Endocrine-disrupting chemicals are mainly discharged into the environment by wastewater treatment plants (WWTPs) and are known to induce adverse effects in aquatic life. Advanced treatment with ozone successfully removes such organic micropollutants, but an increase of estrogenic effects after the ozonation of hospital wastewater was observed in previous studies. In order to investigate this effect, estrogenic and androgenic as well as anti-estrogenic and anti-androgenic activities were observed during treatment of hospital wastewater using three different effect-based reporter gene bioassays. Despite different matrix influences, sensitivities, and test-specific properties, all assays used obtained comparable results. Estrogenic and androgenic activities were mainly reduced during the biological treatment and further removed during ozonation and sand filtration, resulting in non-detectable agonistic activities in the final effluent. An increased estrogenic activity after ozonation could not be observed in this study. Antagonistic effects were removed in the biological treatment by up to 50 % without further reduction in the advanced treatment. Due to the presence of antagonistic substances within the wastewater, masking effects were probable. Therefore, this study showed the relevance of antagonistic activities at hospital WWTPs and illustrates the need for a better understanding about antagonistic effects.

Effluents from municipal wastewater treatment plants (WWTPs) are known to be point sources of micropollutants for surface waters. The aim of this study was to examine a reconstructed full-scale ozonation equipped with a pump-injector system for ozone (O) dosage and a fluidized moving-bed reactor as biological posttreatment at a municipal WWTP utilizing an effect-directed approach. This approach consists of chemical analysis in combination with toxicological tests for the assessment of treatment efficiency of the plant. Chemical analysis showed elimination rates > 80% for pharmaceuticals and industrial chemicals. Analysis of endocrine disruptors was limited due to substance concentrations below the limit of detection (LOD). Estrogenic activity was detected by the Arxula Adeninivorans yeast estrogen screen (A-YES) at low concentrations (pg to ng EEQ/l range). Estrogenic activity was reduced by more than 90% after ozonation. In contrast, androgenic activity (measured in the Adeninivorans yeast androgen screen, A-YAS) was still found after O treatment and after biological posttreatment, which is consistent with the data obtained by chemical analysis. Furthermore, no marked genotoxic or cytotoxic effects were observed after ozonation using the alkaline comet and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assays, respectively. Results suggest that the applied specific O dose of 0.4 mg/mg is a safe operation setup in terms of toxicologically relevant transformation products. In addition, no adverse effects on primary producers, as evidenced by algae growth inhibition tests, were detected. The monitored biofilm growth in the biological posttreatment exhibited a steady state after one month. Based on computational fluid dynamics (CFD) simulations and biomass, one might conclude that O did not apparently enter biological posttreatment to a great extent and that hydraulic retention time in the O reactor was sufficient. Our data demonstrate the effectiveness of a full-scale O treatment in combination with a fluidized moving-bed reactor as biological posttreatment for the reduction of a majority of micropollutants without the release of relevant toxic transformation products as assessed by a chemical and toxicity-based approach.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17β-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17β-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

To gain biologically and chemically safe conditions in aquatic systems, as required by the EU Water Framework Directive, advanced oxidation processes are the only conceivable possibility to remove pharmaceuticals and their metabolites from waste water. Water treatment plants are not designed for the complete mineralization of these substances, so that oxidation by-products occur, whose chemical and toxicological properties are often unknown. In this study an UV-oxidation system with and without H2O2 was tested to optimize the elimination of pharmaceuticals, and replenished with toxicological investigations of untreated and oxidatively treated samples. The substances carbamazepine, metoprolol, amidotrizoic acid, diclofenac, and sulfamethoxazole were used as lead compounds. They were studied separately and as a substance mixture. The UV-system had a low-pressure lamp with an emission wavelength of 254 nm and a maximum UV output of 15 W. The sample volume was 1 L with a flow rate of 0.03 m³ h−1. Different matrices (waste water, ultrapure water) were used to detect possible matrix effects of real samples. The degradation rates of the substances were analyzed and quantified using LC-MS/MS with an API 3000 and a Q TRAP 3200. The comparison revealed that the additional use of H2O2 enhances the degradation significantly, especially in waste water samples. Cyto- and genotoxicity were tested with the MTT Assay and Alkaline Comet Assay, respectively, using Chinese Hamster Ovary cells. Generally, samples treated only with UV showed no toxic effects. Toxic results occurred only for samples with the substance mixture that were treated with UV+H2O2, indicating that combination effects might lead to the formation of toxic by-products.