2.7. In Vitro Recombinant Yeast Estrogen Screen (YES)

The recombinant yeast-based screen followed the protocol described by Sohoni and Sumpter (1998). Saccharomyces cerevisiae transfected with the human estrogen receptor (hER) gene and a plasmid containing an estrogen response element-linked lac-Z gene was used. Successful binding of ligands in the water samples (steroids) to the receptors in the yeast cells initiate the expression of the lac-Z reporter gene that encodes for the enzyme β-galactosidase in the assay. The β-galactosidase then metabolises chlorophenol red galactopyranoside (CPRG), which results in a colour change of the assay medium, indicating a dose-dependent activation of the ligands to bind to the estrogen receptor. The assay medium was prepared as described by [29]. The yeast was incubated in assay medium containing no CPRG for 48 h under 26 °C on an orbital shaker. The concentrated wastewater extracts (500×) were serially diluted and 10 µL was spiked into the 96-well sterile flat-bottomed plates with low evaporation lids (Costar, 3370, Sigma). The previously incubated yeast culture was then included into new assay medium containing CPRG at a concentration of approximately 8 × 105 cells/mL. The seeded assay medium was then added at 200 µL/well into the assay plate to provide a final concentration of the water extracts ranging from 50× to 1.56×. A concentration of 1× was depicted as an un-concentrated water sample. For the raw wastewater samples, serial dilutions of the samples were made with MeOH to obtain a concentration range of each sample ranging from 12.5× to 0.39× in the assay due to cytotoxicity observed in the 50× and 25× concentrated sample. For the effluent (permeate) water samples, serial dilutions of the samples were made with MeOH to obtain a concentration range of each sample ranging from 50× to 6.25× due to the lower observed estrogenicity in these samples compared to raw wastewater samples. All samples were analysed in triplicate in the same assay plate, and each assay was repeated twice. A standard curve for the steroid hormone 17β-estradiol (E₂; CAS 50-28-2; Sigma) was included for each assay plate in 12 serial dilutions, ranging from 1.0 to 2700.0 ng/L. Blank wells were also included in each assay plate containing only assay medium without any hormone spike or water sample extracts. The assay plates were then allowed to incubate on a shaker for 72 h at 30 °C under dark conditions [30].