In the nanoparticle concentration effect experiment, four different Ag-EN concentrations (1.85, 9.27, 27.8, 92.7 µM) were used.
In the lowest GSH concentration treatments, the cell growth was inhibited by 22.5%, 39.8%, and 100% when the Ag-EN concentrations were 139.1, 185.4, and 278.1 µM, respectively, as is consistent with the first toxicity experiment.
To further test whether the toxicity of Ag-ENs we observed could be explained by either of the latter two mechanisms described above, the three highest Ag-EN concentrations in the first toxicty experiment were chosen together with the control treatment.
Furthermore, Ag-EN concentration in the 1 35 nm fraction also decreased abruptly at this high GSH concentration (Fig. 3).
Significant toxicity (p<0.05) to the freshwater alga O. danica was observed in the higher Ag-EN concentration treatments.
Therefore, the different [Ag+]T obtained in the different Ag-EN concentration treatments may also be explained by the surface-area-limited release of Ag+.
However, significantly more Ag (p<0.05) were distributed in the <1 nm fraction while less Ag-ENs were distributed in the 1 35 nm fraction for the lower Ag-EN concentration treatments (Fig. 2).
The Ag+ depletion in the media could be quickly replenished in the two highest Ag-EN concentration treatments, which would be, however, not the case when its concentration is low.
Cell growth was inhibited by 18.8%, 40.3%, and 100% when Ag-EN concentration was 139.1, 185.4, and 278.1 µM, respectively, while their [Ag+]F was kept relatively constant (1.31 1.89 pM).
For this purpose, three different concentrations of GSH, 83.3, 249.8, and 416.3 µM, were used and there were four different Ag-EN concentration treatments (i.e., 0, 185.4, 185.4, and 278.1 µM Ag-ENs) for each GSH concentration group resulting in a total of twelve treatments.
The observation that a higher percentage of Ag-ENs was dissolved at lower nanoparticle concentrations was also found by Laban et al. [9], which can be ascribed to the more substantial difference in Ag-EN concentration than that of [Ag+]T between the different nanoparticle concentration treatments.
