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

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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.; Łuszczyńska, 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; stachybotrylactam 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 (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 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/).

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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 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 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 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 [9,29–33]. 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 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): [13,44–49].

spores

hyphae

Figure 2. Spores and hyphae of S.chartarum (Nikon microscopy).

2.2. Cultures of Stachybotrys chartarum

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Energies 2021 14 , , 1602 2. Materials and Methods 3 of 12 2.1. Sample Collection and Cultivation Samples were collected from the inner surfaces of partition walls with visible mould andfromD. 4 magnaplaces:to the evaluate Palace in the Rakow; toxicity a ofbuildingS. chartarum of the andUniversity assess theof Zielona mycotoxic Góra risks (UZ); for a thetenement residents house of water-damaged in Zielona Góra; buildings and the or whereScout’s there house, are otherZielona factors Góra, contributing in Poland. toS. mouldchartarum formation from the in tenement homes where house residents was selected reported for significantfurther research health (Figure problems. 1). 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 fromFigure 4 places:1. Building the partition Palace in of Rakow; the tenement a building house of (Zielona the University Góra, Poland) of Zielona infested Gó withra (UZ); S. a ten- ementchartarum. house in Zielona Góra; and the Scout’s house, Zielona Góra, in Poland. S. chartarum from the tenement house was selected for further research (Figure1). TheThe wallswalls werewere mademade ofof bricksbricks coveredcovered withwithcement–lime cement–limeplaster. plaster.Acrylic Acrylicpaint paintwas was usedused asas thethe finishingfinishing material.material. TheThe wallwall moisturemoisture contentcontent waswas measuredmeasured atat thethe samplesample collectioncollection sitesite usingusing aa TrotecTrotec T650T650 hygrometer.hygrometer. The The moisture moisture content content of of the the partition partition wall wall waswas assessedassessed accordingaccording toto thethe operatingoperating instructionsinstructions suppliedsupplied withwith thethe device.device. WallWallmass mass moisturesmoistures (%)(%) werewere 0–3:0–3: drydry wall,wall, 3–5:3–5: wallwall withwith lowlow moisturemoisture content,content, 5–8:5–8: wallwall withwith mediummedium moisturemoisture content,content, 8–12:8–12: wallwall withwith highhigh moisturemoisturecontent. content. ForFor thethe mycologicalmycological analysis,analysis, aa methodologymethodology developeddeveloped byby thethe CBSCBS (Centraalbureau(Centraalbureau voorvoor Schimmelcultures)Schimmelcultures) [[13]13] waswas applied.applied. InIn thisthis methodology,methodology, aa materialmaterial containingcontaining mouldsmoulds isis disaggregateddisaggregated intointo smallsmall piecespieces andand insertedinserted toto PetriPetri dishesdishes directlydirectly atat thethe collectioncollection sitesite thatthat werewere pre-preparedpre-prepared withwith cultureculture media media [ 43[43].]. FourFour replicatesreplicates werewere takentaken fromfrom eacheach sample:sample: twotwo usingusing MaltMalt ExtractExtract AgarAgar (MEA)(MEA) asas thethe cultureculture mediummedium andand twotwo usingusing PotatoPotato DextroseDextrose AgarAgar (PDA),(PDA), Merck.Merck. ThenThen thethe samplessamples werewere transportedtransported toto thethe laboratorylaboratory ofof thethe InstituteInstitute ofof EnvironmentalEnvironmental Engineering,Engineering, UniversityUniversity ofof ZielonaZielona GGóra,óra, forfor further mycological analysis. They were covered with white linen and incubated at room further mycological analysis. They◦ were covered with white linen and incubated at room temperaturestemperatures between between 18 18 and and 22 22 °C.C. Day/night Day/night rhythmsrhythms werewere maintained.maintained. FromFromthe themixed mixed starterstarter culturescultures purepure (axenic)(axenic) culturescultures werewere isolatedisolated andand againagain transferredtransferred ontoonto thethe PDAPDA andand MEAMEA media,media, respectively.respectively. TheThe totaltotal cultivationcultivation timetime untiluntil singlesingle isolatedisolated speciesspecies werewere observedobserved waswas 2121 daysdays [[4,7,42].4,7,42]. ForFor identificationidentification andand taxonomictaxonomic classificationclassification ofof isolated isolated strains, Nikon light microscopy was used (Figure2): [13,44–49]. strains, Nikon light microscopy was used (Figure 2): [13,44–49].

spores

hyphae

FigureFigure 2.2. SporesSpores andand hyphaehyphae ofofS.chartarum S.chartarum(Nikon (Nikon microscopy). microscopy).

