The potential environmental risks of silver nanoparticles (AgNPs) require advanced toxicological studies that elucidate the intrinsic and extrinsic cellular responses in organisms, triggered by nanoparticle (NP) exposure. As part of our ongoing efforts to confirm the utility of continuously cultured embryonic zebrafish cells (ZF4) as an in vitro aquatic model for nanotoxicology, we evaluated the molecular mechanisms of cytotoxicity and the cellular repair mechanisms triggered after exposure to three AgNP sizes (10 nm, 30 nm and 100 nm) and ionic Ag (as AgNO₃) under serum conditions. The results demonstrated the crucial role of the adsorbed protein corona in reducing AgNP cytotoxicity and the time dependent AgNP internalisation. At 2 hours, the NPs were likely to be attached to the cell membranes as part of the first NP-cell encounter, whereas after 24 hours AgNPs were found in lysosomes and in close proximity to the nucleus. The cytotoxicity of PVP-coated AgNPs was size-related, as smaller (10 nm) AgNPs and ionic silver displayed major induction of all evaluated responses compared to the 30 nm and 100 nm AgNPs. All treatments demonstrated overgeneration of reactive oxygen species (ROS) and disruption of the intracellular Ca²⁺ balance. Cells were able to activate defence mechanisms in response to the induced damage, such as cell cycle arrest which prevented cells reaching the S phase, thereby providing time to repair DNA damage. The smaller AgNPs and the ionic control triggered massive cell cycle arrest, high percentages of DNA breaks and cell death, while exposure to the 100 nm AgNPs led to activation of G1 and G2 phases suggesting that ZF4 cells can overcome the damage. In addition, we evaluated the sequence of molecular events that lead to the toxic mode of action of the AgNPs in cells, supporting the establishment of adverse outcome pathways (AOP) and the 3Rs framework for the reduction of animal experimentation.