**1. Introduction**

Spalting fungi are a specific group of wood decay fungi that have the ability to internally color wood [1]. The coloration that they cause can be classified into three types: bleaching, zone lines, and pigmentation. The first two types are produced mostly by white-rotting fungi. Pigmentation is caused by ascomycete fungi through the generation of secondary metabolites that cause coloration in wood. Known colors produced by these fungi include blue-green produced by *Chlorociboria* spp. [2–4], red from *Scytalidium cuboideum* (Sacc. and Ellis) Sigler and Kang [5], and yellow from *Scytalidium ganodermophthorum* Sigler and Kang [6], among others.

Beginning in the 15th century, wood stained blue-green by fungi from the genus *Chlorociboria* was a prized commodity in fine woodworking [7–9]. Artworks containing the blue-green pigment, named xylindein [10] (Figure 1), retain their coloration today, attesting to its stability. The structure of this pigment has since been established [11–13], and its impressive UV stability and thermal stability have been the subject of research [14]. The properties of other pigments have also been the subject of recent investigation, including the identification of the napthoquinonic crystal produced by *Scytalidium cuboideum,* called dramada [15]. This red compound has also been isolated from an actinomycete [16].

**Citation:** Almurshidi, B.H.; Van Court, R.C.; Vega Gutierrez, S.M.; Harper, S.; Harper, B.; Robinson, S.C. Preliminary Examination of the Toxicity of Spalting Fungal Pigments: A Comparison between Extraction Methods. *J. Fungi* **2021**, *7*, 155. https://doi.org/10.3390/jof7020155

Academic Editor: Laurent Dufossé

Received: 10 December 2020 Accepted: 15 February 2021 Published: 22 February 2021

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**Figure 1.** Structure of spalting fungal pigments. (**a**) Blue/green pigment xylindein produced by *Chlorociboria spp.*; (**b**) red pigment "dramada" (5,8-Dihydroxy-2,7-dimethoxy-1,4-naphthoquinone) produced by *Scytalidium cuboideum.*

Pigments produced by these fungi have been investigated for their use in a variety of fields, for example, as a coloring agent for wood stain [17], in paint [18], and as a textile dye [19–21]. The use of fungal pigments in these applications could replace current unsustainable industrial practices. For example, conventional textile dyeing practices produce toxic wastewater associated with negative environmental and health consequences [22–25]. The use of sustainably produced fungal pigments instead would allow for the production of desired colored cloth without the toxic tradeoff. In addition to use as a colorant, the pigments have also been the subject of investigation into use as organic semiconductors [14,26], and may allow for fully sustainable energy generation through organic photovoltaic systems. However, before the adoption of these green technologies is possible, especially for those associated with extended human contact, the toxicity of the pigments must be understood.

Many filamentous fungi produce secondary metabolites with bioactive effects. Some have a beneficial effect, with pharmacological uses like antibiotics [27,28]. However, fungi also produce a range of mycotoxins such as aflatoxins and rhizonin, which are severe health hazards [29]. A number of filamentous fungi also produce pigments that are themselves toxic [30]. For example, pigments from *Monascus* spp., used for coloring food and in pharmacological applications, are restricted due to fungal co-production of citrinin [31], which is nephrotic [32]. This has led to research attempting to reduce citrinin production through growth condition variation or strains [33–37].

Spalting fungi have received attention for their potential associated health risks, most notably by woodturners who have spread fear about their supposed toxicity. Theories spread have included spalted wood causing allergic reactions, releasing "carloads" of spores, and implications of brain infection, which have likely been driven through limited understanding of fungal biology [38]. These and more urban myths around any potential threats that spalted wood might pose compared to non-decayed wood have been debunked, as spalted wood is not inherently more dangerous or toxic than non-decayed wood [39].

To determine the potential hazard of toxins in humans, a broadly used method involves the testing of compounds in zebrafish (*Danio rerio*) embryos. Zebrafish are a tropical freshwater fish that have been highly studied and used as a model organism for rapid and low-cost research in the fields of toxicology, genetics and developmental biology [40–43]. Zebrafish assays are used to indicate bioactive drugs and therapeutic compounds for pharmaceutical applications and to understand effects on developmental mechanisms [44–47]. The use of the zebrafish embryo model for toxicology research is accepted internationally as an alternate animal model on testing hazard and risk assessment [48,49]. The zebrafish model has also been used to evaluate the toxicity of natural products from plants and other organisms such as secondary metabolite extracts [50–52], making it an appropriate model for preliminary examination of the toxicity of spalting fungal pigments.

This study sought to characterize the potential toxic effects of spalting fungal pigments and identify whether they came from the pigments themselves or due to the presence of other compounds in the extract. Understanding the toxicity of these pigments, as well as

the cause (whether the pigment *itself* is toxic or the accompanying secondary metabolites are toxic), will enable the determination of what products they are appropriate for use in, and what level of processing after fungal production they require to be safely used. This information will inform future research and industrial adoption of these unique, sustainably sourced pigments.
