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Testing the Toxicity of Stachybotrys chartarum in Indoor Environments—A Case Study

Institute of Environmental Engineering, University of Zielona Góra, Licealna 9, PL 65-417 Zielona Góra, Poland
Author to whom correspondence should be addressed.
Energies 2021, 14(6), 1602;
Submission received: 3 February 2021 / Revised: 8 March 2021 / Accepted: 11 March 2021 / Published: 13 March 2021
(This article belongs to the Special Issue Life Cycle Thinking for a Sustainable Built Environment)


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) and lower for stachybotrylactam (27,100 on PDA and 46,300 on MEA). Both may explain the result observed through the bioindicators. Highly toxic compounds such as satratoxins were not found in the sample. This confirms the applicability of the two bioindicators, which also show mutual compatibility, as suitable tools to assess the toxicity of moulds.

1. Introduction

The problem of moulds on partition walls in buildings occurs worldwide in all climate zones. Mycological studies in buildings point to two of the most dangerous mycotoxigenic species of moulds. These are Stachybotrys chartarum and Aspergillus versicolor [1,2,3,4,5,6,7]. Bloom et al. [8] demonstrated that several mycotoxins synthesized by S. chartarum (macrocyclic 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 between the growth of this mould in residential buildings and primary idiopathic pulmonary hemosiderosis (IPH) was confirmed [9,10,11,12]. S. chartarum in buildings can always be found in areas characterized by excessive humidity. S. chartarum is a “hydrophilic” fungus with a preference for moist conditions [13]. It is a tertiary colonizer on partition walls in building interiors, occurring at water activity (aw) as high as 0.98. It grows on materials with a high content of cellulose, e.g., plasterboard, wood and wood panelling, natural fibre carpets, insulation pipe coverings, etc. A frequent cause of infestation of the partitions is excessive humidity caused by flooding, leaks, or water condensation [14,15,16,17]. 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 chemotypes do not demonstrate any differences; nevertheless, what differentiates them is the type of secondary metabolites produced [9,18]. S. chartarum synthesizes macrocyclic trichothecenes 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 verrucarin 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), [9,19,20,21,22,23,24]. 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 different 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, inhibition of TNF-α liberation, cytotoxicity and neurotoxicity, and stimulation of plasminogen, fibrinolysis, and thrombolysis [9,29,30,31,32,33].
In mycological research in the Lubuskie Province, 82 species of moulds were identified in more than 280 residential and public buildings. S. chartarum was found sporadically (only in 4 cases), [4,7,34,35]. In the presented studies tests were conducted on S. chartarum, which was isolated from an infested partition wall of a tenement house in Zielona Góra (Figure 1) from a site where residents complained of health problems such as allergic diseases, frequent eye and ear inflammations, headaches, and coughs. In another three cases, such mould occurred in buildings intended for repair after technological failures. Understanding occurrence and health relevance of moulds is critical, especially in the case of thermal modernization of buildings.
Research conducted by Gravesen and Flannigan in Danish residential buildings proved that S. chartarum belonged to a species which can be encountered most frequently on the walls in that country [36,37,38]. However, despite extensive research [18,21,22,39,40,41], the harmfulness of this mould has not been clearly established, and the strains found in residential buildings still need to be subjected to analytical–toxicological and ecotoxicological tests. The aim of this research was the application of ecotoxicological tests using D. tigrina and D. magna to evaluate the toxicity of S. chartarum and assess the mycotoxic risks for the residents of water-damaged buildings or where there are other factors contributing to mould formation in homes where residents reported significant health problems.
D. tigrina is a sensitive bioindicator for mycotoxins. It has been used previously on Aspergillus versicolor Tiraboschi - sterigmatocystin and is a more sensitive organism than D. magna [4,7,42]. Ecotoxicological analyses of biomass of A. versicolor showed that less than 50% of strains can produce significant amounts of sterigmatocistin, whereby significant amounts were detected in only three out of 17 samples [42]. Therefore, the presence of toxic mould biomasses and mycotoxins could be detected by applying the D. tigrina bioassay. Here, the first report on its application on S. chartarum is presented. Introducing a different indicator allows for the strengthening of the evidence regarding the toxicity of the observed moulds.

2. Materials and Methods

2.1. Sample Collection and Cultivation

Samples were collected from the inner surfaces of partition walls with visible mould from 4 places: the Palace in Rakow; a building of the University of Zielona Góra (UZ); a tenement house in Zielona Góra; and the Scout’s house, Zielona Góra, in Poland. S. chartarum from the tenement house was selected for further research (Figure 1).
The walls were made of bricks covered with cement–lime plaster. Acrylic paint was used as the finishing material. The wall moisture content was measured at the sample collection site using a Trotec T650 hygrometer. The moisture content of the partition wall was assessed according to the operating instructions supplied with the device. Wall mass moistures (%) were 0–3: dry wall, 3–5: wall with low moisture content, 5–8: wall 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 between 18 and 22 °C. Day/night rhythms were maintained. From the mixed starter cultures pure (axenic) cultures were isolated and again transferred onto the PDA and MEA media, respectively. The total cultivation time until single isolated species were observed was 21 days [4,7,42]. For identification and taxonomic classification of isolated strains, Nikon light microscopy was used (Figure 2): [13,44,45,46,47,48,49].

