Back-transformation and dual-type metabolites associated with pharmaceutical negative removal and residual ecological risks in biological treatment.

Pharmaceuticals entering wastewater treatment plants undergo complex biological transformations to generate transformation products (TPs). However, the formation mechanisms and environmental impacts of these TPs remain insufficiently understood. In this study, an integrated LC-HRMS monitoring workflow combining target, suspect, and non-target screening (SNTS) was applied to characterize pharmaceutical biotransformation within a biological reactor (BR). A total of 37 pharmaceuticals and 39 TPs were detected, including 6 TPs quantified by target screening and 33 tentatively identified through SNTS. Major reactions included dealkylation and hydroxylation, and several previously unreported TPs were detected, including losartan-derived products (M435, M437) and fenbufen-derived products (FBF-M225). Fold-change analysis enabled classification of TPs into human-derived, microbial-derived, and dual-type metabolites. Notably, human- and dual-type metabolites were closely associated with negative removal phenomena and secondary TP formation, suggesting overlapping processes of back-transformation and in situ microbial conversion within the BR. Structure-based PMT/PBT assessments and risk quotient analysis indicated that several TPs, including CFN-M209, LST-M421, and LST-M435, exhibited higher persistence, mobility, bioaccumulation potential, or ecological risk than their parent compounds. In addition, parent compounds such as olmesartan and climbazole exhibited RQ values greater than 1, indicating potential ecological risks to aquatic organisms. Mixture risk assessment further highlighted cumulative ecological concerns in wastewater effluent, particularly for CBZ and its associated TPs. These findings indicate that the modified sequencing batch reactor (MSBR) examined in this study inherently generates environmentally relevant TPs, and that environmental risk assessment should extend beyond parent compounds to include both human-derived metabolites and treatment-induced TPs, thereby supporting more comprehensive and sustainable wastewater management strategies.