Assessment of silver nanoparticle toxicity for common carp (Cyprinus carpio) fish embryos using a novel method controlling the agglomeration in the aquatic media

Formation of agglomerates and their rapid sedimentation during aquatic ecotoxicity testing of nanoparticles is a major issue with a crucial influence on the risk assessment of nanomaterials. The present work is aimed at developing and testing a new approach based on the periodic replacement of liquid media during an ecotoxicological experiment which enabled the efficient monitoring of exposure conditions. A verified mathematical model predicted the frequencies of media exchanges which checked for formation of agglomerates from silver nanoparticles AgNP with 50 nm average size of the original colloid. In the model experiments, embryos of common carp Cyprinus carpio were exposed repeatedly for 6 h to AgNPs (5-50 mu m Ag L-1) either under semistatic conditions (exchange of media after 6 h) or in variants with frequent media exchanges (varying from 20 to 300 min depending on the AgNP colloid concentration and the desired maximum agglomerate size of 200 or 400 nm). In contrast to other studies, where dissolved free metals are usually responsible for toxic effects, our 144-h experiments demonstrated the importance of AgNP agglomerates in the adverse effects of nanosilver. Direct adsorption of agglomerates on fish embryos locally increased Ag concentrations which resulted in pronounced toxicity particularly in variants with larger 400 nm agglomerates. The present study demonstrates the suitability of the novel methodology in controlling the conditions during aquatic nanomaterial toxicity testing. It further provided insights into the mechanisms underlying the effects of AgNP, which rank on a global scale among the most widely used nanomaterials.