Single and mixture toxicity effects of legacy and emerging per- and polyfluoroalkyl substances on submerged plants and epiphytic biofilms.

Although per- and polyfluoroalkyl substances (PFASs), often referred to as "forever chemicals," pose persistent threats to aquatic ecosystems, the effects of multiple PFAS types on submerged macrophyte-biofilm symbiotic systems remain poorly understood. In this study, we systematically investigated the impacts of single and combined exposures to both legacy and emerging PFAS on submerged macrophytes, biofilms, and associated microbial risks. Our results show that the growth of Vallisneria natans was inhibited under both single and mixed PFAS stress. Photosynthetic performance and nutrient uptake in V. natans were variably affected depending on the PFAS type. Emerging PFAS were more likely to induce oxidative stress, with malondialdehyde content increasing by 36.7 % under hexafluoropropylene oxide dimer acid exposure. Notably, PFAS stress significantly altered biofilm morphology and microbial community composition, including enrichment of several human bacterial pathogens. Additionally, PFAS exposure promoted the enrichment of antibiotic resistance genes (ARGs), and the increased abundance of mobile genetic elements suggested a higher potential for horizontal gene transfer. Co-occurrence network analysis further revealed that potential ARG hosts were affected under PFAS stress.