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

Abstract

Endocrine active substances (EAS), which are commonly used in pharmaceuticals and person-al care products, cannot be completely removed during conventional treatment in wastewater treatment plants (WWTPs) and are consequently released into surface water through WWTP effluents. EAS have been shown to cause adverse effects in aquatic organisms. Hence, they need to be evaluated with suitable test methods to achieve a good quality of water bodies, which is demanded by national and international directives (e.g. EU water framework directive). In wastewater, a variety of EAS with different hormonal activities is present, which can lead to mixture toxicity. These combination effects might result in additive or masking effects. The aspect of hormonal combination effects was investigated in this project, with a focus on estro-gen and androgen-modulators.
Wastewater samples were spiked with selected model substances, namely 17α-ethinylestradiol (estrogenic), toremifene (anti-estrogenic), 17α-methyltestosterone (androgenic) and bicalutam-ide (anti-androgenic), in concentrations related to their EC50 value. The individual effect of each substance as well as the effect of mixtures in hospital and municipal wastewaters was analysed using ER- and AR-CALUX assays to determine the respective endocrine activity.
Endocrine activities could be determined in municipal as well as hospital wastewater and were modified by adding the endocrine model substances. Independent from the used wastewater the estrogenic activity was reduced when the estrogen-antagonist toremifene was added to the samples. Likewise, the androgenic effect of methyltestosterone decreased by adding the antag-onist bicalutamide, but to a lesser extent compared to estrogens. Alterations of estrogenic ef-fects by adding the androgenic model substances were negligible and vice versa. Though a sim-ilar trend in altered endocrine activities was observed after substance addition, the intensities of altered activation or inhibition differed between the wastewaters.
This leads to the conclusion that masking effects are of significant importance in the assess-ment of complex water samples. The intensity of a measured hormonal activity caused by spe-cific EAS cannot be transferred from one wastewater to another. Furthermore, the results show that the occurrence of a specific endocrine substance in a sample does not allow a direct con-clusion on the final biological effect. This emphasises the relevance of bioassays in the assess-ment of water samples.