2.2. Cultures of Stachybotrys chartarum

Based on the isolated strains, mass cultures of S. chartarum were established in order to obtain the mould biomass needed for ecotoxicological and physico-chemical tests using two different growth media (Figure 3a,b): MEA and PDA according to the CBS [13].
A total of 5 mL of the respective media, PDA, MEA (Merck), were poured onto 30 Petri dishes with ø of 9 cm. Mould spores were applied centrally onto the growth media using a preparation needle. Again, samples were incubated after having been covered with white linen and cultivated at room temperature (18–22 °C) while keeping the circadian rhythm. Then, cultivation of the isolated culture lasted 3 months in order to allow growth, sporulation, and regrowth to extend over a significant part of the Petri dish. The strain aged and the mycelium was allowed to air-dry. The method of the cultivation of the mould according to Piontek [4,7,42] reflects the conditions prevalent on partition walls in residential housing. With a scalpel, the dried moulds (still containing remains of the culture medium) were scraped off the Petri dishes. The material extracted was weighed and transferred for storage to glass jars, which were closed with a ground stopper (Figure 4). Further analyses (see below) were performed using methanol extracts of these materials.

2.3. Methanol Extracts for Ecotoxicological Tests

The method developed and refined by Piontek [4,42,50] was applied to extract analytes from the samples for further mycotoxin testing. Prepared pure (single species) mould biomass samples (see Section 2.2 above) were collected in the form of 1 g of air-dry extracts and held in 100 mL of 80% methanol for 96 h (room temperature). Methanol extracts were prepared in duplicate. The extracts were then filtered through 47 mm fiberglass filter discs (Whatman GF/C), and the supernatant was collected for ecotoxicological analysis in which D. tigrina and D. magna were used as the indicator organisms. In this procedure, 1 mL of extract was obtained from 10.0 mg of an air-dried sample (moulds + medium).

2.4. Ecotoxicological Tests Using Dugesia Tigrina

Following the concepts of Piontek [51], planarians were cultivated and used in toxicological tests (Figure 5). Starting from the two methanol extracts, solutions were diluted to different concentrations, and 40 mL of a test solution was stored in beakers with a capacity of 50 mL. Each concentration level was prepared in three repetitions; furthermore, control tests were added. Ten cut organisms were inserted into each beaker so as to have thirty organisms per concentration level. The mortality of the planarians was determined after ten days (240 h) in order to establish the lethal concentration for half of the organisms (240 h LC 50), [52,53,54,55]. A graphical method (probit analysis) was applied in this interpretation step. The χ2-test was used to check whether the empirical results matched the normal distribution. Agreement was considered sufficient when the χ2-test resulted in probabilities higher than 0.7 [56]. The toxicity, according to the ecotoxicological tests, was established following Liebmann’s classification [57] (Table 1). According to the standard [58], the control tests ascertain whether any foreign factors, apart from the toxicity of the tested substances, interfere with the test or not, as evidenced by the mortality of the control organisms not exceeding 10%. This also applies when checking the condition of the bio-indicators used in the research.

2.5. Ecotoxicological Tests Using Daphnia Magna

The daphnia used for ecotoxicological testing were bred in the laboratory of the Institute of Environmental Engineering at the Zielona Góra University. In order to conduct the toxicological tests, 3-day-old organisms of equal size and condition were collected. Two methanol extracts obtained from the dry biomass of S. chartarum cultivated on two different media were used. A total of 10 concentrations were prepared. Tubes with a volume of 50 mL were filled with the test solution (about 45 mL) at varying concentrations, and then the daphnia (10 organisms in each case) were added using a dropper. The test was performed in three repetitions including the control test. The test results are considered reliable if the percentage of daphnia mortality in the contaminated sample is 10% or less [58]. The prepared samples were left for 48 h, and then the mortality of the test organisms was checked. This was performed for the purpose of the calculation of the 48h LC 50. In order to calculate the values of the LC 50 concentrations, the method of graphic interpretation (probit analysis) was applied just as in the case of Dugesia tigrina.

2.6. Determination of Secondary Metabolites of S. chartarum Using the LC-MS/MS Method

The S. chartarum biomass samples (PDA and MEA basis) were analysed using the LC-MS/MS method [59,60]. The samples were diluted with acetonitrile/water/acetic acid solvent (79:20:1, v/v/v), resulting in a sample-to-solvent ratio of 1:8, then centrifuged and transferred to autosampler vials in aliquots of 100 µL. Liquid chromatography was used for separation on a Gemini® C18-column, 150 × 4.6 mm i.d., 5 µm particle size, protected by a C18 guard cartridge, 4 × 3 mm i.d. (all from Phenomenex, Torrance, CA, USA). A binary gradient mode with 5 mM ammonium acetate in a methanol/water/acetic acid mixture was used for elution [60]. Mass spectrometric detection took advantage of the scheduled multiple reaction monitoring (sMRM) mode. Two separate runs were made for each sample, one in positive and one in negative polarity, by scanning two fragmentation reactions per analyte. Metabolites were identified unambiguously by comparing retention times and sMRM ion ratio of standards according to the criteria established for mycotoxins [61]. The tests were carried out in the Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU).

2.7. Statistical Analysis

Statistical analysis was performed using Excel 2010. In order to show the significance of differences between the results of the metabolites of S. chartarum grown on various media, the PDA and MEA analysis of variance at a significance level of α = 0.01 was calculated.

3. Results

3.1. Results of Mycological Analysis

Mycological analysis allowed for the determination of the species which accompanied S. chartarum on the infested building partitions (Table 2). Studies showed that S. chartarum always occurs in the presence of Penicillum chrysogenum and a large amount of bacteria due to high humidity. In the tenement house these were Penicillium chrysogenum, Mucor hiemalis, and bacteria. Results regarding the moisture content level of the partition wall and air humidity on the day during which samples were collected were as follows: partition wall—11% water content, hence this is a wall with a high moisture content. The air humidity was only 53%. The conditions prevailing at the sample collection site confirmed that the described mould grew well in damp locations (three other cases).

