This investigation applied novel techniques for characterizing and fractionating nanosilver particles and aggregates and relating these measurements to toxicological endpoints. The acute toxicity of eight nanosilver suspensions of varying primary particle sizes (10-80 nm) and coatings (citrate, polyvinylpyrrolidone, EDTA, proprietary) was assessed using three aquatic test organisms (Daphnia magna, Pimephales promelas, Pseudokirchneriella subcapitata). When 48-h lethal median concentrations (LC50) were expressed as total silver, both D. magna and P. promelas were significantly more sensitive to ionic silver (Ag+) as AgNO3 (mean LC50 = 1.2 and 6.3 mu g/L, respectively) relative to a wide range in LC50 values determined for the nanosilver suspensions (2-126 mu g/L). However, when LC50 values for nanosilver suspensions were expressed as fractionated nanosilver (Ag+ and/or <4 nm particles), determined by utiracentrifugation of particles and confirmed field-flow-fractograms, the LC50 values (0.3-5.6 mu g/L) were comparable to the values obtained for ionic Ag+ as AgNO3. These results suggest that dissolved Ag+ plays a critical role in acute toxicity and underscores the importance of characterizing dissolved fractions in nanometal suspensions.