Structure-dependent regulation of nanoplastic uptake by humic substances in a freshwater protozoan

Micro- and nanoplastic (MNP) pollution has become a global concern due to its persistence and interactions with aquatic biota. However, the environmental mechanisms governing their cellular uptake under natural conditions remain poorly understood. Natural waters contain diverse humic substances (HS) spanning a wide range of molecular structures, aromaticity, and molecular weights, yet how such heterogeneity modulates MNP uptake has never been systematically assessed. Here, focusing on nanoplastics (NPs) as a highly bioavailable and biologically reactive fraction of MNPs, we investigated how six structurally distinct HS regulate NP uptake by the freshwater protozoan Tetrahymena thermophila. By integrating physiological assays, transcriptomics, and quartz crystal microbalance with dissipation monitoring, we demonstrate that HS markedly reduce NP internalization through coupled biological and physicochemical pathways, with uptake rate inhibition ranging from 5.6% to 98.2%. Biologically, HS exposure was associated with altered membrane dynamics, Ca²⁺ regulation, and cellular energy status, which may contribute to reduced phagocytic uptake. Physicochemically, several HS formed surface eco-coronas that imposed modest steric inhibition, whereas unbound HS appeared to make only a limited contribution. Correlation analyses further revealed that inhibition strength was positively associated with HS molecular weight and aromatic carbon content, suggesting structure-dependent differences in HS effects. These results identify HS as active regulators of NP-cell interactions rather than passive surface modifiers, and provide a mechanistic framework for incorporating natural organic matter heterogeneity into bioavailability assessments of NPs in aquatic systems.