3.2. Toxicological Tests

Ecotoxicological tests using two bioindicators were conducted in the laboratory of the Institute of Environmental Engineering at the Zielona Góra University (test methodology). The results obtained for the LC 50 are listed in Table 3.
Toxicological tests have confirmed a moderate to low total toxicity of samples. The LC 50 values from the respective bioindicators show good agreement, confirming that D. tigrina is a sensitive bioindicator for Stachybotrys metabolites (the calculations of LC 50 are included in Supplementary Materials).

3.3. Chromatographic Analysis

In order to carry out the chromatographic analysis, two air-dried biomass surfaces of S. chartarum on PDA and MEA media were analysed as the background for the obtained results (Table 4). The MS/MS chromatograms of the identified analytes were included in the Supplementary Materials. According to [62], the expanded measurement uncertainty close to a 95% confidence interval for the method is 50%. From the variability of the results for all species, we derived a generally applicable relative standard deviation of the measurements of 20%.
S. chartarum produced secondary metabolites on PDA, in the following quantities: stachybotryamide—109,000 ng/g, stachybotrylactam—27,100 ng/g, antibiotic F 1839 A—6470 ng/g, aurantin—67.1 ng/g, orsellinic acid—21,500 ng/g. On the other hand, the quantity of stachybotryamide on MEA was smaller and amounted to 62,500 ng/g. The quantity of stachybotrylactam and antibiotic F 1839 A increased and amounted to 46,300 ng/g and 10,200 ng/g, respectively. S. chartarum on MEA: aurantine and orsellinic acid were below the detection limit. Regarding the occurrence of unspecific metabolites from S. chartarum on the PDA medium samples, eight metabolites were found, including five at concentrations below the limit of detection (LOD): brevianamid F, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), N-benzoyl-phenylalanine, and rugulusovin. In the samples on the MEA medium N-benzoyl-phenylalanine did not occur, instead tenuazonic acid was detected. Asperglaucide and tryptophol were not detected in either medium. The analysis of variance showed highly significant differences between the amounts of metabolites of S. chartarum grown on media PDA and MEA (Fcalc 8,46 Fcrit 4,16; p > 0.01).

4. Discussion

S. chartarum is a species which can be very dangerous to the health of the dwellers of infested premises; therefore, toxicity analysis of this species is necessary. In this case, the occupants of the buildings with infected partitions complained of various health problems associated with massive indoor moisture and mould problems. Two bioindicators were used to carry out the toxicological tests: D. tigrina and D. magna, and Liebmann’s classification [57] was used to allocate the toxicity class. The analysed mould strain was shown to be moderately toxic (toxicity class III) for the S. chartarum strain cultivated on the PDA medium and slightly toxic (toxicity class IV) for the S. chartarum strain cultivated on MEA. The results of the bioassays confirmed that the used organisms were sensitive to the presence of mycotoxins.
Studies by other authors [63] have shown the effect of the culture medium on the production of Stachybotrys chartarum mycotoxins. The highest concentrations of macrocyclic trichothecenes were determined on the PDA medium; the MEA medium was the intermediate medium. The lowest amounts of mycotoxins were detected on moulds grown on glucose–yeast–peptone–agar and Sabouraud–dextrose–agar media. The research carried out with the use of D. tigrina and D. magna confirmed that growing Stachybotrys chartarum on PDA was more toxic than on the MEA medium.
Testing of the dry biomass of S. chartarum conducted by means of the liquid chromatography technique combined with tandem mass spectrometry (LC-MS/MS) demonstrated the presence of 13 of them in different quantities, depending on the medium on which the mould grew (PDA and MEA). Satratoxins are included in the method but were not detected. The quantity of stachybotryamide synthesized by S. chartarum on PDA was higher than on MEA, which indicates that this compound may be responsible for the medium toxicity of the sample (toxicity class III according to Liebmann). The lower quantity of stachybotryamide (on MEA, just half the concentration) caused the extract to be less toxic according to the bio-indicators (toxicity class IV according to Liebmann). Additionally, according to the chromatographic test, S. chartarum on MEA did not produce any orsellinic acid or aurantine. The tests carried out by Gaylarde et al. [64] on moulds of painted surfaces showed that S. chartarum synthesized on the substrate to an amount of MEA stachybotryamide (3167 ng/sample), while stachybotrylactam came to an amount of 914 ng/sample. Neither orsellinic acid nor aurantine were found.
Nielsen [9] distinguishes the important drimanes detected as coming from S. chartarum, which are stachybotryamide, stachybotrylactams, and di-aldehydes. These metabolites have been detected at significantly higher quantities in plasterboard samples of this species compared to those found in other moulds [3,18,22,65,66,67], using methods such as LC-UV, LC-MS, and bioassays. In the samples of S. chartarum on MEA, tenuazonic acid was also detected. Tenuazonic acid is known for its antitumor and antiviral activities. It inhibits protein synthesis in vivo and in vitro and protects in vitro cells against 1-β-D-arabinofuranosylcytosine. This protection has been ascribed to its protein synthesis inhibition properties [68]. Furthermore, tenuazonic acid has been linked to the heamatologic disease onyalai [13,69].
Tests using the HPLC method conducted on cultures of S. chartarum isolated from apartment walls in Cleveland demonstrated that trichothecenes can be present in very different quantities. Additionally, the MTT (tetrazolim reduction assay) cytotoxicity tests were conducted on the cell lines of cat lung cells. Spirodrimanes in greater concentrations than trichothecenes were detected in all the samples. Additionally, in this case no relationship was found between their concentration and the cytotoxicity of the samples [21].
The results of the tests for mycotoxins in building materials conducted by Gutarowska [70] have confirmed the high quantity of the spirodrimanes produced on MEA by S. chartarum (stachybotrylactam at a quantity of 156,800 ng/g); on the other hand, the quantity of stachybotrylactam on building materials was one hundred times lower (plaster mortar at a quantity of 1312 ng/g, plasterboard—2584 ng/g). The cytotoxicity tests for S. chartarum conducted by the author using XTT (cell proliferation assay) demonstrated that S. chartarum was not toxic for mouse fibroblasts; neither was the genotoxicity of S. chartarum proven (MLA test) after 21 days of mould growth both on building materials and on MEA.
Pieckova et al. [71] analysed indoor-originated S. chartarum from an office. Tests conducted with male rats have shown that this mould can generate metabolites in extracellular products that can be associated with lung cytotoxicity. Some authors argue that several analytical techniques should be used to investigate building-related health hazards [3].

