3.2.3. Mortality and Morphology

Dramada was the only tested compound that exhibited significant morbidity in tested endpoints, with 50 μM concentrations associated with near 100% mortality by 24 hpf (Figure 4). Deformations found to be significantly associated with exposure included caudal fin deformity at 10.7 and 23.2 μM, abnormal pigmentation at 23.2 μM, and modest body length shortening at 10.7 and 23.2 μM. Concentration response modeling for dramada showed that the LC50 for mortality at 24 hpf was estimated to be 38.2 μM (±1.4), and mortality (LD50) at 120 hpf was estimated to be 25.5 μM (±3.4). This higher bioactivity is not surprising, as many other naphthoquinones from various sources are known to have bioactive properties [72], and the red pigment has been previously described as having modest bioactivity against Gram-negative bacteria and fungi [16]. Other red naphthoquinonic pigments extracted from filamentous fungi have shown cytotoxicity against cell lines [73,74], and one of these compounds, erythreostominone, has also been shown to induce malformations and impair locomotor activity [75,76].

In contrast, xylindein and aniline showed non-significant toxicity responses without a clear dose-response relationship (Figure 4). Aniline is associated with known toxic and potential mutagenic effects [77], and has been shown to have developmental and sublethal effects in zebrafish [78] with reported LD50 values at 96 h of 618 (±43.0) μmol/L [79], higher than the concentrations tested here.

Based on the ratio of LD50 value to body mass, it would take 510.4 kg (2040 M at 250.20 g/mol) of dramada for an 80 kg (~176 lbs) human to experience equivalent exposure to the 25.5 μM experienced by a 1 mg embryo – while also being fully immersed in pigment. For reference, the LD50 of table salt taken orally is 3 g/kg for a rat [80], equivalent to 240 g for an 80 kg human. Previous work on the toxicity of the red pigment when isolated from an actinomycete showed it to be non-toxic at 600 mg/kg administered intraperitoneally to mice [16]. These factors suggest that while dramada may present some toxicity, it may not present a human health risk except at extreme quantities.

The applications developed based on dramada from *Scytalidium cuboideum* have focused on its use as a coloring agent for textiles and bamboo [20,21,58], using a standard concentration of extracted pigment. Estimates of the pigment concentration in this "standard" pigment solution range from 0.73 to 2.4 mM [55], making the standard used much higher than the calculated LD50 of 25.5 μM (±3.4) at 120 hpf. This suggests that solutions used in current methods likely contain quantities of pigment that may pose a risk for developing embryos, if the pigments were transferred to water to allow for exposure.

However, while dramada has demonstrated a toxic effect on zebrafish, this result does not necessarily prevent its use in industry. Many dyes currently in use in the textile industry have been shown to have toxic effects [81], and the development of natural dyes to replace toxic synthetics has been a focus of research [82]. Additionally, as the processes currently used for dyeing with dramada do not produce wastewater [59,83,84], there is a limited likelihood of effluent reaching watersheds and impacting fish development. In addition, dramada has limited solubility in water and strongly binds to materials such as textiles, wood, and glass, making transfer and high levels of exposure unlikely. While any toxicity suggests that caution should be taken in regard to the use of the pigment for applications where consistent exposure to humans is expected, further testing of prepared textiles and other materials should be carried out to determine bioavailability and the likelihood of exposure.

While the red pigment produced by *S. cuboideum* demonstrates some toxicity, the green pigment produced by *Chlorociboria* spp. in solid form has limited bioactivity. However, pigmented fungal media solutions and, in some cases, extraction using DCM were associated with significant mortality. This suggests the co-production of other toxic secondary metabolites, while xylindein itself may be non-toxic. Purer *Chlorociboria* extract has also been shown to have improved semiconductive capabilities [56], further supporting the need for improved methods of xylindein purification. As xylindein is under investigation for use in solar cells, an industry associated with toxic byproducts and materials [85–87],

the adoption of xylindein as an alternative could improve the environmental friendliness of energy generation in addition to its sustainability. Future work should focus on the identification and removal of possible mycotoxins and the use of purified pigments. This research supports previous research into the use of these pigments for a variety of applications, allowing for future industrial adoption. The use of these sustainably sourced pigments with limited toxicity has the potential to improve sustainability and replace toxic byproducts across multiple industries.

**Figure 4.** Mortality and Morphology Endpoint Counts for Dramada and Xylindein. Presence/absence data across 32 replicates with points above and at threshold for binomial significance in red. Concentration for xylindein is approximate due to lack of pure compound for testing. Control incidence of all morphological and touch response endpoints below 20% cutoff for biological validity.
