Heteroaggregation with microalgae masks charge-dependent accumulation but amplifies charge-dependent toxicity of...

In natural waters, nanoplastics (NPLs) often form heteroaggregates (HAs) with microalgae. Despite surface charge being a primary driver of NPL behavior, its role in governing HA toxicokinetics and impacts in bivalves remains largely unknown. Here, the effects of positively charged polystyrene NPLs (NPL+), their negative charged counterparts (NPL-), and the pre-formed HAs (HA+ and HA-) with the microalga Chlorella salina were evaluated in the green mussel (Perna viridis) during a 21-day exposure followed by 7-day elimination. HA+ and HA- enhanced NPL uptake in digestive gland and peripheral tissues, with uptake rate constants 1.5 - 4.0 times those of NPLs alone. Although 70 - 95% of accumulated NPLs were eliminated within 24 h, higher initial burdens under HA+ and HA- produced greater residual levels. NPL+ showed stronger uptake than NPL- due to greater electrostatic affinity with negatively charged epithelial surfaces. However, this charge-related uptake advantage was attenuated in HAs, concurrent with algal-like ζ-potentials of the heteroaggregates. Biological responses paralleled toxicokinetics, with HA+ inducing the strongest effects, including hemocyte dysfunction, oxidative stress, and fungal community disruption. Overall, HAs and surface charge jointly regulated NPL accumulation and toxicity in P. viridis, highlighting the need for ecological risk assessments to consider charge effects and HA formation to avoid underestimating NPLs hazards in filter-feeding bivalves.