5. Conclusions

This study confirmed the scientific and practical usefulness of biotesting with D. tigrina for analysing the risks from moulds in the human residential environment. All biomass extracts were toxic to D. tigina.
Toxicological studies of moulds from partition walls using test organisms from our own culture (a laboratory of the Institute of Environmental Engineering, University of Zielona Góra) and using our own published methods [6,65] were carried out to determine the toxicity of moulds from S. chartarum. The conducted studies are a continuation of studies on the toxicity of moulds in partition walls using Dugesia tigrina as a bioindicator. Studies on Aspergillus versicolor strains have shown that there are strains whose toxicity ranges from low to high [7,22,23,65]. The ecotoxicological tests using bioindicators such as planarians (D. tigrina) or daphnia (D. magna) demonstrate the sample toxicity through the total value. These tests allow for the assessment of the actual risk from moulds in buildings. The simplicity of the cultivation of the organisms and the method of performance of the test including the low financial outlay make them readily applicable for the evaluation of the mycotoxic hazard in residential housing. Every single case of the occurrence of S. chartarum is dangerous to the health of the residents. Instrumental analysis provides results that allow for the explanation of the toxicities observed using these bioindicators. Mycological and ecotoxicological testing in residential housing must still be continued.

Supplementary Materials

The following are available online at Table S1. Calculation of 240-h LC 50 for Dugesia tigrina Girard [Weber, 1972]—PDA; Table S2. Calculation of 48-h LC 50 for Daphnia magna [Weber, 1972]—PDA; Table S3. Calculation of 240-h LC 50 for Dugesia tigrina Girard [Weber, 1972]—MEA; Table S4. Calculation of 48-h LC 50 for Daphnia magna [Weber, 1972]—MEA.

Author Contributions

Conceptualization, M.P. and K.Ł.; methodology, M.P.; software, M.P.; validation, M.P. and K.Ł.; formal analysis, M.P.; investigation, M.P. and K.Ł.; resources, K.Ł.; data curation, K.Ł.; writing—original draft preparation, K.Ł.; writing—review and editing, M.P. and K.Ł.; visualization, M.P.; supervision, M.P.; project administration, M.P.; funding acquisition, M.P. All authors have read and agreed to the published version of the manuscript.


This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable; own research results.


