Evaluation of mixture effects of endocrine active substances in wastewater using CALUX reporter-gene assays

Endocrine active substances (EAS), which are commonly used in pharmaceuticals and personal care products, are released into surface water mainly through WWTP effluents and have been shown to cause adverse effects in aquatic organisms. In wastewater, a variety of EAS with different hormonal activities is present, which can lead to additive effects or mask an endocrine activity. To investigate hormonal combination effects, with a focus on estrogen and androgen-modulators, influent samples from municipal and hospital wastewater treatmenr plants were spiked with 17α-ethinylestradiol, toremifene, 17α-methyltestosterone and bicalutamide and analyzed using in vitro reporter gene CALUX assays. All wastewaters caused endocrine activities in human cells, which were modified by adding one or several endocrine active substances. As expected, estrogenic activity was reduced in presence of the anti-estrogenic toremifene and androgenic activity decreased with the anti-androgen bicalutamide. In general, substance addition caused a similar trend in altered endocrine activities; however, their intensities differed between the wastewaters. Our results indicate that masking effects, leading to a suppressed biological signal, are of significant importance in the assessment of complex water samples, and combination effects rather than single substances determine the final biological effect. This emphasizes the need of effect-based tools in the assessment of water samples.