Long-term effects of Ag NPs on denitrification in sediment: Importance of Ag NPs exposure ways in aquatic ecosystems

•Dynamic responses of denitrifiers to Ag NPs were highly relative to exposure way.•The obvious inhibition of Ag NPs pulsing exposure decreased with time.•The irreversible negative effects on nitrogen cycling would be caused by Ag NPs.•The recovery of microbial function did not mean the restoration of water ecosystem.•The long-term risks of Ag NPs releasing on ecosystems should not be ignored. The widespread use of silver nanoparticles (Ag NPs) inevitably leads to their increasing release into aquatic systems, with studies indicating that the mode of Ag NPs entry into water significantly affects their toxicity and ecological risks. However, there is a lack of research on the impact of different exposure ways of Ag NPs on functional bacteria in sediment. This study investigates the long-term influence of Ag NPs on denitrification process in sediments by comparing denitrifies responses to single (pulse injection of 10 mg/L) and repetitive (1 mg/L × 10 times) Ag NPs treatments over 60-day incubation. Results showed that a single exposure of 10 mg/L Ag NPs caused an obvious toxicity on activity and abundance of denitrifying bacteria on the first 30 days, reflecting by the decreased NADH amount, ETS activity, NIR and NOS activity, and nirK gene copy number, which resulted in a significant decline of denitrification rate in sediments (from 0.59 to 0.64 to 0.41–0.47 μmol15N L−1 h−1). While inhibition was mitigated with time and denitrification process recovered to the normal at the end of the experiment, the accumulated nitrate generated in the system showed that the recovery of microbial function did not mean the restoration of aquatic ecosystem after pollution. Differently, the repetitive exposure of 1 mg/L Ag NPs exhibited the evident inhibition on metabolism, abundance, and function of denitrifiers on Day 60, due to the accumulated amount of Ag NPs with the increased dosing number, indicating that the accumulated toxicity on functional microorganic community of repetitive exposure in less toxic concentration. Our study highlights the importance of Ag NPs entry pathways into aquatic ecosystem on their ecological risks, which affected dynamic responses of microbial function to Ag NPs. [Display omitted]