We would like to express our gratitude to M. Sulyok from the Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU) and to Piotr Jedziniak and Marta Piątkowska from the Department of Pharmacology and Toxicology, National Veterinary Research Institute in Puławy for their help in carrying out chromatographic tests using the LC-MS/MS method.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Dales, R.E.; Zwanenburg, H.; Burnett, R.; Franklin, C.A. Respiratory health effects of home dampness and molds among Canadian children. Am. J. Epidemiol. 1991, 134, 196–206. [Google Scholar] [CrossRef]
  2. Hendry, K.M.; Cole, E.C. A review of mycotoxins in indoor air. J. Toxicol. Environ. Health 1993, 38, 138–198. [Google Scholar] [CrossRef] [PubMed]
  3. Johanning, E.; Gareis, M.; Chin, Y.S.; Hintikka, E.L.; Jarvis, B.B.; Dietrich, R. Toxicity screening of materials from buildings with fungal indoor air quality problems (Stachybotrys chartarum). Mycotoxin Res. 1998, 14, 60–73. [Google Scholar]
  4. Piontek, M. Grzyby Pleśniowe i Ocena Zagrożenia Mikotoksycznego w Budownictwie Mieszkaniowym (Moulds and Estimation of Mycotoxic Threat in Dwelling Buildings); Wydawnictwo Uniwersytetu Zielonogórskiego: Zielona Góra, Poland, 2004; 174p. [Google Scholar]
  5. Piontek, M.; Jasiewicz, M.; Bednar, K. Biodeterioracja pleśniowa wywołana występowaniem wad technologicznych w budownictwie (Mould biodeterioration caused by technological defects in dwelling buildings). Ochr. Przed Korozją 2010, 1, 8–13. [Google Scholar]
  6. Piontek, M.; Jasiewicz, M.; Łuszczyńska, K. Thermal Modernization and Biodeterioration of Prefabricated Elements of Buildings—A Case Study—W: Management of Indoor Air Quality; Dudzińska, M.R., Ed.; Taylor & Francis Group: London, UK, 2011; pp. 109–122. [Google Scholar]
  7. Piontek, M.; Łuszczyńska, K.; Lechów, H. Occurrence of the toxin-producing Aspergillus versicolor Tiraboschi in residential buildings. Int. J. Environ. Res. Public Health 2016, 13, 862. [Google Scholar] [CrossRef] [Green Version]
  8. Bloom, E.; Bal, K.; Nyman, E.; Must, A.; Larsson, L. Mass spectrometry-based strategy for direct detection and quantification of some mycotoxins produced by Stachybotrys and Aspergillus spp. in indoor environments. Appl. Environ. Microbiol. 2007, 73, 4211–4217. [Google Scholar] [CrossRef] [Green Version]
  9. Nielsen, K.F. Mycotoxin production by indoor molds. Fungal Genet. Biol. 2003, 39, 103–117. [Google Scholar] [CrossRef]
  10. Dearborn, D.G.; Infeld, M.D.; Smith, P.G. Update. Pulmonary hemorrhage/hemosiderosis among infants 1993–1996. Morb. Mortal. Wkly. Rep. 1997, 46, 33–35. [Google Scholar]
  11. Dearborn, D.G.; Yike, I.; Sorenson, W.G.; Miller, M.J.; Etzel, R.A. Overview of investigations into pulmonary hemorrhage among infants in Cleveland, Ohio. Environ. Health Perspect. 1999, 107 (Suppl. 3), 495–499. [Google Scholar] [CrossRef] [Green Version]
  12. Vesper, S.J.; Vesper, M.J. Stachylisin may be a cause of hemorrhaging in humans exposed to Stachybotrys chartarum. Infect. Immun. 2002, 70, 2065–2069. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Samson, R.A.; Hoekstra, E.S.; Frisvad, J.C. Introduction to Food and Airborne Fungi, 7th ed.; Centralbureau voor Schimmercultures (CBS): Utrecht, The Netherlands, 2004; 389p. [Google Scholar]
  14. Nelson, B.D. Stachybotrys chartarum: The Toxic Indoor Mold. APSnet Features. 2001. Available online: (accessed on 11 February 2021). [CrossRef]
  15. Nielsen, K.F.; Holm, G.; Uttrup, L.P.; Nielsen, P.A. Mould growth on building materials under low water activities. Influence of humidity and temperature on fungal growth and secondary metabolism. Int. Biodeterior. Biodegrad. 2004, 54, 325–336. [Google Scholar] [CrossRef]
  16. Andersen, B.; Thrane, U.; Szaro, T.; Taylor, J.W.; Jarvis, B.B. Molecular and phenotypic descriptions of Stachybotrys chlorohalonata sp. nov. and two chemotypes of Stachybotrys chartarum found in water-damaged buildings. Mycologia 2003, 95, 1227–1238. [Google Scholar] [CrossRef] [PubMed]
  17. Andersen, B.; Frisvad, J.C.; Søndergaard, I.; Rasmussen, I.S.; Larsen, L.S. Associations between Fungal Species and Water-Damaged Building Materials. Appl. Environ. Microbiol. 2011, 77, 4180–4188. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Nielsen, K.F. Mould Growth on Building Materials. Secondary Metabolites, Mycotoxins and Biomarkers. Ph.D. Thesis, Hørsholm DK, By og Byg, Statens Byggeforskningsinstitut, Danish Building and Urban Research, Copenhagen, Denmark, 2002; 106p. [Google Scholar]
  19. Andersen, B.; Nielsen, K.F.; Jarvis, B.B. Characterisation of Stachybotrys from water-damaged buildings based on morphology, growth and metabolite production. Mycologia 2002, 94, 392–403. [Google Scholar] [CrossRef] [PubMed]
  20. Croft, W.A.; Jarvis, B.B.; Yatawara, C.S. Airborne outbreak of Trichothecene Mycotoxicosis. Atmos. Environ. 1986, 20, 549–552. [Google Scholar] [CrossRef]
  21. Jarvis, B.B.; Sorenson, W.G.; Hintikka, E.-L.; Nikulin, M.; Zhou, Y.; Jirang, J.; Wang, S.; Hinkley, S.; Etzel, R.A.; Dearborn, D.G. Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants. Appl. Environ. Microbiol. 1998, 64, 3620–3625. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Nielsen, K.F.; Thrane, U.; Larsen, T.O.; Nielsen, P.A.; Gravesen, S. Production of mycotoxins on artificially inoculated building materials. Int. Biodeterior. Biodegrad. 1998, 42, 9–16. [Google Scholar] [CrossRef]
