energies Article Testing the Toxicity of Stachybotrys chartarum in Indoor Environments—A Case Study Marlena Piontek and Katarzyna Łuszczy ´nska* Institute of Environmental Engineering, University of Zielona Góra, Licealna 9, PL 65-417 Zielona Góra, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-683-282-679 Abstract: Infestation of interior walls of buildings with fungal mould is a reason for health concern which is exacerbated in energy-efficient buildings that limit air circulation. Both mycological and mycotoxicological studies are needed to determine the potential health hazards to residents. In this paper, a rare case of the occurrence of Stachybotrys chartarum in an apartment building in the Lubuskie Province in Poland has been described. Isolated as the major constituent of a mixed mycobiota, its specific health relevance still needs to be carefully analyzed as its biochemical aptitude for the synthesis of mycotoxins may be expressed at different levels. Therefore, ecotoxicological tests were performed using two bioindicators: Dugesia tigrina Girard and Daphnia magna Straus. D. tigrina was used for the first time to examine the toxicity of S. chartarum. The ecotoxicological tests showed that the analyzed strain belonged to the third and fourth toxicity classes according to Liebmann’s classification. The strain of S. chartarum was moderately toxic on Potato Dextrose Agar (PDA) as a culture medium (toxicity class III), and slightly toxic on Malt Extract Agar (MEA) (toxicity class IV). Toxicity was additionally tested by instrumental analytical methods (LC-MS/MS). This method allowed for the identification of 13 metabolites (five metabolites reported for Stachybotrys and eight for unspecific metabolites). Spirocyclic drimanes were detected in considerable quantities (ng/g); a higher concentration was observed for stachybotryamide (109,000 on PDA and 62,500 on MEA) Citation: Piontek, M.; Łuszczy´nska, and lower for stachybotrylactam (27,100 on PDA and 46,300 on MEA). Both may explain the result K. Testing the Toxicity of Stachybotrys observed through the bioindicators. Highly toxic compounds such as satratoxins were not found chartarum in Indoor Environments— A Case Study. Energies 2021, 14, 1602. in the sample. This confirms the applicability of the two bioindicators, which also show mutual https://doi.org/10.3390/en14061602 compatibility, as suitable tools to assess the toxicity of moulds. Academic Editor: Franco Cotana Keywords: biotests; moulds; indoor contamination; spirocyclic drimanes; stachybotryamide; stachy- botrylactam Received: 3 February 2021 Accepted: 11 March 2021 Published: 13 March 2021 1. Introduction Publisher’s Note: MDPI stays neutral The problem of moulds on partition walls in buildings occurs worldwide in all climate with regard to jurisdictional claims in zones. Mycological studies in buildings point to two of the most dangerous mycotoxi- published maps and institutional affil- genic species of moulds. These are Stachybotrys chartarum and Aspergillus versicolor [1–7]. iations. Bloom et al. [8] demonstrated that several mycotoxins synthesized by S. chartarum (macro- cyclic trichothecenes) and A. versicolor (sterigmatocystins) may be present in the majority of samples collected from the construction materials of damp apartments and from samples of dust deposits. Interest in S. chartarum in buildings increased when a relationship be- Copyright: © 2021 by the authors. tween the growth of this mould in residential buildings and primary idiopathic pulmonary Licensee MDPI, Basel, Switzerland. hemosiderosis (IPH) was confirmed [9–12]. S. chartarum in buildings can always be found This article is an open access article in areas characterized by excessive humidity. S. chartarum is a “hydrophilic” fungus with a distributed under the terms and preference for moist conditions [13]. It is a tertiary colonizer on partition walls in building conditions of the Creative Commons interiors, occurring at water activity (aw) as high as 0.98. It grows on materials with Attribution (CC BY) license (https:// a high content of cellulose, e.g., plasterboard, wood and wood panelling, natural fibre creativecommons.org/licenses/by/ carpets, insulation pipe coverings, etc. A frequent cause of infestation of the partitions 4.0/). Energies 2021, 14, 1602. https://doi.org/10.3390/en14061602 https://www.mdpi.com/journal/energies Energies 2021, 14, 1602 2 of 12 is excessive humidity caused by flooding, leaks, or water condensation [14–17]. Lack of proper ventilation in energy-efficient buildings may contribute to the problem. However, studies have shown that not all strains of S. chartarum are highly toxic [9]. S. chartarum is present in two chemotypes: S and A. In terms of morphology, these chemo- types