3.1.2. Pigment from Liquid Culture

Pigments from liquid cultures were associated with high rates of mortality, with all pigments across test conditions resulting in mortality significantly different than the control by 120 hpf (Figure 2). *Scytalidium cuboideum* liquid culture pigment was especially lethal, with all embryos dead at 24 h in every test condition except SPE purified pigment (Figure 2). This was a contrast compared to DCM-extracted pigments, which did not show the same consistent toxicity. The differences in toxicity seen between the liquid cultures and the solid cultures are likely explained by variation in differential metabolite production. Variation in growth conditions is known to alter the secondary metabolite production of fungi generally [67–69], and variation in metabolite production in spalting fungi specifically [70]. Increased co-production of bioactive compounds in the liquid cultures compared to that

in the wood chip plates is likely, especially as pigmented metabolite production is often seen earlier in the lab in liquid compared to solid cultures. In addition to this effect, differences between liquid cultures and DCM extracted pigment were likely influenced by the differences in pigment concentration, the presence of growth media in liquid cultures, and the likelihood that DCM extract contains fewer products of fungal metabolism due to its polarity limiting transfer of compounds.

At 24 hpf there were differences between fungal species, with *Chlorociboria* species showing no significant toxicity in live, sterilized, or filtered media. However, *C. aeruginascens* showed relatively high sublethal effects at 24 hpf despite the lack of significant mortality (Figure S2). This variation in the production of secondary metabolites in addition to target pigment was also observed in DCM-extracted cultures, while both *Chlorociboria* spp. showed high levels of toxicity in DCM- and liquid media solutions. This suggests that other fungal metabolites may have been responsible for the effects seen, especially as toxicity was also lower in SPE-purified samples compared to liquid media and there appeared to be variation in toxicity between species. *Chlorociboria aeruginosa* showed lower percent mortality than *C. aeruginascens* in multiple tests. Notably, in live media testing at 120 hpf, *C. aeruginosa* showed only half the mortality of *C. aeruginascens*, and in sterilized media at 24 hpf it showed no toxicity while *C. aeruginascens* had 100% deformities. Variation in pigment production between the two species has been seen in other studies [70,71], including variation in the production of a yellow pigment in addition to differential production of xylindein [54].

SPE purification resulted in no mortality seen at 24 hpf, though by 120 hpf there was significant mortality for all tested pigments (Figure 2) and high levels of total sublethal effects. Deformations observed included pericardial edema, yolk sac edema, trunk, axis, and craniofacial malformations.
