Combined effects of estrogenic chemicals with the same mode of action using an estrogen receptor binding bioassay

•The joint action of estrogenic mixtures prepared by different designs was evaluated.•CA and EEQ predict estrogenic effect well with single mode of action.•We offer evidence to the application of EEQ in the non-equi-effect mixing scenario.•Estrogenicity should be assessed based on both concentration and estrogenic potency.

The increasing amounts of various estrogenic chemicals coexisting in the aquatic environment may pose environmental risks. While the concept of estradiol equivalent (EEQ) has been frequently applied in studying estrogenic mixtures, few experiments have been done to prove its reliability. In this study, the reliability of EEQ and the related model concentration addition (CA) was verified based on the two-hybrid recombinant yeast bioassay when all mixture components had the same mode of action and target of action. Our results showed that the measured estrogenic effects could be well predicted by CA and EEQ for all laboratory-made mixtures using two designs, despite the varying estrogenic activity, concentration levels and ratios of the test chemicals. This suggests that when an appropriate endpoint and its relevant bioassay are chosen, CA should be valid and the application of EEQ in predicting the effect of non-equi-effect mixtures is feasible.