*2.4. AgNPs Ecosafety Tests*

The intrinsic toxicity of AgNPs was investigated through standardized 72 h algal growth inhibition tests [62,63] using two di fferent algal species, belonging to the freshwater and to the marine water environment, *Raphidocelis subcapitata* and *Phaeodactylum tricornutum*. Algae were cultured, respectively, in TG 201 medium and F/2 medium, in axenic and exponential growth conditions, at 18 ± 1 ◦C and 16/8 h light-dark cycle photoperiod. In order to reduce the introduction of chelating agents which have been shown to interfere with heavy metals toxicity [64,65], algal toxicity tests were run with the same medium used for algal culturing modified only in the concentration of ethylenediaminetetraacetic acid (EDTA) (0.05 mg/<sup>L</sup> and 0.8 mg/<sup>L</sup> respectively as previously proved to ensure acceptable algal growth). PS single-use sterile multiwell were used for toxicity tests, with 2 mL volume capacity for each well. Algae were acclimated 72 h before running the test under the following conditions: 22 ± 2 ◦C and 16/8 light-dark photoperiod. Initial algal concentration was 1 × 10<sup>4</sup> cells/mL and tests were manually aerated eVery 24 h using a pipette with sterile tips. Exposure concentrations were as follows: 10, 25, 50, 100, 200, 500 μg AgNPs/L. AgNO3 was used as positive control at the following concentrations of 3.5, 7, 14, 21, 35 μg/L. In order to exclude any possible role of coating components the toxicity of the AgNPs, citrate and L-Cysteine were tested separately at the following concentrations of 0.5, 1, 2, 5, 10 mg/L.
