Impact of low-dose free chlorine on the conjugative transfer of antibiotic resistance genes in wastewater effluents: Identifying key environmental factors for predictive modeling

Reclaimed water disinfection results in the coexistence of antibiotic resistance genes (ARGs) and low-dose free chlorine in receiving environments. However, the impact of low-dose free chlorine on ARGs conjugative transfer and the key factors influencing the transfer under complex environmental conditions remain unclear, hindering the establishment of an effective monitoring system for resistance pollution in reclaimed water. This study investigated ARGs conjugative transfer under the influence of free chlorine at environmentally relevant concentrations and key interactive factors using machine learning models. The results showed that low-dose free chlorine (0.05–0.3 mg/L) promoted ARGs conjugative transfer, with 0.15 mg/L having a greater promoting effect than free chlorine concentrations of 0.05 and 0.3 mg/L. Additionally, different exposure patterns of low-dose chlorine affected ARGs conjugative transfer, with intermittent exposure posing a higher risk of ARGs dissemination. SVM linear model performed best in predicting ARGs conjugative transfer ( RMSE =0.012, R 2 =0.975), and the SHapley Additive Explanations (SHAP) method revealed that key factors such as HCO 3 - , SAA, NO 3 - , and HA had positive SHAP values, indicating a positive influence on ARGs transfer under low-dose chlorine, making them the key features for predicting the ARGs conjugative transfer under the low-dose chlorine exposure. This study also revealed potential mechanisms of ARGs transfer under continuous low-dose free chlorine exposure, including intracellular reactive oxygen species (ROS), enzyme activity , cell membrane permeability , and gene expression. The integration of the machine learning model and post-hoc interpretation methods clarified the key drivers of ARGs conjugative transfer in reclaimed water-replenished environments, providing new insights for the safe reuse of reclaimed water and the development of river monitoring indicators.