Polyethylene terephthalate (PET) nanoplastics target carbonic anhydrase and alter physiological functions in Daphnia magna

Polyethylene terephthalate (PET) nanoplastics are increasingly recognized as relevant contaminants in freshwater ecosystems, yet the molecular mechanisms underlying their sublethal biological effects remain poorly understood. The aim of this study was to investigate whether true-to-life polyethylene terephthalate (PET) nanoplastics, generated from post-consumer PET bottles, affect carbonic anhydrase (CA) activity in Daphnia magna and whether such molecular alteration is associated with physiological dysfunction at the organism level. Label-free PET nanoplastics were generated from post-consumer bottles through a solvent-free mechanical fragmentation process designed to mimic environmental degradation and were characterized by a bimodal size distribution, with peaks centered at 185 and 538 nm. Using a pharmacological loss-of-function approach with acetazolamide, we first demonstrated that CA contributes to hemolymph pH regulation, gastrointestinal acidification, feeding activity, heart rate, and appendage movement in D. magna. In vitro exposure of whole-body homogenates to PET nanoplastics (0.5-5 mg/L) caused a clear dose-dependent inhibition of CA esterase activity, reaching about 54% at 5 mg/L. Short-term in vivo exposure (24 h) confirmed significant CA inhibition, with reductions of up to about 33%, and was accompanied by parallel decreases in heart rate and thoracic appendage beating frequency. By integrating particle characterization with in vitro and in vivo evidence, this study provides mechanistic support for CA-mediated disruption as a relevant pathway of PET nanoplastic toxicity in freshwater zooplankton. Our findings highlight CA as a promising mechanistic biomarker for nanoplastic exposure and contribute to the ecotoxicological assessment of PET nanoplastics in freshwater environments.