In aqueous solutions, silver nanoparticle (AgNP) behavior is affected by a variety of factors which lead to altered AgNP size and toxicity. Our research aims to explore the effect of media composition on citrate-coated AgNP (cit-AgNP) behavior and toxicity to the cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. Three different exposure media (L15/ex, L15/ex w/o Cl and d-L15/ex) were used. These were characterized by varying ionic strength and chloride content, both of which had a dominant effect on the behaviour of cit-AgNP. Comparing the behaviour and toxicity of cit-AgNP in the different media, stronger agglomeration of cit-AgNP correlated with higher toxicity. Deposition of cit-AgNP on cells might explain the higher toxicity of agglomerated cit-AgNP compared to that of suspended cit-AgNP. The cit-AgNP concentration-response curves as a function of dissolved silver ions, and the limited prevention of toxicity by silver ligands, indicated that cit-AgNP elicited a particle-specific effect on the cells. Furthermore, the lysosomal membrane integrity was significantly more sensitive to cit-AgNP exposure than cellular metabolic activity or cell membrane integrity and showed the weakest protection by silver ligands. This revealed that cit-AgNP toxicity seems to particularly act on RTgill-W1 cell lysosomes. The newly developed low ionic strength medium, d-L15/ex, which can stabilize cit-AgNP and better mimic the freshwater environment, offers an excellent exposure solution to study cellular and molecular effects of NP to gill cells.