Removal of pharmaceutical' estrogenic activity of sequencing batch reactor effluents assessed in the T47D-KBluc reporter gene assay

Various water treatment processes may be ineffective to remove pharmaceutical compounds (PhCs) and their by-products, leading to endocrine-disruptive activity that might be detrimental to wildlife and human health. This study investigated the degradation of carbamazepine (CBZ), diclofenac (DCF), ibuprofen (IBP), and their intermediates, as well as estrogenic activity that is not effectively removed by conventional methods. A consortium of isolated South African indigenous fungi A. niger, M. circinelloides, T. polyzona, T. longibrachiatum and R. microsporus, was used in a sequencing batch reactor (SBR) to remove PhCs, their intermediates and strongly reduce their estrogenic activity. The fungal ligninolytic enzymatic activity was determined for laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) using a spectrophotometric method. The biodegradation of PhCs and their intermediates was monitored by SPE-UPLC/MS. The in vitro estrogenic activity was assessed in the T47D-KBluc reporter gene assay. Lac, MnP and LiP production appeared to be biomass growth dependent. During a lag phase of growth, a constant biomass of about 122.04 mg/100 mL was recorded with average enzymatic activity around 63.62 U/L for Lac, 151.91 U/L for MnP and 42.12 U/L for LiP. The exponential growth phase from day 7 to day 17, was characterised by a biomass increase of 124.46 units, and an increase in enzymatic activity of 9.91 units for Lac, 99.03 units for MnP and 44.24 units for LiP. These enzymes played an important synergistic role in PhCs degradation in the cytochrome P450 system. A decrease of 13.89%, 29.7% and 16.15% in PhC concentrations was observed for CBZ, DCF and IBP, respectively, and their intermediates were identified within 4 h of incubation. The removal efficiency achieved after 24 h in the SBR was about 89.77%, 95.8% and 91.41% for CBZ, DCF and IBP, respectively. The estradiol equivalent (EEq) values of 1.71 +/- 0.30 ng/L and 2.69 +/- 0.17 ng/L were recorded at the start-up time and after 4 h, respectively. The presence of intermediates was found to induce estrogenic activity. The EEq values after 24 h incubation was found to be below the LoQ and below the LoD of the assay. None of the samples exhibited any anti-estrogenic activity. The fungal consortium inoculum was found to induce toxicity at a 0.4 x concentration, as observed under a microscope. This study revealed that the use of the fungal consortium can remove the estrogenic activity of pharmaceutical metabolites, which appeared to be the most significant contributors to the endocrine-disrupting activity of the wastewater treatment plant effluents.