2.2.1. Pigment from Solvent Extraction

The pigment bioactivity testing procedure using zebrafish embryos followed methods laid out in Truong, Harper and Tanguay [60]. Standardized pigments in DCM were placed in Zinser 96-well glass petri dishes and the solvent was allowed to evaporate under a fume hood for 24 h or until the DCM had evaporated completely, with 100 μL of each pigment extract used per well. At 8 hpf, 200 μL of fish water containing 0.3 g/L of aquatic salt was transferred with a VWR disposable wide-bore glass pipette in the Zinser 96-well glass petri dish, and one dechorionated embryo was added per well. These were then incubated at 28 ◦C until 24 hpf, then the appropriate assessments were performed as described below.

Extracted pigment toxicity was compared across multiple conditions for pigments from all tested fungal species. First, embryos were exposed to standardized pigments from fungi grown on aspen wood chip amended malt agar plates in one 96-well plate at 100% concentration (*n* = 12 for *Chlorociboria* species and *n* = 24 for *Scytalidium cuboideum*). Next, extracted pigment from fungi grown in maple amended wood chip plates at standard concentration was carried out in individual plates (*n* = 72 per pigment). Testing across concentrations was then carried out using only pigment extracts from amended maple wood chip plates. Each pigment extract was tested in separate 96-well plates using 72 embryos (*n* = 72 per pigment), with separate tests for 100%, 200%, and 400% concentrations. This range of concentrations was used to allow for comparison with previous publications using standardized pigment extract.
