The occurrence of endocrine-disrupting compounds (EDCs) consisting of natural and synthetic estrogens, namely estrone (E₁), 17β-estradiol (E₂), estriol (E₃) and 17α-ethinylestradiol (EE₂) was quantified in wastewater samples. The aim of this study was to assess the removal efficiency for the selected estrogens (E₁, E₂, E₃ and EE₂) and reduction of estrogenic activity in wastewater samples from wastewater treatment plants (WWTPs) using different processes. Solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to quantify the selected estrogens in wastewater samples. Estrogenic activity was assessed using the T47D-KBluc gene reporter assay. Results revealed a decrease in estrogen concentrations observed in the effluents of all the WWTPs, except for E₂ at Daspoort where no removal was noted. In general, the highest removal for total estrogens was observed at Phola (84%) combining three processes (AP, BF and wetland). The AS at Daspoort had a highest removal of 75% for E₃; while at Zeekoegat the highest removal reached 61% for EE₂. The PST at Daspoort had no removal recorded for all the compounds, except for the EE₂ (33%). The AP and BF systems at Phola contributed to a higher removal of selected compounds. Downstream of the wetland at Phola no removal was recorded for E₃; while the highest removal reached 61% for E₁. The best performance in terms of the overall influent-to-effluent removal efficiency was observed at Phola WWTP, where E₁ removal of 85% was recorded.

The highest estrogenic activity in the effluent was reported at Phola, with an average estradiol equivalent (EEQ) value of 6.3 ± 6.7 ng/L. However, no anti-estrogenic activity was detected in any of the samples. The daily mass load discharged from the effluent of the three WWTPs was higher for E₁ recorded at Zeekoegat (8002.3 ± 6416.3 mg/d), followed by Daspoort (3509.8 ± 849.0 mg/d) and finally Phola (176.1 ± 34.9).