2.2.2. Pigment from Liquid Culture

Three liquid culture solutions (live, autoclaved, and filtered, as described above) from each fungal species and SPE column purified pigment from *Chlorociboria* spp. and *S. cuboideum* were tested. These forms were tested in order to compare the toxicity of the extracted pigment solution to the full panel of compounds present in fungal cultures used to produce the pigments. For each, 100 μL of pigment solution was transferred into a Falcon sterile 96-well plate and fish water was added to give a final working volume of 250 μL, with controls of fresh fish water and sterile liquid malt extract. At 8 hpf, a dechorionated embryo was transferred into each individual well of the 96-well plate using a VWR disposable wide-bore glass pipette. The plates were incubated at 28 ◦C until 24 hpf, then assessed as described below.

First, pigmented liquid media taken directly from fungal cultures were applied to 12 embryos per pigment. This was then repeated using media autoclaved at 120 ◦C for 30 min. Next, pigment solutions were filtered to remove fungal cells from the medium using 0.2 μm EMD Millipore filters. This experiment was run twice, with 24 embryos per test condition each time. Finally, green pigment from *C. aeruginosa* and *C. aeruginascens* and red pigment from *S. cuboideum* collected from liquid media samples and purified using SPE columns (as described in Section 2.1.2) were tested on 72 embryos each.
