Harmonizing nanomaterial exposure methodologies in ecotoxicology: the effects of two innovative nanoclays in the freshwater microalgae Raphidocelis subcapitata .

Layered double hydroxides (LDHs) are innovative nanomaterials (NMs) with a typical nanoclay structure (height <40 nm) consisting of layers of metallic cations and hydroxides stabilized by anions and water molecules. Upon specific triggers, anions can exchange by others in the surrounding environment. Due to this stimuli-responsive behavior, LDHs are used as carriers of active ingredients in the industrial or pharmaceutical sectors. Available technical guidelines to evaluate the ecotoxicity of conventional substances do not account for the specificities of NMs, leading to inaccuracies and uncertainty. The present study aimed to assess two different exposure methodologies (serial dilutions of the stock dispersion vs. direct addition of NM powder to each concentration) on the ecotoxicological profile of different powder grain sizes of Zn-Al LDH-NO 3 and Cu-Al LDH-NO 3 (bulk, <25, 25-63, 63-125, 125-250, and >250 µm) in the growth of the freshwater microalgae Raphidocelis subcapitata . Results revealed that the serial dilutions methodology was preferable for Zn-Al LDH-NO 3 , whereas for Cu-Al LDH-NO 3 both methodologies were suitable. Thus, the serial dilutions methodology was selected to assess the ecotoxicity of different grain sizes for both LDHs. All Zn-Al LDH-NO 3 grain sizes yielded similar toxicity, while Cu-Al LDH-NO 3 powders with smaller grain sizes caused a higher effect on microalgae growth; thus, grain size separation might be advantageous for future applications of Cu-Al LDH-NO 3 s. Considering the differences between exposure methodologies for the Zn-Al LDH-NO 3 , further research involving other NMs and species must be carried out to achieve harmonization and validation for inter-laboratory comparison.