*3.4. AgNPs Ecosafety*

Algal toxicity tests with *R. subcapitata* and *P. tricornutum* exposed to AgNPs showed no inhibition of growth rate at all tested concentrations, as reported in Figure 5. Furthermore, algae showed a slight increase in growth rate compared to the control (negative growth rate inhibition), at almost all tested concentrations. Such increase, however, was not observed by positive control tests carried out with Cit and L-cys, which showed no e ffect on algal growth.

Reference toxicant (AgNO3) showed a 60% inhibition of growth rate compared to the control of *R. subcapitata* (freshwater), already at the lowest concentration tested (3.5 μg AgNO3/L), confirming the toxicity of Ag for microalgae. On the opposite, no e ffect on growth was observed for *P. tricornutum* (marine water) exposed in the same conditions, confirming the of drop in free Ag+ ions in solution due to complexation with Cl− ions in seawater as measured by Gunsolus et al. [73].

The absence toxicity for our AgNPs confirms chemical analysis results showing insu fficient release of Ag+ ions to exert a toxic e ffect on both microalgae (Table 1). Such findings further validate the hypothesis by which AgNP toxicity is closely bound to the release of Ag+ ions [44,46,47,54]. In fact, the dissolution of Ag+ from AgNPs, either pristine or coated (chitosan, lactate, polyvinylpyrrolidone, polyethelene glycol, gelatin, sodium dodecylbenzenesulfonate, Cit, dexpanthenol, carbonate), has been reported by many studies [44–48,52,54,80,81] and recognized to be linked to the observed toxicity to

microalgae [44,46,47,54]. The covalently-bound L-cys coating of AgNPs might prevent the dissolution of Ag+ ions, since previous studies revealed that Cit coating was not able to avoid such release in aqueous media [44,46,47,73].

**Figure 5.** Percentage of growth rate inhibition compared to control of *R. subcapitata* and *P. tricornutum* exposed to AgNPs (10, 25, 50, 100, 200, 500 μg/L) for 72 h. Data are shown as mean ± standard deviation.

Molecules with a high reduced sulphur content are known to bind metal ions; L-cys, thanks to the presence of a thiol group, is able to act as a strong Ag+ ligand [82]. Thiol groups could act both either binding Ag+ ions, blocking their release in the media, or by excluding oxidizing agents to come in contact with the particles surface, preventing their dissolution and consequent release of Ag+ ions in the medium [83]. Gunsolus et al., [73] reported a significant reduction of Ag+ ions release and of bactericidal activity of citrate-AgNPs upon incubation with natural organic matter rich in thiol groups. Some studies observed a reduction in the toxic effect of Ag-releasing AgNPs after addition of L-cys in solution, probably as a consequence of L-cys complexation of free Ag+ ions [45–47].

However, information on L-cys effects on AgNPs stability and dissolution are conflicting. The presence of free L-cys in solution might also enhance Ag+ ions release from AgNPs [84]. In our study, however, the absence of toxicity to microalgae and low Ag levels in exposure media confirmed that Cit/L-cys coating prevents the release Ag+ ions from the NP. Similar results were obtained by Mu et al. [85] for graphene oxide (GO) nanosheets with covalently-bound L-cys, compared to pristine GO nanosheets. In terms of toxicity, for particles smaller than 20 nm an additional dissolution-independent effect seems to play a role [44,53]. However, despite being very small, AgNPs further confirm their integrity in terms of low Ag+ release. Since the main concern regarding the environmental application of AgNP is due to potential toxic effect to non-target aquatic species [44], our findings clearly showed the ecosafety of AgNPs and promote their use in the aquatic environment.