do not demonstrate any differences; nevertheless, what differentiates them is the type of secondary metabolites produced [9,18]. S. chartarum synthesizes macrocyclic tri- chothecenes that are highly cytotoxic, such as satratoxin H, G, F, and iso-F, or roridin L-2. Additionally, several roridin E epimers have been identified: hydroxyroridin E and verru- carin J and B. However, not all strains from residential housing synthesize these harmful mycotoxins (only 30–40% of chemotype S strains: usually satratoxin H and roridins E and L-2), [9,19–24]. With other isolates, diterpenoid atranones have been found, as well as their dolabellane precursors [25] and simple (non-macrocyclic) trichothecenes in small amounts. S. chartarum chemotype A does not produce macrocyclic trichothecenes (in 70–80% of strains) [9,20]. Both chemotypes synthesize many metabolites which belong to the family of spirocyclic drimanes (stachybotryamide, stachybotrylactam) in quantities much greater than trichothecenes and atranones [9,17,25,26]. There are about 140 known compounds coming from Stachybotrys sp. [26]. S. chartarum has been shown to produce large quantities of spirocyclic drimanes, of which up to 40 dif- ferent species have been found [9,19,27]. Production pathways are through a terpenoid structure (generating two lower rings under the spiro bond) and from polyketides that produce the upper part of the molecule [9,27,28]. The harmful biological properties of spirocyclic drimanes include enzyme inhibition, disruption of the complement system, inhi- bition of TNF-α liberation, cytotoxicity and neurotoxicity, and stimulation of plasminogen, fibrinolysis, and thrombolysis [9,29–33]. In mycological research in the Lubuskie Province, 82 species of moulds were identified Energies 2020, 13, x FOR PEER REVIEWin more than 280 residential and public buildings. S. chartarum was found sporadically4 of 13 (only in 4 cases), [4,7,34,35]. In the presented studies tests were conducted on S. chartarum, which2. Materials was isolated and Methods from an infested partition wall of a tenement house in Zielona Góra (Figure2.1. Sample1) from Collection a site and where Cultivation residents complained of health problems such as allergic diseases,Samples frequent were eye collected and ear from inflammations, the inner surfac headaches,es of partition and coughs. walls with In another visible mould three cases,from 4 such places: mould the occurredPalace in inRakow; buildings a building intended of the for University repair after of technological Zielona Góra failures. (UZ); a Understandingtenement house occurrence in Zielona and Góra; health and relevance the Scout’s of moulds house, is critical,Zielona especially Góra, in inPoland. the case S. ofchartarum thermal modernizationfrom the tenement of buildings. house was selected for further research (Figure 1). FigureFigure 1. 1.Building Building partition partition of of the the tenement tenement house hous (Zielonae (Zielona G óGóra,ra, Poland) Poland) infested infested with withS. chartarumS. chartarum. Research conducted by Gravesen and Flannigan in Danish residential buildings provedThe that wallsS. chartarum were madebelonged of bricks to covered a species with which cement–lime can be encountered plaster. Acrylic most frequently paint was onused the as walls the infinishing that country material. [36– 38The]. However,wall moisture despite content extensive was research measured [18 ,at21 the,22, 39sample–41], thecollection harmfulness site using of this a Trotec mould T650 has not hygrometer. been clearly The established, moisture andcontent the strainsof the partition found in wall res- identialwas assessed buildings according still need to the to be operating subjected instru to analytical–toxicologicalctions supplied with the and device. ecotoxicological Wall mass tests.moistures The aim (%) of were this research0–3: dry waswall, the 3–5: application wall with of low ecotoxicological moisture content, tests using 5–8: D.wall tigrina with medium moisture content, 8–12: wall with high moisture content. For the mycological analysis, a methodology developed by the CBS (Centraalbureau voor Schimmelcultures) [13] was applied. In this methodology, a material containing moulds is disaggregated into small pieces and inserted to Petri dishes directly at the collection site that were pre-prepared with culture media [43]. Four replicates were taken from each sample: two using Malt Extract Agar (MEA) as the culture medium and two using Potato Dextrose Agar (PDA), Merck. Then the samples were transported to the laboratory of the Institute of Environmental Engineering, University of Zielona Góra, for further mycological analysis. They were covered with white linen and incubated at room temperatures