The uptake and toxicity of 2 poly(methylmethacrylate)‐based plastic nanoparticles (PNPs) with different surface chemistries (medium and hydrophobic) were assessed using aquatic organisms selected for their relevance based on the environmental behavior of the PNPs. Pure poly(methylmethacrylate) (medium; PMMA PNPs) and poly(methylmethacrylate‐co‐stearylmethacrylate) copolymer (hydrophobic; PMMA–PSMA PNPs) of 86 nm to 125 nm were synthesized using a miniemulsion polymerization method. Fluorescent analogs of each PNP were also synthesized using monomer 7‐4‐(trifluoromethyl)coumarinacrylamide and studied. Daphnia magna, Corophium volutator, and Vibrio fischeri were employed in a series of standard acute ecotoxicity tests, being exposed to the PNPs at 3 different environmentally realistic concentrations (0.01 mg/L, 0.1 mg/L, and 1.0 mg/L) and a high concentration 500 mg/L to 1000 mg/L. In addition, sublethal effects of PNPs in C. volutator were determined using a sediment reburial test, and the uptake and depuration of fluorescent PNPs was studied in D. magna. The PNPs and fluorescent PNPs did not exhibit any observable toxicity at concentrations up to 500 mg/L to 1000 mg/L in any of the tests except for PMMA–PSMA PNPs and fluorescent PNPs following 48‐h exposure to D. magna (median lethal concentration values of 879 mg/L and 887 mg/L, respectively). No significant differences were observed between labeled and nonlabeled PNPs, indicating the suitability of using fluorescent labeling. Significant uptake and rapid excretion of the fluorescent PNPs was observed in D. magna. Environ Toxicol Chem 2016;35:1641–1649. © 2015 SETAC