 days. Total bar length indicates the total number of eggs per tank (mean ± SEM; top error bar). The lighter bar indicates total survival of viable eggs at 14 hpf (mean ± SEM; bottom error bar). The black section indicates the number of nonviable eggs at 14 hpf. : *Significant decrease in egg number and 14 hpf viability when compared with the control group for each period, and increase in rate of nonviable eggs when compared with the same group in (A); ANOVA, all cases n = 12, p < 0.01, post hoc analysis against control treatments by Dunnet’s test with p > 0.05.

Figure 3. Reproductive success of the F₁ generation of zebrafish after 7 months (210 dpf) exposure to 0.5 and 5 ng/L EE₂, 5 ng/L E₂, and unexposed controls for two successive 5-day periods: (A) 1–5 days, and (B) 6–10 days. Total bar length indicates the total number of eggs per tank (mean ± SEM; top error bar). The lighter bar indicates the total number of viable eggs at 14 hpf (mean ± SEM; bottom error bar). The black section indicates the proportion of eggs nonviable at 14 hpf. : *Long-term exposure to 5 ng/L EE₂ resulted in a reduced fecundity (n = 12, p < 0.01) and no survival past 14 hpf. **The proportion of nonviable eggs was significantly higher for all treatments when compared with the control rates (n = 12, p < 0.05).

Figure 4. Effects of life-long exposure to 5 ng/L EE₂ on gonad development in adult zebrafish (314 dpf). (A) Persisting juvenile undifferentiated (ovary-type) gonad in presumptive males. (B) Intersex fish with one ovary and one testis. (C) Intersex fish with two testes and smaller juvenile (ovary-type) tissue. (D) Ciliated sperm duct in testis of mature male (found only in adults when exposure to EE₂ was stopped at 75 dpf).

Figure 5. Reproductive success (5-day means) in the F₁ generation of zebrafish at 240 dpf after lifelong exposure to 5 ng/L EE₂, with subsequent manipulation of males in the populations (all experiments done under no direct exposure). In the controls, males were either retained (control; six tanks) or removed (control – males; six tanks). In the EE₂ treatment, EE₂-exposed males were either retained (5 ng/L EE₂, six tanks) or two males were substituted with two healthy control males (EE₂ + males; six tanks). Total bar length indicates the total number of eggs per tank (mean ± SEM) for the six replicate tanks in each group. The lighter bar indicates the total number of viable eggs (mean ± SEM) to 14 hpf. The black section indicates the percentage of nonviable eggs at 14 hpf. : *Significantly different (p < 0.05) from control in the proportion of viable eggs at 14 hpf. **Significantly different (p < 0.05) from control in total egg numbers. ^#Significantly different (p < 0.05) number of eggs laid with the addition of healthy males compared with controls (F = 16.5, p < 0.001, n = 6); the proportion of nonviable eggs was still significantly higher (F = 177, p < 0.001, n = 6) than in the control group.

Figure 6. Whole blood VTG concentrations (mean ± SEM) in male and female zebrafish in the F₀ generation after 40 days exposure to 0.5, 0.5, 5, or 50 ng/L EE₂ (A) and in the F₁ generation after life-long (310 dpf) exposure to 0, 0.5, or 5 ng/L EE₂. : *Dose-dependent induction of VTG (p < 0.05 compared with controls of the same sex).