Concerns about possible environmental implications of nano- and micro-plastics are continuously raising. Hence, comprehensive understanding of their behaviour, bioaccumulation and toxicity potential is required. Nevertheless, systematic studies on their fate and possible effects in freshwaters, as well as the influence of particle-specific and environmental factors on their behaviour and impacts are still missing. The aims of the present study are thus two-fold: (i) to examine the role of the surface charge on nanoplastic stability and acute effects to freshwater zooplankton; (ii) to decipher the influence of the refractory natural organic matter (NOM) on the nanoplastic fate and effects. Amidine and carboxyl-stabilized polystyrene (PS) spheres of 200 nm diameter characterized by opposite primary surface charges and neutral buoyancy were selected as model nanoplastics. The results demonstrated that the surface functionalization of the polystyrene nanoplastics controls their aggregation behaviour. Alginate or Suwannee River humic acid (SRHA) modified significantly the surface charge of positively-charged amidine PS nanoplastic and the aggregation state, while had no significant influence on the negatively-charged carboxyl PS nanoplastic. Both amidine and carboxyl PS nanoplastics were ingested by the zooplankton and concentrated mainly in the gut of water flea Daphnia magna and larvae Thamnocephalus platyurus, and the stomach of rotifer Brachionus calyciflorus. Amidine PS nanoplastic was more toxic than carboxyl one. The toxicity decreased in the order D. magna (48 h -immobilization) > B. calyciflorus (24 h - lethality) > T. platyurus (24 h - lethality). Alginate or SRHA reduced significantly the toxicity of both amidine and carboxyl PS nanoplastics to the studied zooplankton representatives. The implications of this laboratory study findings to natural environment were discussed.