2.2. Cultures of Stachybotrys chartarum

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Energies 2020, 13, x FOR PEER REVIEW2.2. Based Cultures on of the Stachybotrys isolated strains, chartarum mass cultures of S. chartarum were established in 5order of 13 to obtainBased the onmould the isolated biomass strains, needed mass for ecotox culturesicological of S. chartarum and physico-werechemical established tests in using order Based on the isolated strains, mass cultures of S. chartarum were established in order twoto obtain different the growth mould biomassmedia (Figure needed 3a,b): for ecotoxicological MEA and PDAand according physico-chemical to the CBS tests[13]. using to obtain the mould biomass needed for ecotoxicological and physico-chemical tests using two different growth media (Figure3a,b): MEA and PDA according to the CBS [13]. two different growth media (Figure 3a,b): MEA and PDA according to the CBS [13].

(a) (b)

Figure 3. Growth(a )of S. chartarum on different growth(b) media after a three-month incubation (a) PDA, (b) MEA. FigureFigure 3. 3.GrowthGrowth of ofS.S. chartarum chartarum onon different different growth growth media media after after a athree-month three-month incubation incubation (a (a) ) PDA, PDA, (b) MEA. (b) MEA.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 A total of 5 mL of the respective media, PDA, MEA (Merck), were poured onto 30 using Aa totalpreparation of 5 mL ofneedle. the respective Again, samples media, PDA, were MEA incubated (Merck), after were having poured been onto covered 30 Petri Petri dishes with ø of 9 cm. Mould spores were applied centrally onto the growth media withdishes white with linen ø of 9and cm. Mouldcultivated spores at wereroom appliedtemperature centrally (18–22 onto °C) the growthwhile keeping media using the using a preparation needle. Again, samples were incubated after having been covered circadiana preparation rhythm. needle. Then, Again, cultivation samples of the were isolated incubated culture after lasted having 3 months been covered in order with to with white linen and cultivated at room temperature (18–22◦ °C) while keeping the allowwhite growth, linen and sporulation, cultivated and at roomregrowth temperature to extend (18–22over a significantC) while keepingpart of the the Petri circadian dish. circadian rhythm. Then, cultivation of the isolated culture lasted 3 months in order to Therhythm. strain Then, aged cultivationand the mycelium of the isolated was allowed culture to lasted air-dry. 3 months The method in order of tothe allow cultivation growth, allowsporulation, growth, sporulation, and regrowth and to regrowth extend over to extend a signiﬁcant over a significant part of the part Petri of dish. the Petri The dish. strain of the mould according to Piontek [4,7,42] reflects the conditions prevalent on partition Theaged strain and aged the myceliumand the mycelium was allowed was allowed to air-dry. to air-dry. The method The method of the of cultivation the cultivation of the walls in residential housing. With a scalpel, the dried moulds (still containing remains of ofmould the mould according according to Piontek to Piontek [4,7, 42[4,7,42]] reﬂects reflects the conditions the conditions prevalent prevalent on partition on partition walls the culture medium) were scraped off the Petri dishes. The material extracted was wallsin residential in residential housing. housing. With With a scalpel, a scalpel, the the dried dried moulds moulds (still (still containing containing remains remains of of the weighed and transferred for storage to glass jars, which were closed with a ground theculture culture medium) medium) were were scraped scraped off the off Petri the dishes. Petri Thedishes. material The extractedmaterial wasextracted weighed was and stopper (Figure 4). Further analyses (see below) were performed using methanol extracts weighedtransferred and fortransferred storage to for glass storage jars, whichto glass were jars, closed which with were a ground closed stopper with a (Figureground4 ). of these materials. stopperFurther (Figure analyses 4). (seeFurther below) analyses were performed(see below) using were methanolperformed extracts using methanol of these materials. extracts of these materials.

