Functionalized Polystyrene Nanoplastics Drive Distinct Mitochondrial Bioenergetic and Phenotypic Responses in Rainbow Trout Gill Epithelia

ABSTRACT Nanoplastics are emerging contaminants of concern in freshwater ecosystems and have been shown to be ingested and assimilated in fish gill tissues. Understanding the interactions of nanoplastics in fish gill cells and how these interactions influence bioenergetics is essential. In particular, the mitochondria are vulnerable due to their critical role in energy production, lipid metabolism, redox regulation, apoptosis, and much more. Surface charge is a key determinant in nano‐bio interactions, mediating mechanisms of toxicity by influencing uptake and subsequent subcellular organelle responses. In our previous study, we found a possible mode of action governing the phenotypic response in positively charged nanoparticles specific to the mitochondria. In this study, we investigated the bioenergetic and morphological effects of surface functionalized polystyrene nanoplastics (PS NPs)—amine‐functionalized (PS‐NH 2 ) and carboxyl‐modified (PS‐COOH) PS NPs—in rainbow trout gill epithelia (RTgill‐W1). Using the Seahorse XF Mito Stress Test and high‐content imaging, we demonstrated that PS‐NH 2 NPs, which are positively charged, caused dose‐dependent declines in basal and maximal oxygen consumption rates (OCRs), increased proton leak, and reduced ATP‐linked respiration, indicating disruption of mitochondrial respiration. Conversely, PS‐COOH NPs exhibited minimal effects on mitochondrial function. Extracellular acidification rate (ECAR) analysis revealed impaired glycolytic compensation in sublethal PS‐NH 2 ‐treated cells. Phenotypic profiling demonstrated that morphological data distinguished treatment groups by distinct clusters based on similarity and revealed a more comprehensive understanding of nano‐induced cellular stress than bioenergetics measurements alone. Future nanotoxicological studies should implement multiparametric approaches to expand beyond traditional endpoints, including the use of other environmentally relevant nanoplastics in aquatic organisms.