Estrogenic activity in wastewater treatment plants through in vitro effect-based assays: Insights into extraction phase

Effluents of wastewater treatment plants can abundantly spread endocrine disrupting chemicals in the environment. To improve water quality monitoring, the use of effect-based tools that measure estrogenic activity has been suggested, however their results could be influenced by different factors. This study compared the estrogenic activity of wastewater samples extracted with two stationary phases and tested with two in vitro effect-based assays to investigate whether and how stationary phases and assays could influence biomonitoring data. During four seasonal periods, the effluents of six WWTPs located in northern Italy were sampled. After the extraction using two different stationary phases (HLB, C18), the samples (n = 72) were tested using two effect-based assays: a gene reporter luciferase assay on mammalian cells (MELN) and yeast estrogen screen assay (YES). The results showed that estrogenic activity of HLB extracts was significantly different from the activity of C18 extracts, suggesting that extraction phase can influence biomonitoring data. Moreover, the estrogenic activity was overall higher using gene reporter MELN assay than using YES assay, suggesting that, due to difference in cell membrane permeability and metabolic activation, the applied cell model can affect the biomonitoring results. Finally, from the comparison between the activity of the final effluent and the environmentally safe estrogenic levels in surface waters, MELN data suggested that the activity of this effluent may pose an environmental risk, while YES data showed that it should not be considered a threat to the receiving surface waters. This study pointed out that a standardized approach is needed to assess the estrogenic activity of waters; it reported important data to select the most suitable stationary phase for samples extraction (samples extracted with C18 sorbent showed higher estradiol equivalent concentration values) and the most appropriate bioassay (gene reporter luciferase MELN assay was more sensitive than YES assay) to assess the environmental risk, thus protecting human health.