(a) (b)

Figure 4. Stachybotrys chartarumFigure mass 4. cultureStachybotrys after chartarum3 months(a) ofmass incubation culture afteron the 3 monthsPDA medium of incubation (a)( band) ondry the biomasses PDA medium of (a) S. chartarum on PDA medium in a glass jar closed with a ground stopper (b). Figure 4. Stachybotrys chartarumand mass dry biomassesculture after of 3S. months chartarum of incubationon PDA medium on the inPDA a glass medium jar closed (a) and with dry a groundbiomasses stopper of (b). S. chartarum on PDA medium in a glass jar closed with a ground stopper (b). 2.3.2.3. Methanol Methanol Extracts Extracts for for Ecotoxicological Ecotoxicological Tests Tests 2.3. MethanolTheThe method method Extracts developeddeveloped for Ecotoxicological and and reﬁned refined Tests by Piontekby Pion [tek4,42 [4,42,50],50] was appliedwas applied to extract to analytesextract analytesfrom the from samples the samples for further for further mycotoxin mycoto testing.xin testing. Prepared Prepared pure (single pure (single species) species) mould The method developed and refined by Piontek [4,42,50] was applied to extract mouldbiomass biomass samples samples (see Section (see Section 2.2 above) 2.2 above) were collected were collected in the form in the of form 1 g of of air-dry 1 g of air-dry extracts analytes from the samples for further mycotoxin testing. Prepared pure (single species) extracts and held in 100 mL of 80% methanol for 96 h (room temperature). Methanol mould biomass samples (see Section 2.2 above) were collected in the form of 1 g of air-dry extracts were prepared in duplicate. The extracts were then filtered through 47 mm 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

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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 Energies 2021, 14, 1602 toxicological tests (Figure 5). Starting from the two methanol extracts, solutions5 ofwere 12 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; andfurthermore, held in 100 control mL of tests 80% methanolwere added. for Ten 96 h cut (room organisms temperature). were inserted Methanol into extracts each beaker were preparedso as to have in duplicate. thirty organisms The extracts per concentration were then filtered level. through The mortality 47 mm fiberglassof the planarians filter discs was (Whatmandetermined GF/C), after ten and days the supernatant (240 h) in order was collectedto establish for the ecotoxicological lethal concentration analysis for in whichhalf of D.the tigrina organismsand D. (240 magna h LCwere 50), used[52-55]. as theA graphical indicator method organisms. (probit In thisanalysis) procedure, was applied 1 mL of in 2 extractthis interpretation was obtained step. from The 10.0 mgχ -test of anwas air-dried used to sample check (moulds whether + medium).the empirical results matched the normal distribution. Agreement was considered sufficient when the χ2-test 2.4.resulted Ecotoxicological in probabilities Tests Usinghigher Dugesia than 0.7 Tigrina [56]. The toxicity, according to the ecotoxicological tests,Following was established the concepts following of Piontek Liebmann’s [51], planarians classification were [57] cultivated (Table 1). and According used in toxico- to the logicalstandard tests [58], (Figure the 5control). Starting tests from ascertain the two whether methanol any extracts, foreign solutions factors, were apart diluted from tothe differenttoxicity concentrations,of the tested substances, and 40 mL ofinterfere a test solution with the was test stored or innot, beakers as evidenced with a capacity by the ofmortality 50 mL. Each of the concentration control organisms level was not prepared exceeding in three10%. repetitions;This also applies furthermore, when checking control teststhe condition were added. of the Ten bio-indicators cut organisms used were in the inserted research. into each beaker so as to have thirty organisms per concentration level. The mortality of the planarians was determined after tenTable days 1. (240Toxicity h) in classes order of to poison establish substances the lethal [57]. concentration for half of the organisms (240 h LCResult 50), [ 52of –Toxicity55]. A graphical Test–LC method (probit analysis) was applied in this interpretation χ2 step. The 50-test Value was used to checkToxicity whether Classes the empirical results matched Classes the normal distribution. Agreement was considered sufficient when the χ2-test resulted in probabilities (mg∙L−1) higher than 0.7 [56]. The toxicity, according to the ecotoxicological tests, was established fol- <1 highly toxic I lowing Liebmann’s classification [57] (Table1). According to the standard [ 58], the control tests ascertain1–10 whether any foreign factors,potently apart toxic from the toxicity of the tested II substances, interfere with10–100 the test or not, as evidencedmoderately by the toxic mortality of the control organismsIII not exceeding 10%.100–1000 This also applies when checkingslightly toxic the condition of the bio-indicators IV used in the research.>1000 barely toxic V

