ZnO nanoparticles (ZnO-NPs) are emerging contaminants that raise the concerns of potential risk in the aquatic environment. It has been estimated that the environmental ZnO-NPs concentration is 76 μg/l in the aquatic environment. Our aim was to determine the aquatic toxicity of ZnO-NPs with chronic exposure at environmentally relevant concentrations using the nematode Caenorhabditis elegans. Two simulated environmentally relevant mediums—moderately hard reconstituted water (EPA water) and simulated soil pore water (SSPW)—were used to represent surface water and pore water in sediment, respectively. The results showed that the ZnO-NPs in EPA water has a much smaller hydrodynamic diameter than that in SSPW. Although the ionic release of Zn ions increased time-dependently in both mediums, the Zn ions concentrations in EPA water increased two-fold more than that in SSPW at 48 h and 72 h. The ZnO-NPs did not induce growth defects or decrease head thrashes in C. elegans in either media. However, chronic exposure to ZnO-NPs caused a significant reduction in C. elegans body bends in EPA water even with a relatively low concentration (0.05 μg/l); similar results were not observed in SSPW. Moreover, at the same concentrations (50 and 500 μg/l), body bends in C. elegans were reduced more severely in ZnO-NPs than in ZnCl2 in EPA water. The ATP levels were consistently and significantly decreased, and ROS was induced after ZnO-NPs exposure (50 and 500 μg/l) in EPA water. Our results provide evidences that chronic exposure to ZnO-NPs under environmentally relevant concentrations causes metabolic and locomotive toxicities implicating the potential ecotoxicity of ZnO-NPs at low concentrations in aquatic environments.