The presence of ZnO NPs in waste streams can negatively affect the efficiency of biological nutrient removal from wastewater. However, details of the toxic effects of ZnO NPs on microbial activities of wastewater biofilms have not yet been reported. In this study, the temporal and spatial inhibitory effects of ZnO NPs on the O-2 respiration activities of aerobic wastewater biofilms were investigated using an O-2 microelectrode. The resulting time-course microelectrode measurements demonstrated that ZnO NPs inhibited O-2 respiration within 2 h. The spatial distributions of net specific O-2 respiration were determined in biofilms with and without treatment of 5 or 50 mg/L ZnO NPs. The results showed that 50 mg/L of nano-ZnO inhibited the microbial activities only in the outer layer (similar to 200 mu m) of the biofilms, and bacteria present in the deeper parts of the biofilms became even more active. Scanning electron microscopy (SEM) analysis showed that the ZnO NPs were adsorbed onto the biofilm, but these NPs had no adverse effects on the cell membrane integrity of the biofilms. It was found that the inhibition of O-2 respiration induced by higher concentrations of ZnO NPs (50 mg/L) was mainly due to the release of zinc ions by dissolution of the ZnO NPs. (C) 2014 Elsevier B.V. All rights reserved.