*2.4. Toxicity Studies*

The concentrations of ELT suspensions tested in the acute assays were chosen based on both OECD guidelines and on results of the range-finding tests (performed at 1.0, 10.0 and 100.0 mg/L of ELT-dg and ELT-dp), in which we did not observe significant effects. For this reason, as defined by OECD guidelines, we performed the limit tests only at 100.0 mg/L. For the subsequent chronic tests, the concentrations were determined in accordance with the results of acute tests. The ecotoxicity methods followed in this study are described in detail in the OECD guidelines [47–50,52], and for this reason we report only a brief description of them below.

#### 2.4.1. Growth Inhibition Test on *P. subcapitata*

A chronic toxicity test was conducted on *P. subcapitata* with 72 h exposure under static conditions to 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L of both ELT-dg and ELT-dp suspensions [47]. We evaluated the percentage of cell growth rate and yield inhibition at 24, 48 and 72 h. The organisms were cultured in the facility of ChemService Controlli e Ricerche s.r.l. in a climatic chamber at 24 ± 2 ◦C, 4440–8880 lx in the spectral range of 400–700 nm and maintained under continuous shaking to guarantee both the cell suspension and the correct concentration of carbon dioxide (CO2). Only the algal cultures showing an exponential growth were used in the exposure, and the procedures were conducted under a laminar flow hood to avoid algal contamination. For the exposure, 100 mL capacity conical glass flasks with algal medium (see Supplementary Materials for composition) were used, with 3 replicates for each concentration and 6 replicates for the control. In each flask, 50 mL of test solution was poured and inoculated with the algae (10<sup>4</sup> cells/mL). Algal density was measured every 24 h by diluting an aliquot in 9 g/L sodium chloride (NaCl) solution and reading the algal cell amount with a cell counter (Beckman coulter Z2).
