Short-chain per/polyfluoroalkyl substances alternatives enhance horizontal gene transfer risks in nitrification systems under quaternary ammonium compounds antimicrobials co-stress despite lower acute toxicity than perfluorooctanoic acid.

The ecological risks posed by per/polyfluoroalkyl substances (PFAS) and quaternary ammonium compounds (QACs), as emerging contaminants, to the aquatic environment have recently attracted considerable attention. However, it is still unclear whether and how the combined stress of PFAS and QACs affects wastewater treatment system performance and modulates the transmission of resistance genes (RGs). In this paper, it was investigated that the ecological impacts of perfluorooctanoic acid (PFOA) and its alternatives, perfluorobutanesulfonic acid (PFBS) and perfluorohexanoic acid (PFHxA), on nitrification systems with/without diallyl dimethylammonium chloride (DADMAC), a typical QACs disinfectant, during 120 days. Results showed that 3 mg/L PFOA significantly reduced ammonia removal efficiency, while 0-3 mg/L PFBS and PFHxA had no significant impacts. Interestingly, the addition of 0.3 mg/L DADMAC mitigated the inhibitory effect of PFOA on ammonia oxidation and elevated the abundance of complete ammonia oxidizers amoA and ammonia-oxidizing bacteria amoA genes by 15.8 %-52.9 % and 45.0 %-113.9 %, respectively, through looser protein structures of extracellular polymeric substances and more RGs activated. Under single stress, the abundance of total RGs exhibited first decreasing and then increasing trends with increasing concentrations of all three PFAS, and 3 mg/L PFOA enriched the highest. Under combined stress, PFOA led to the highest abundance of RGs by adding 0.3 mg/L DADMAC, while PFBS resulted in the highest abundance of RGs by adding 3 mg/L DADMAC. Notably, the system with PFBS was observed to have the highest abundance of mobile genetic elements (MGEs), followed by PFHxA, particularly inducing intracellular MGEs in sludge to maintain richness and continuity during combined stress stages. Moreover, MGEs were found to have the most positive contribution to the multiplication of antibiotic resistance genes in all three systems. Overall, although PFBS and PFHxA are regarded as typical alternatives to PFOA and are significantly less toxic to the nitrification systems compared with PFOA, both alternatives resulted in higher levels of MGEs, especially posing a more severe risk of horizontal gene transfer in the combined stress environment. Thus, this requires a focus on the RGs transmission risks of using PFAS and its alternatives in disinfectant-intensive environments.