While the importance of nanoparticle (NP) characterization under relevant test conditions is widely recognized in nanotoxicology, few studies monitor NPs behavior in the presence of exposed organisms. Here we studied the behavior of nine types of silver nanoparticles (AgNPs) during the 48 h algal toxicity test. In particular, we investigated NP aggregation and dissolution by time-resolved inductively coupled plasma mass spectrometry and ultrafiltration and performed mass balance measurements to study the distribution of Ag in the test system. We also determined the amount of extra- and intracellular Ag by chemically etching AgNPs on the surface of algal cells and used dark field microscopy for their imaging. We observed that positively charged branched polyethilenimine (bPEI)-coated AgNPs tend to aggregate in the presence of algae and interact with test vessels and algal cells, while citrate-coated AgNPs have a tendency to dissolve. On the other hand, with large variation of half-maximum effective concentration (EC50) across tested NPs (5.4 to 300 ngAg mL−1), Ag internalized by the algal cells at EC50 was similar (0.8 to 3.6 ngAg mL−1) for all AgNP types. These data show that while sorption to the vessels, dissolution, and aggregation impact on the distribution of AgNPs in the test system and on interactions with algal cells, AgNP toxicity is strongly correlated with the NP-cell surface interaction and internalization of Ag.