4.4.2. Comet Assay

Damage to DNA was evaluated using an adaptation of the alkaline single-cell gel electrophoresis (Comet) assay, developed by Singh et al. [33], adapted by Raimundo et al. [34] for molluscan solid tissue. Freshly harvested gill samples were minced with pliers in 700 mL of cold PBS and centrifuged for 1 min at 1200 rpm. The supernatant (clear cell suspension) was diluted in 1% *m*/*v* molten (37–40 ◦C) low melting point agarose (LMPA) prepared in PBS. Afterwards, two 80-μL drops of LMPA cell suspensions were placed on slides pre-coated with 1.2% *m*/*v* of normal melting point agarose (dried for at least 48 h) and covered with a coverslip. After LMPA solidification (15 min, 4 ◦C) coverslips were removed and the slides immersed in cold lysis buffer (0.45 M NaCl (*m*/*v*); 40 mM EDTA (*m*/*v*); 5 mM Tris pH 10) for 1 h. Slides were then placed in cold electrophoresis buffer (0.1 mM EDTA; 0.3 M NaOH, pH 13) for 40 min to allow DNA-unwinding and expression of alkali-labile sites. Electrophoresis was run at 25 V for 30 min at 4 ◦C. Then, the slides were neutralized in 0.2 M Tris-HCl, pH 7.5, and dried with methanol for archiving before analysis. Rehydrated slides were stained with GreenSafe (Nzytech, Portugal) [35] and analyzed with a DM 2500 LED microscope adapted for epifluorescence with an EL 6000-light source (Leica Microsystems). Scoring was done with CometScore 1.6 (Tritek), with 100 nucleoids being analyzed per slide. The percentage of DNA in tail was considered as a direct measure of DNA damage [36].

### 4.4.3. Acute Toxicity Assay with *Daphnia pulex*

Based on standard guidelines for toxicity testing with *Daphnia* sp. [37–39], daphnids with less than 24 h were selected for the assay. For the purpose, several females with mature eggs in the brood pouch were transferred to a 90-mm Petri dish containing with distilled water 12 h before the experiments. Hatched juveniles (daphnids) were afterwards collected and used for the tests. The test replicate consisted of a well of a 24-well clear bottom microplate (2-mL well) containing 20 daphnids. Each well contained 0.2 mL of extract and 1.8 mL of distilled water. Plates were then exposed under dark (D) of light (L) conditions, for 1 h, to dilutions D1, D2, or D3 of P and S pigment extracts, plus controls, which consisted of adding 0.2 mL of PBS to the test water (*n* = 6) and afterwards incubated in unchanged medium, in the dark, until analysis. The number of immobilized daphnids was evaluated after 24 and 48 h from the beginning of the exposure.
