Contrasting toxic effects and recovery potential of AnGS exposed to polystyrene micro- and nanoplastics: Modulating roles of particle size and surface functionalization

Anaerobic ammonium oxidation (Anammox) systems are highly sensitive biological nitrogen removal processes, yet the acute effects and recovery potential of anaerobic granular sludge (AnGS) exposed to micro-/nanoplastics with different surface functionalizations remain insufficiently understood. This study systematically investigated the acute toxic effects and short-term recovery responses of AnGS exposed to polystyrene microplastics (PS-MP), pristine polystyrene nanoplastics (PS-NP), and surface-functionalized polystyrene nanoplastics (PS-NH 2 and PS-COOH) under a 12 h exposure and 12 h recovery period.The results showed that the toxicity of PS-MNPs was jointly influenced by particle size and surface functionalization, with an inhibition intensity order of PS-NH 2 > PS-NP > PS-MP > PS-COOH. PS-NH 2 caused the strongest inhibition of nitrogen removal and showed incomplete recovery within the tested recovery period, accompanied by marked oxidative stress. Protein secondary structure analysis further suggested that PS-NH 2 induced pronounced conformational disturbance of proteins in the TB-EPS layer, as indicated by a decrease in α-helix proportion and an increase in aggregated strands. In contrast, PS-COOH caused substantial EPS protein loss but showed relatively weaker functional inhibition, indicating that EPS quantity alone could not fully explain the recovery of AnGS function. The recovery results further suggested that protein conformational integrity may be more important than total protein content for restoring system performance. Overall, this study reveals that PS-MNPs toxicity to AnGS involves both biological stress and EPS structural disturbance, providing a molecular-level perspective for assessing the ecological risks of micro-/nanoplastics in Anammox-based wastewater treatment systems.