FigureFigure 5. 5.Breeding Breedingof of Dugesia Dugesia tigrina tigrinaused used for for ecotoxicological ecotoxicological tests. tests.

Table2.5. Ecotoxicological 1. Toxicity classes Tests of poison Using substances Daphnia Magna [57]. The daphnia used for ecotoxicological testing were bred in the laboratory of the Result of Toxicity Test–LC 50 Value Institute of Environmental Engineering at theToxicity Zielona Classes Góra University. Classes In order to (mg·L−1) conduct the toxicological tests, 3-day-old organisms of equal size and condition were collected. Two methanol<1 extracts obtained from the highly dry toxicbiomass of S. chartarum I cultivated 1–10 potently toxic II 10–100 moderately toxic III 100–1000 slightly toxic IV >1000 barely toxic V

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 Energies 2021, 14, 1602 6 of 12

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 (Table2). 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 Table3. 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). Energies 2021, 14, 1602 7 of 12

Table 2. Mycological and moisture analysis of samples.

Coexisting Moulds and No. Sampling Place Wall Finishing Material Wall Humidity [%] Air Humidity [%] Organisms Stachybotrys chartarum, Ulocladium botrytis, Penicillium 1. Palace in Rakow, Poland glue paint 8 72 chrysogenum, Bacteria Stachybotrys chartarum, Building of UZ, Zielona Penicillium chrysogenum, 2. acrylic paint 10 65 Góra, Poland Actinobacteria Bacteria Stachybotrys chartarum *, Penicillium chrysogenum, 3. Tenement house, Poland acrylic paint 11 53 Mucor hiemalis Bacteria Stachybotrys chartarum, Scout’s house, Zielona Penicillium chrysogenum, 4. wallpaper 10 58 Góra, Poland Actinobacteria Bacteria * 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.

Type of LC 50 Toxicity Class Medium/Bioindicator [mg·L−1] According to Liebmann PDA D. tigrina 67.6 class III (moderately toxic) D. magna 75.9 class III (moderately toxic) MEA D. tigrina 169.8 class IV (slightly toxic) D. magna 190.5 class IV (slightly toxic)

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 (Table4). 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). Energies 2021, 14, 1602 8 of 12

Table 4. Results of chromatographic analyses performed by means of liquid chromatography combined with tandem mass spectrometry (LC-MS/MS).

S. chartarum S. chartarum Metabolites on PDA on MEA of Stachybotrys chartarum [ng/g] [ng/g] Metabolites Reported for Stachybotrys in Antibase Stachybotryamide 109,000 62,500 Stachybotrylactam 27,100 46,300 Antibiotic F 1839A 6470 10,200 Aurantine 67.1

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). Ad- ditionally, according to the chromatographic test, S. chartarum on MEA did not produce Energies 2021, 14, 1602 9 of 12

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 [3,18,22,65–67], 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 heamato- logic 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. Energies 2021, 14, 1602 10 of 12

Supplementary Materials: The following are available online at https://www.mdpi.com/1996-107 3/14/6/1602/s1. 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. Funding: 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. Acknowledgments: We would like to express our gratitude to M. Sulyok from the Center for Analyt- ical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU) and to Piotr Jedziniak and Marta Pi ˛atkowskafrom 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.

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