3.1.1. Pigment from Solvent Extraction (No Purification)

Dichloromethane-extracted pigment resulted in significantly more mortality than the control across a number of test conditions (Figure 2), including tested pigments, exposure concentrations, and fungal growth substrate. Mortality results were also reflected in the number of embryos with sublethal effects, presented in Figure S1. Deformations seen included pericardial edema, yolk sac edema, reduced total length, and axis problems, with deflated swim bladder and bent tail/trunk seen in 400× standard concentration.

**Figure 2.** Percent Embryo Death After Exposure to Dichloromethane Extracted Pigments from Fungi at 24 and 120 hpf. Asterisks denote significant difference from control, with all significant values having a *p*-value of <0.0001 as determined by Fisher's exact test. Zeros indicate there was no fish mortality in that condition. DCM-extracted pigment is shown to result in significantly more mortality than control across a number of test conditions, with higher embryo death rates after longer exposure. Aspen media resulted in less mortality than maple media, and higher concentrations of maple media resulted in more deaths for pigments from most fungal species.

> The toxicity of pigments varied based on if fungi were grown in wood chips amended with either maple or aspen. Pigments from fungi grown in maple media resulted in

significantly more deaths at 24 hpf than the control for all fungal species, most notably in the case of *S. cuboideum* where exposure resulted in 100% mortality. In contrast, no death or deformity was seen in extracts from any fungi grown on aspen at 24 hpf, though by 120 hpf both species of *Chlorociboria* showed significant deaths and high levels of deformity (Figures 2 and 3). The two wood varieties have differing extractive profiles [65], with maple containing bioactive compounds such as resorcinol [66], which likely accounts for these differences. *Chlorociboria aeruginascens* resulted in complete mortality by 120 hpf in all tested conditions, with *C*. *aeruginosa* showing similar toxicity. Both species also showed high rates of sublethal effects, with deformations seen in all or nearly all embryos by 120 hpf (Supplemental Figure S1). This suggests that other bioactive secondary metabolites are likely produced by *Chlorociboria* species in amended plate cultures. Finally, *S. cuboideum* extract showed total mortality by 24 h at 100% concentration; however, it did not have significantly different mortality than the control at the same time under 200% exposure (*p* = 1.000) and did not show complete mortality by 120 hpf. This suggests that there may be variation in embryo response, leading to inconsistency in the lethality of pigment extract, though the eventual significance of embryo mortality indicates overall toxicity.

**Figure 3.** Percent Embryo Death After Exposure to Pigments from Fungi Grown in Liquid Media at 24 and 120 hpf. Asterisks denote significant differences from control at the α = 0.05 level, with all significant values having a *p*-value of <0.0001, apart from live media *C. aeruginosa* at 120 hpf which had a *p*-value of 0.0028. Zeros indicate there was no embryo mortality in that condition.