  23. Nielsen, K.F.; Huttunen, K.; Hyvärinen, A.; Andersen, B.; Jarvis, B.B.; Hirvonen, M.-R. Metabolite profiles of Stachybotrys isolates from water-damaged buildings and their induction of inflammatory mediators and cytotoxicity in macrophages. Mycopathologia 2001, 154, 201–205. [Google Scholar] [CrossRef] [PubMed]
  24. Vesper, S.J.; Dearborn, D.G.; Yike, I.; Allen, T.; Sobolewski, J.; Hinkley, S.F.; Jarvis, B.B.; Haugland, R.A. Evaluation of Stachybotrys chartarum in the house of an infant with pulmonary hemmorrhage: Quantitative assessment before, during, and after Remediation. J. Urban Health 2000, 77, 68–85. [Google Scholar] [CrossRef] [Green Version]
  25. Hinkley, S.F.; Mazzola, E.P.; Fettinger, J.C.; Lam, Y.K.T.; Jarvis, B.B. Atranones A–G, from the toxigenic mold Stachybotrys chartarum. Phytochemistry 2000, 55, 663–673. [Google Scholar] [CrossRef]
  26. Došen, I.; Andersen, B.; Phippen, C.B.; Clausen, G.; Nielsen, K.F. Stachybotrys mycotoxins: From culture extracts to dust samples. Anal. Bioanal. Chem. 2016, 408, 5513–5526. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Pestka, J.J.; Yike, I.; Dearborn, D.G.; Ward, M.D.W.; Harkema, J.R. Stachybotrys chartarum, Trichothecene Mycotoxins, and Damp Building–Related Illness: New Insights into a Public Health Enigma. Toxicol. Sci. 2008, 104, 4–26. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Jansen, B.J.; de Groot, G.A. Occurrence, biological activity and synthesis of drimane sesquiterpenoids. Nat. Prod. Rep. 2004, 2, 449–477. [Google Scholar] [CrossRef] [PubMed]
  29. Hasumi, K.; Ohyama, S.; Kohyama, T.; Ohsaki, Y.; Takayasu, R.; Endo, A. Isolation of SMTP-3, 4, 5 and -6, novel analogs of staplabin, and their effects on plasminogen activation and fibrinolysis. J. Antibiot. 1998, 51, 1059–1068. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  30. Kaneto, R.; Dobashi, K.; Kojima, I.; Sakai, K.; Shibamoto, N.; Yoshioka, T.; Nishid, A.H.; Okamoto, R.; Akagawa, H.; Mizuno, S. Mer-nf5003b, mer-nf5003e and mer-nf5003f, novel sesquiterpenoids as avian-myeloblastosis virus protease inhibitors produced by Stachybotrys sp. J. Antibiot. 1994, 47, 727–730. [Google Scholar] [CrossRef]
  31. Kohyama, T.; Hasumi, K.; Hamanaka, A.; Endo, A. SMTP-1 and -2, novel analogs of staplabin produced by Stachybotrys microspora IFO30018. J. Antibiot. 1997, 50, 172–174. [Google Scholar] [CrossRef] [Green Version]
  32. Nozawa, Y.; Yamamoto, K.; Ito, M.; Sakai, N.; Mizoue, K.; Mizobe, F.; Hanada, K. Stachybotrin C and parvisporin, novel neuritogenic compounds. 1. Taxonomy, isolation, physico-chemical and biological properties. J. Antibiot. 1997, 50, 635–640. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Došen, I.; Andersen, B.; Nielsen, K.F. LC-MS Based Analysis of Secondary Metabolites from Chaetomium and Stachybotrys Growth in Indoor Environments. Ph.D. Thesis, Department of Systems Biology, Technical University of Denmark, Copenhagen, Denmark, 2016; 239p. [Google Scholar]
  34. Piontek, M. Moulds occurring in buildings of western district of Poland Lubuskie e. In Proceedings of the II Conference on Microbial Biodegradation and Biodeterioration of Technical Materials, Łódź, Poland, 30–31 May 2001; pp. 86–94. [Google Scholar]
  35. Piontek, M. Moulds occurring in buildings of the Lubuskie province, Poland. Int. Biodeterior. Biodegrad. 2004, 53, 185. [Google Scholar]
  36. Flannigan, B.; Beardwood, K.; Ricaud, P.M.; Kirsch, J. Growth and toxin production in moulds isolated from houses. In Biodeterioration and Biodegradation; Rossmore, H.W., Ed.; Elsevier: London, UK, 1991; Volume 8, pp. 487–488. [Google Scholar]
  37. Flannigan, B. Microbial aerosols in buildings: Origin, health implications and controls. In Proceedings of the II Conference on Microbial Biodegradation and Biodeterioration of Technical Materials, Łódź, Poland, 30–31 May 2001; pp. 11–27. [Google Scholar]
  38. Gravesen, S.; Nielsen, P.A.; Nielsen, K.F. SBI Report 282, Microfungi in Water Damaged Buildings; Danish Building Research Institute: Hørsholm, Denmark, 1997. [Google Scholar]
  39. Jarvis, B.B.; Nielsen, K.F. Stachybotrys–An Unusual Mold Associated with Water–Damaged Buildings; 22 Mycotoxin–Workshop: Bonn, Germnay, 2000. [Google Scholar]
  40. Hodgson, M.J.; Morey, P.R.; Leung, W.Y.; Morrow, L.; Miller, J.D.; Jarvis, B.B.; Robbins, H.; Halsey, J.F.; Storey, E. Building–associated pulmonary disease from exposure to Stachybotrys chartarum and Aspergillus versicolor. J. Occup. Environ. Med. 1998, 40, 241–249. [Google Scholar] [CrossRef]
  41. Nikulin, M.; Reijula, K.; Jarvis, B.B.; Veijalainen, P.; Hintikka, E.-L. Effects on intranasal exposure to spores of Stachybotrys atra in mice. Fundam. Appl. Toxicol. 1997, 35, 182–188. [Google Scholar] [CrossRef] [PubMed]
  42. Piontek, M. Strains of Aspergillus versicolor Tiraboschi synthesizing sterigmatocistin and the differentiation of mycotoxic risk dependent on their productivity in housing buildings. Mycotoxin Res. 2007, 23, 34–38. [Google Scholar] [CrossRef] [PubMed]
