Cumulative effects of sulfamethoxazole and its metabolite on nitrogen reduction and antibiotic resistance in constructed wetlands: Microbial mechanisms and ecological risks.

Antibiotic residues in the tailwater of wastewater treatment plants (WWTPs) threaten nitrogen removal in constructed wetlands (CWs), yet the long-term impacts of fluctuating sulfamethoxazole (SMX) and its metabolite N-acetylsulfamethoxazole (N-SMX) remain unclear. Here, the dual effects of dynamic recovery in microbial nitrogen-removal functions and irreversible accumulation of antibiotic resistance genes (ARGs) under gradient SMX + N-SMX exposure (10-1000 μg L with 30-day stepwise increments) in lab-scale CWs were systematically revealed. At ≤ 100 μg L, denitrification and anammox rates could recover to baseline levels, whereas 1 mg L exposure triggered a short-term surge and then cumulative inhibition of nitrogen reduction (e.g., denitrification rates was 34.7 % lower than the controls even after the SMX + N-SMX concentration reduction). Notably, sulfonamide resistance genes (sul1/sul2) increased steadily over 180 days despite the decline in SMX + N-SMX exposure (from 1 mg L to 10 μg L from day 120 to day 180), probably driven by horizontal gene transfer. Microbial analysis identified Burkholderia and Anaerolineales as dual-functional taxa linking nitrogen metabolism with ARGs propagation. Furthermore, sustained exposure suppressed the expression of denitrification genes (narG/nirK) of Methylotenera, despite its role in degrading SMX/N-SMX. These findings highlight a critical threshold: exposures <100 μg L allow the functional recovery of nitrogen reduction, but ≥1 mg L induces irreversible ARGs enrichment and disrupts microbial nitrogen cycling. This study provides mechanistic insights into the ecological risks of antibiotic fluctuations, advocating stricter control of high-concentration SMX/N-SMX in WWTP tailwater to mitigate the dissemination of resistance genes.