*2.2. Zebrafish Preparation and Exposure for Pigment Extracts*

All experiments were performed in compliance with national care and use guidelines, and were approved by the Institutional Animal Care and Use Committee (IACUC) at Oregon State University (ACUP 5113). Adult wild-type D5 zebrafish (*Danio rerio*) embryos were raised at the Sinnhuber Aquatic Research Laboratory (SARL) at Oregon State University (Corvallis, Oregon, USA). Fish were maintained in fish water, consisting of reverse osmosis water supplemented with 0.3 g/l Instant Ocean salts (Aquatic Ecosystems, Apopka, FL) with pH adjusted with sodium bicarbonate to pH 7 ± 0.2, with a temperature of 28 ◦ C and a 14 h light to 10 h dark photoperiod. After group spawn and egg collection, an Olympus-SZ51 stereomicroscope was used to select and remove the abnormal and non-fertilized eggs. Six hours post-fertilization (hpf), all normal embryos were dechorionated to ensure contact with test materials [60]. Embryos were placed into a 60 mm glass petri dish with 25 mL fish water and exposed to 50 μL of 50 mg/mL pronase enzyme (Sigma-Aldrich, cat # 81750, St. Louis, MO, USA) to degrade the outer chorionic layer. After chorion deflation (~7 min) solution was diluted with fresh fish water and recovered in a petri dish at room temperature until 8 hpf, when waterborne exposure testing was carried out. At this time, each embryo was placed in its own well in a prepared 96-well plate containing fish water and tested pigment condition. The embryos were incubated at 28 ◦C for 24 hfp for the first assessment.
