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The increasing application of silver nanoparticles (nAg) in various consumer products has raised concerns regarding toxicological impacts in the environment. It is unclear at present whether the toxicity of nAg is mainly the result of the release of ionic Ag(+) in mussels. The freshwater mussel Elliptio complanata was exposed to increasing concentrations of 20-nm nAg, 80-nm nAg, and dissolved Ag(+) for 48 h at 15°C. The following biomarkers were used to determine the mode of action of nAg-induced adverse effects: metallothioneins (MT) (ionic Ag(+) release), lipid peroxidation (LPO) (ionic Ag(+) and nanosurface interactions), heat-shock proteins (HSP) (size-related effects), protein-ubiquitin levels (size-related effects), and DNA strand breaks (ionic Ag(+) and size effects). Results revealed that the response pattern of 80 nm nAg was more closely related to ionic Ag(+) than 20 nm nAg, suggesting a more important release of dissolved Ag from 80 nm nAg. Data showed that all forms of Ag were able to increase the levels of MT and LPO, which suggests the presence of ionic Ag(+) leads to oxidative stress. However, nanoparticles were also able to induce changes in protein-ubiquitin and to a lesser extent actinomyosin-ATPase, MT, and DNA strand breaks in the digestive gland in a manner different from Ag(+), which permitted discrimination of the forms of Ag. Moreover, LPO was closely associated with DNA strand breaks in the digestive gland and was not entirely explained by induction of MT, suggesting another type of toxic interaction. It was concluded that the presence of nAg not only increases the toxic loadings of released Ag ions but also generates other and perhaps cumulative effects of nanoparticle-induced toxicity related to size and surface properties.

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