The occurrence and removal of antibiotic residues and antibiotic resistance genes in the largest European constructed wetland at Orhei (Moldova)

Constructed wetlands (CWs) are increasingly promoted as low-cost, nature-based solutions for wastewater treatment, particularly in low- and middle-income countries (LMICs), yet their performance in removing pharmaceutical compounds, antibiotic resistance genes (ARGs), and bacterial pathogens remains insufficiently characterized under real-field-scale conditions. Here, we investigated the fate of pharmaceutical compounds (including antibiotic residues), wastewater bacterial communities, and the associated ARGs in the largest European passive treatment system, the vertical-flow CW of Orhei (Moldova), serving nearly 26,000 inhabitants. Metagenomic profiling revealed 51 bacterial families, with a reduction from 33 families in raw sewage to 25 in the final effluent and clear enrichment of autochthonous wetland taxa. A total of 150 ARG types conferring resistance to 16 antibiotic classes were detected. The cumulative ARG load decreased by approximately 82% from influent to effluent. Complete removal (100%) was observed for ARGs conferring resistance to fosfomycin, nitroimidazoles, rifamycins, streptothricin, oxazolidinones, and pleuromutilins, while sulfonamide resistance genes (sul1, sul2) persisted across all stages. Out of 25 antibiotic residues analyzed, 13 (including two sulfamethoxazole metabolites) were detected, together with 14 non-antibiotic pharmaceuticals. Mean removal of individual antibiotics reached 93%, and 83% for other pharmaceuticals, although some compounds (e.g., carbamazepine, 10,11-epoxycarbamazepine, alprazolam) showed negative removals. Environmental risk assessment (risk quotients, RQ) indicated no significant risk to freshwater biota (RQ < 0.1) for all detected compounds in the treated effluent. Results demonstrated that a large-scale CW in the LMIC context can substantially reduce antibiotic residues and ARGs, supporting its role as an effective, nature-based component of One Health-oriented wastewater management.