The massive production and use of silver nanoparticles (Ag NPs) have led to their increasing release into the environment. Even though the antimicrobial and cytotoxic effects of native nanoparticles have been well studied, the environmental impacts of transformation products such as silver sulfide nanoparticles (Ag2S NPs) have not been elucidated. In the present study, we assessed the toxicity of Ag2S NPs and silver nitrate (AgNO3), as a source of Ag, to the earthworm Eisenia andrei using a nominal concentration of 5 mg Ag kg-1 soil. We used the OECD guidelines to assess effects on weight loss and mortality for 14 days. After exposure, we also extracted the immune effector cells (coelomocytes) and conducted a battery of biomarker tests. To ensure the quality of the toxicological results, the structural changes of NPs during the experiment and the uptake of silver by the earthworms were monitored. During the experiment, mortality effects were not detected, but a weight loss was observed in the earthworms exposed to Ag2S NPs. Altough Ag2S NPs were engulfed by E. andrei cells, neither phenoloxidase activity nor lipid peroxidation differed from the untreated control group. Cells from earthworms treated with Ag2S NPs exerted very broad value range of nitric oxide (NO) generation, suggesting an imbalance in the NO metabolism. Overall, this study suggests minimal risks associated with Ag2S NPs exposure to earthworms. However, further studies are needed to assure no immunotoxicological or chronic effects on a wider range of terrestrial organisms.

Applied Nanoparticles S L C Alaba 88 08018 Barcelona Spain

Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia c Catedrático José Beltrán 2 46980 Paterna Valencia Spain; IMDEA Water Institute Science and Technology Campus of the University of Alcalá Punto Com 2 28805 Alcalá de Henares Madrid Spain

Department of Modern Immunotherapy Institute of Hematology and Blood Transfusion U Nemocnice 2094 128 20 Prague 1 Czech Republic

Institute of Hydrodynamics of the Czech Academy of Sciences Pod Patankou 30 5 166 12 Prague 6 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 00 Prague 4 Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 00 Prague 4 Czech Republic; Cavanilles Institute of Biodiversity and Evolutionary Biology University of Valencia c Catedrático José Beltrán 2 46980 Paterna Valencia Spain

Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 00 Prague 4 Czech Republic; Institute for Environmental Studies Faculty of Science Charles University Benatska 2 128 01 Prague 2 Czech Republic

Regional Centre of Advanced Technologies and Materials Czech Advanced Technology and Research Institute Palacký University Šlechtitelů 27 783 71 Olomouc Czech Republic

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