**4. Conclusions**

Pigments from spalting fungi show varying levels of toxicity to zebrafish embryos across species and growth/extraction methodology. The red pigment dramada from *S. cuboideum* was the only solidified and pure pigment associated with significant toxicity in zebrafish, though with a relatively high LD50 value, making it unlikely to affect humans. Solidified and washed xylindein did not demonstrate toxicity. However, significant mortality was associated with impure solutions of fungal metabolites containing pigments from all tested species. This suggests the co-production of mycotoxins or toxicity related to media components. The development of improved purification methodologies, especially for xylindein from *Chlorociboria* species, is therefore of paramount importance for future industrial adoption. However, the low levels of toxicity seen in the solidified xylindein are sufficient to suggest that future technologies are likely to be both sustainable and environmentally safe. The adoption of these sustainably produced pigments has the potential to replace conventional technologies currently associated with toxicity, allowing for a greener future.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2309-608 X/7/2/155/s1, Figure S1: Percent of Embryos Showing Sublethal Effects After Exposure to DCM-Extracted Pigments from Fungi at 24 and 120 hpf, Figure S2: Percent of Embryos Showing Sublethal Effects After Exposure to Pigments from Fungi Grown in Liquid Media at 24 and 120 hpf.

**Author Contributions:** Conceptualization, B.H.A. and S.C.R.; methodology, B.H.A., S.M.V.G., S.H. and B.H.; validation, S.H., B.H.; formal analysis, B.H.A.; investigation, B.H.A. and S.M.V.G.; resources, S.H. and S.C.R.; writing—original draft preparation, R.C.V.C.; writing—review and editing, S.C.R., S.H.; visualization, R.C.V.C.; supervision, S.C.R.; project administration, S.C.R.; funding acquisition, S.C.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Oregon BEST Innovation Grant (not Vertue Lab) for partial funding of this research. This work was also supported by the USDA National Institute of Food and Agriculture, McIntire Stennis project number 1009811 (SR) and partially supported by NIH grants ES017552 and ES016896 (SH).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by approved by the Institutional Animal Care and Use Committee (IACUC) at Oregon State University (ACUP 5113).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank the Tanguay Lab at Sinnhuber Aquatic Research Laboratory at Oregon State University for carrying out the experiment and analysis described in the Solid Pigments and Behavior Response Testing. The authors would also like to thank Ariel Muldoon (College of Forestry, Oregon State University) for help with statistical analysis review.

**Conflicts of Interest:** The authors declare no conflict of interest.
