Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals

Abstract

Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures. We investigated the implications of this by analyzing the combined effects of a multicomponent mixture of five estrogenic chemicals using vitellogenin induction in male fathead minnows as an end point. The mixture consisted of estradiol, ethynylestradiol, nonylphenol, octylphenol, and bisphenol A. We determined concentration-response curves for each of the chemicals individually. The chemicals were then combined at equipotent concentrations and the mixture tested using fixed-ratio design. The effects of the mixture were compared with those predicted by the model of concentration addition using biomathematical methods, which revealed that there was no deviation between the observed and predicted effects of the mixture. These findings demonstrate that estrogenic chemicals have the capacity to act together in an additive manner and that their combined effects can be accurately predicted by concentration addition. We also explored the potential for mixture effects at low concentrations by exposing the fish to each chemical at one-fifth of its median effective concentration (EC50). Individually, the chemicals did not induce a significant response, although their combined effects were consistent with the predictions of concentration addition. This demonstrates the potential for estrogenic chemicals to act additively at environmentally relevant concentrations. These findings highlight the potential for existing environmental risk assessment procedures to underestimate the hazard posed by mixtures of chemicals that act via a similar mode of action, thereby leading to erroneous conclusions of absence of risk.

Figure 1. Pooled concentration–response data and best-fit regression curves for each of the individual mixture components. (A) EE₂. (B) E₂. (C) NP. (D) OP. (E) BPA. Each point represents the VTG response of one fish, with each color representing an independent exposure study. The solid line represents the best-fit curve, and the dashed lines represent the 95% confidence interval.

Figure 2. Best-fit regression curves for the individual mixture components plotted on the same concentration scale.

Figure 3. Comparison between the observed and CA-predicted mixture effects of five estrogenic chemicals in the male fathead minnow. Each point represents the VTG response of one fish, with each color representing an independent exposure study. The solid black line represents the best-fit of the observed effect data, and the solid red line represents the CA prediction. Dashed lines represent the 95% confidence intervals. The predicted effect of the mixture falls within the 95% confidence interval of the observed data across the entire dose–response curve.

Figure 4. Mixture effects at low-effect concentrations (one-fifth of EC₅₀) of five estrogenic chemicals. Error bars indicate SEM. Individual concentrations were 0.12 ng/L EE₂, 5 ng/L E₂, 1.4 μg/L NP, 9 μg/L OP, and 30 μg/L BPA. The mixture treatment contained all five chemicals at the aforementioned concentrations, resulting in an overall mixture concentration of 40.4 μg/L. Analysis of variance detected a significant difference between treatments (F_6,19 = 4.05, p < 0.01). Post hoc tests revealed no difference between the response of fish exposed to each of the chemicals individually and that of the control fish. In contrast, the mixture elicited a response that was significantly different from that of the controls.