  43. Hoekstra, E.S.; Samson, R.A.; Summerbell, R.C. Methods for the detection and isolation of fungi in the indoor environments. In Introduction to Food and Airborne Fungi, 7th ed.; Samson, R.A., Hoekstra, E.S., Frisvad, J.C., Eds.; Centralbureau voor Schimmercultures (CBS): Utrecht, The Netherlands, 2004. [Google Scholar]
  44. Fassatiova, O. Moulds in Technical Microbiology; WNT: Warszawa, Poland, 1983; 255p. [Google Scholar]
  45. Seifert, K.; Morgan-Jones, G.; Gams, W.; Kendrick, B. The Genera of Hyphomycetes; CBS-KNAW Fungal Biodiversity Centre: Utrecht, The Netherlands, 2011; 997p. [Google Scholar]
  46. Piontek, M. Moulds Atlas; Wydawnictwo Politechniki Zielonogórskiej: Zielona Góra, Poland, 1999; 113p. [Google Scholar]
  47. De Hoog, G.S.; Guarro, J. Atlas of Clinical Fungi, 2nd ed.; Centralbureau voor Schimmelcultures (CBS): Utrecht, The Netherlands, 2000. [Google Scholar]
  48. Pitt, J.I. A Laboratory Guide to Common Penicillium Species, 3rd ed.; Commonwealth Scientific and Industrial Research Organisation: North Ryde, Australia, 2000; 197p. [Google Scholar]
  49. Samson, R.A.; Houbraken, J.; Thrane, U.; Frisvad, J.C.; Andersen, B. Food and Indoor Fungi; CBS Manual Series 2; CBS KNAW Biodiversity Center: Utrecht, The Netherlands, 2010; 390p. [Google Scholar]
  50. Piontek, M. Use of planarian Dugesia tigrina Girard bioassay for assessing the toxicity of sterigmatocistin produced by Aspergillus versicolor Tiraboschi. Environ. Prot. Eng. 2010, 36, 65–71. [Google Scholar]
  51. Piontek, M. The regenerative ability of the planarian Dugesia tigrina (Girard) and the possibility of its use in reproduction of this species. Acta Hydrobiol. 1984, 25, 81–88. [Google Scholar]
  52. Piontek, M. Application of Dugesia tigrina Girard in toxicological studies of aquatic environments. Pol. Arch. Hydrobiol. 1998, 45, 565–572. [Google Scholar]
  53. Piontek, M. Use of the planarian Dugesia tigrina Girard in studies of acute intoxication. Pol. Arch. Hydrobiol. 1999, 46, 41–48. [Google Scholar]
  54. Piontek, M. Use of a planarian Dugesia tigrina Girard in the studies of acute toxicity of organic substances. Pol. Arch. Hydrobiol. 1999, 46, 331–338. [Google Scholar]
  55. Piontek, M. Application of the Dugesia tigrina Girard bioassay in mycotoxicological investigations. Part, I. Acute toxicity. In Proceedings of the VII International Scientific Conference, Bydgoszcz, Poland, 28–30 June 2004; pp. 149–155. [Google Scholar]
  56. Weber, E. Grundriss der Biologischen Statistik für Naturwissenschaftler, Landwirte und Mediziner; Jena, G., Ed.; Fischer Verlag: Frankfurt am Main, Germany, 1972; 674p. [Google Scholar]
  57. Liebmann, H. Handbuch der Frischwasser und Abwasserbiologie. Bd. 1, 2; Jena, G., Ed.; Fischer Verlag: Frankfurt am Main, Germany, 1962; 588p, as cited by Breitig and Tümpling, Ausgewählte Methoden der Wasseruntersuchung, Vol. II; Jena, G., Ed.; Fischer Verlag: Frankfurt am Main, Germany, 1970. [Google Scholar]
  58. PN-EN ISO 6341. Water Quality. Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea)–Acute Toxicity Test. Available online: (accessed on 15 October 2012).
  59. Vishwanath, V.; Sulyok, M.; Labuda, R.; Bicker, W.; Krska, R. Simultaneous determination of 186 fungal and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry. Anal. Bioanal. Chem. 2009, 395, 1355–1372. [Google Scholar] [CrossRef]
  60. Malachová, A.; Suylok, M.; Beltran, E.; Berthiller, F.; Krska, R. Optimization and validation of a quantitative liquid chromatography–tandem mass spectrometric method covering 295 bacterial and fungal metabolites including all regulated mycotoxins in four model food matrices. J. Chromatogr. A 2014, 1362, 145–156. [Google Scholar] [CrossRef] [Green Version]
  61. SANTE/12089/2016. Guidance Document on Identification of Mycotoxins in Food and Feed. Available online: (accessed on 1 January 2017).
  62. Stadler, D.; Sulyok, M.; Schuhmacher, R.; Berthiller, F.; Krska, R. The contribution of lot-to-lot variation to the measurement uncertainty of an LC-MS-based multi-mycotoxin assay. Anal. Bioanal. Chem. 2018, 410, 4409–4418. [Google Scholar] [CrossRef] [Green Version]
  63. Urlich, S.; Schäfer, C. Toxin Production by Stachybotrys chartarum Genotype S on Different Culture Media. J. Fungi 2020, 6, 159. [Google Scholar] [CrossRef]
  64. Gaylarde, C.; Otlewska, A.; Celikkol-Aydin, S.; Skóra, J.; Sulyok, M.; Pielech-Przybylska, G.J.; Beech, I.; Gutarowska, B. Interactions between fungi of standard paint test method BS3900. Int. Biodeterior. Biodegrad. 2015, 104, 411–418. [Google Scholar] [CrossRef]
  65. Johanning, E.; Biagini, R.E.; Hull, D.; Morey, P.R.; Jarvis, B.B.; Landsbergis, P. Health and immunology study following exposure to toxigenic fungi (Stachybotrys chartarum) in a waterdamaged office environment. Int. Arch. Occup. Environ. Health 1996, 68, 207–218. [Google Scholar] [PubMed]
  66. Anderson, M.A.; Nikulin, M.; Köljalg, U.; Anderson, M.C.; Rainey, F.; Reijula, K.; Hintikka, E.-L.; Salkinoja-Salonen, M. Bacteria, moulds, and toxins in water-damaged building materials. Appl. Environ. Microbiol. 1997, 63, 387–393. [Google Scholar] [CrossRef] [Green Version]
