2.3.2. Analytical Measurements

For OFL analysis at mg <sup>L</sup>−1, a UV-vis UVmini-1240 spectrophotometer (Shimadzu Corporation) was used. The instrument was set at 287 nm, corresponding to the maximum OFL absorption. Calibration in the range of 1–10 mg L−<sup>1</sup> yielded optimal linearity (R<sup>2</sup> > 0.9988). The quantification limit was 0.8 mg L−1.

HPLC system consisting of a pump Series 200 (Perkin Elmer, Milano, Italy) equipped with a vacuum degasser and a programmable fluorescence detector (FD) was used for OFL analysis at μg L−1. The fluorescence excitation/emission wavelengths selected were 280/450 nm. Fifty μL of each sample were filtered (0.22 μm nylon syringe filter) and injected into a 250 × 4.6 mm, 5 μm Ascentis RPAmide (Supelco-Merck Life Science, Milano, Italy) coupled with a similar guard-column. The mobile phase was 25 mM H3PO4—ACN (85:15), and the flow rate 1 mL min−1. Calibration in the range 1–20 μg L−<sup>1</sup> yielded optimal linearity (R<sup>2</sup> > 0.9988). The quantification limit was 0.9 μg L−1.

### *2.4. Acute Toxicity Tests with Daphnia magna*

The potential acute toxicity of the different materials, i.e., HNT, Fe3O4-C, and HNT/Fe3O4- C, was tested on the freshwater Cladoceran *Daphnia magna* according to the *Daphnia* sp. Acute Immobilization Test, OECD 202 guideline (OECD, 2004). Adult *Daphnia magna* individuals were cultured (30 individuals/L) in a commercial mineral water (San Benedetto®) under controlled laboratory conditions reported elsewhere [42]. Five replicates containing ten daphnids (i.e., <24 h old individuals) each were performed per each experimental condition, including control. In detail, daphnids were exposed for 48 h at 20 ± 0.5 ◦C and 16 h light: 8 h dark photoperiod under static, non-renewal conditions to 0.2 g L−<sup>1</sup> of the materials. A single concentration mimicking the amount of residues in waters after depollution treatment was tested. This concentration reflected the amount of each material used in the experiments aimed at investigating their capability in the removal of OFL. The viability of individuals was tested after 24 and 48 h of exposure. Individuals were considered dead when they did not swim for over 15 s after a slight stirring of the solutions. After checking for viability, all the individuals were observed under a Leica Microsystem EZ4 Stereoscopic microscope to check for the ingestion of materials by daphnids.