  67. Nielsen, K.F.; Gravesen, S.; Nielsen, P.A.; Andersen, B.; Thrane, U.; Frisvad, J.C. Production of mycotoxins on artificially and naturally infested building materials. Mycopathologia 1999, 45, 43–56. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  68. Cole, R.J.; Cox, R.H. Handbook of Toxic Fungal Metabolites; Academic Press: New York, NY, USA, 1981; 937p. [Google Scholar]
  69. Steyn, P.S.; Rabie, C.J. Characterisation of magnesium and calcium tenuazonate from Phoma sorgina. Phytochemistry 1976, 15, 1977–1979. [Google Scholar] [CrossRef]
  70. Gutarowska, B. Grzyby Strzępkowe Zasiedlające Materiały Budowlane: Wzrost Oraz Produkcja Mikotoksyn i Alergenów (Filamentous Fungi Colonizing Building Materials: Growth and Production of Mycotoxins and Allergens); Zeszyty Naukowe Politechniki Łódzkiej Nr 1074; Politechnika Łódzka: Łódź, Poland, 2010. [Google Scholar]
  71. Piecková, E.; Hurbánková, M.; Černá, S.; Pivovarová, Z.; Kováčiková, Z. Pulmonary cytotoxicity of secondary metabolites of Stachybotrys chartarum (Ehrenb.) Hughes. Ann. Agric. Environ. Med. 2006, 13, 259–262. [Google Scholar]
Figure 1. Building partition of the tenement house (Zielona Góra, Poland) infested with S. chartarum.
Figure 1. Building partition of the tenement house (Zielona Góra, Poland) infested with S. chartarum.
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Figure 2. Spores and hyphae of S.chartarum (Nikon microscopy).
Figure 2. Spores and hyphae of S.chartarum (Nikon microscopy).
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Figure 3. Growth of S. chartarum on different growth media after a three-month incubation (a) PDA, (b) MEA.
Figure 3. Growth of S. chartarum on different growth media after a three-month incubation (a) PDA, (b) MEA.
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Figure 4. Stachybotrys chartarum mass culture after 3 months of incubation on the PDA medium (a) and dry biomasses of S. chartarum on PDA medium in a glass jar closed with a ground stopper (b).
Figure 4. Stachybotrys chartarum mass culture after 3 months of incubation on the PDA medium (a) and dry biomasses of S. chartarum on PDA medium in a glass jar closed with a ground stopper (b).
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Figure 5. Breeding of Dugesia tigrina used for ecotoxicological tests.
Figure 5. Breeding of Dugesia tigrina used for ecotoxicological tests.
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Table 1. Toxicity classes of poison substances [57].
Table 1. Toxicity classes of poison substances [57].
Result of Toxicity Test–LC 50 Value
Toxicity ClassesClasses
<1highly toxicI
1–10potently toxicII
10–100moderately toxicIII
100–1000slightly toxicIV
>1000barely toxicV
Table 2. Mycological and moisture analysis of samples.
Table 2. Mycological and moisture analysis of samples.
No.Sampling PlaceCoexisting Moulds and OrganismsWall Finishing MaterialWall Humidity [%]Air Humidity [%]
1.Palace in Rakow, PolandStachybotrys chartarum,
Ulocladium botrytis, Penicillium chrysogenum,
glue paint872
2.Building of UZ, Zielona Góra, PolandStachybotrys chartarum, Penicillium chrysogenum,
acrylic paint1065
3.Tenement house, PolandStachybotrys chartarum *, Penicillium chrysogenum,
Mucor hiemalis
acrylic paint1153
4.Scout’s house, Zielona Góra, PolandStachybotrys chartarum, Penicillium chrysogenum,
* S. chartarum tested.
Table 3. Results of the ecotoxicological tests using D. tigrina and D. magna for two methanol extracts prepared from biomasses of S. chartarum on different growth media.
Table 3. Results of the ecotoxicological tests using D. tigrina and D. magna for two methanol extracts prepared from biomasses of S. chartarum on different growth media.
Type of Medium/BioindicatorLC 50
Toxicity Class
According to Liebmann
D. tigrina67.6class III (moderately toxic)
D. magna75.9class III (moderately toxic)
D. tigrina169.8class IV (slightly toxic)
D. magna190.5class IV (slightly toxic)
Table 4. Results of chromatographic analyses performed by means of liquid chromatography combined with tandem mass spectrometry (LC-MS/MS).
Table 4. Results of chromatographic analyses performed by means of liquid chromatography combined with tandem mass spectrometry (LC-MS/MS).
of Stachybotrys chartarum
S. chartarum
on PDA
S. chartarum
on MEA
Metabolites Reported for Stachybotrys in Antibase
Antibiotic F 1839A647010,200
Orsellinic acid21,500<LOD
Unspecific metabolites
Brevianamid F62.71697
Tenuazonic acid<LOD48.7
LOD—limit of detection.
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Piontek, M.; Łuszczyńska, K. Testing the Toxicity of Stachybotrys chartarum in Indoor Environments—A Case Study. Energies 2021, 14, 1602.

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Piontek M, Łuszczyńska K. Testing the Toxicity of Stachybotrys chartarum in Indoor Environments—A Case Study. Energies. 2021; 14(6):1602.

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Piontek, Marlena, and Katarzyna Łuszczyńska. 2021. "Testing the Toxicity of Stachybotrys chartarum in Indoor Environments—A Case Study" Energies 14, no. 6: 1602.

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