key: cord-0977658-l2nv7uqg authors: Szulc, Justyna; Okrasa, Małgorzata; Majchrzycka, Katarzyna; Sulyok, Michael; Nowak, Adriana; Ruman, Tomasz; Nizioł, Joanna; Szponar, Bogumiła; Gutarowska, Beata title: Microbiological and Toxicological Hazards in Sewage Treatment Plant Bioaerosol and Dust date: 2021-09-28 journal: Toxins (Basel) DOI: 10.3390/toxins13100691 sha: b0013789b7b154e687fdf62b87b46f82377e22e6 doc_id: 977658 cord_uid: l2nv7uqg Despite the awareness that work in the sewage treatment plant is associated with biological hazards, they have not been fully recognised so far. The research aims to comprehensively evaluate microbiological and toxicological hazards in the air and settled dust in workstations in a sewage treatment plant. The number of microorganisms in the air and settled dust was determined using the culture method and the diversity was evaluated using high-throughput sequencing. Endotoxin concentration was assessed with GC-MS (gas chromatography-mass spectrometry) while secondary metabolites with LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry). Moreover, cytotoxicity of settled dust against a human lung epithelial lung cell line was determined with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and UHPLC-Q-ToF-UHRMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight ultrahigh-resolution mass spectrometry) analysis was performed to determine the source of cytotoxicity. The total dust concentration in the sewage treatment plant was low and ranged from 0.030 mg m(−3) to 0.044 mg m(−3). The highest microbiological contamination was observed in sludge thickening building and screenings storage. Three secondary metabolites were detected in the air and sixteen in the settled dust. They were dominated by compounds typical of lichen and plants and Aspergillus, Penicillium and Fusarium genera mould. The settled dust from the sludge thickening building revealed high cytotoxicity to human lung epithelial cells A-549 (IC(50) = 6.98 after 72 h). This effect can be attributed to a biocidal compound—didecyldimethylammonium chloride (DDAC-C10) and seven toxic compounds: 4-hydroxynonenal, carbofuran, cerulenin, diethylphosphate, fenpropimorph, naphthalene and onchidal. The presence of DDAC-C10 and other biocidal substances in the sewage treatment plant environment may bring negative results for biological sewage treatment and the natural environment in the future and contribute to microorganisms’ increasing antibiotics resistance. Therefore, the concentration of antibiotics, pesticides and disinfectants in sewage treatment plant workstations should be monitored. Over 300 km 3 of sewage is produced globally every year, which, combined with the progressing urbanisation, leads to an increasing number of wastewater treatment plants (WWTPs) [1, 2] . Wastewater treatment plants are complexes of technological buildings where industrial wastewater and sewage are treated, i.e., dissolved substances, colloids and suspensions are removed from wastewater before discharge to water or the ground [3] . The European Environment Agency (EEA) published a complete database of the existing urban WWTPs in Europe describing 25,906 such facilities [4] . In Poland, there are over 3257 WWTPs [3] . The wastewater treatment plants can be divided into a few different categories depending on the applied wastewater treatment methods and the related processes. In mechanical ones, only non-soluble contaminants (e.g., sedimented or floating solids and fats) are removed with grates, screens and grit chambers. In the chemical WWTPs, wastewater is treated through precipitation of some soluble compounds and neutralised with chemical methods (e.g., coagulation, sorption on active carbon). In biological WWTPs, organic contaminants and biogenic and refractory compounds are removed from wastewater through biological decomposition using microorganisms. Wastewater treatment plants with high removal of biogens enable higher nitrogen and phosphorus reduction [2, 3] . Wastewater biological treatment systems include around a billion active microorganism species, and activated sludge can contain about 4 × 10 8 cells mL −1 . Wastewater biological treatment broadly uses aeration, mixing and compressing to intensify the growth and proliferation of the microorganisms. Wastewater turbulences, intensive flow rate and aeration are considered to promote bioaerosol formation and release into the wastewater treatment plant's atmosphere [5, 6] . The bioaerosol formed in the wastewater treatment plants includes numerous bacilli (Klebsiella pneumoniae, Escherichia coli, Enterobacter agglomerans, Aeromonas hydrophila, Pseudomonas spp.), haemolytic staphylococci (e.g., Staphylococcus aureus), haemolytic streptococci (Streptococcus faecalis, Streptococcus pneumoniae) and Gram-negative coccobaccilli such as Acinetobacter, some Bacillus genera and coliform bacteria [7, 8] . Li et al. detected 300 bacteria species, including opportunistic pathogens such as Comamonas testosteroni and Moraxella osloensis in bioaerosol from a wastewater treatment plant [9] . Han et al. also point out the potential harmfulness of Acinetobacter lwoffii, Aeromonas caviae, Arcobacter spp., Bacillus spp., Bacteroides spp., Flavobacterium spp., Mycobacterium spp., Pseudomonas fluorescens, Zoogloea spp., Kluyvera intermedia, Staphylococcus lentus, Clostridium spp. and Enterobacter spp. bacteria to the health of wastewater treatment plant employees [10, 11] . Wastewater and bioaerosol generated in a WWTPs were also reported as the source of drug-resistant microorganisms and genes that induce antibiotic resistance [12] . The dominant fungi identified in the wastewater treatment plant air included Cephalotrichum spp., Alternaria spp., Penicillium spp., Monilia spp. and Aspergillus spp. [13] . In addition to the potentially pathogenic microorganism species, wastewater treatment plant staff are exposed to inhalation of bacterial endotoxin from the Gram-negative bacteria cell wall. Smit et al. discovered a positive, dose-dependent relationship between exposure to endotoxins and adverse respiratory effects in humans, including wheezing breath, dyspnoea and cough [14] . The wastewater treatment plant staff often complain about ill-health, cough and breathing problems [15] . The most common diseases caused by exposure to harmful biological factors in wastewater include allergic alveolitis, rhinitis and pharyngitis, conjunctivitis, diarrhoea and other infections of the alimentary tract and central nervous system lesions [16] . Friis et al. discovered a higher number of stomach cancer cases among wastewater treatment plant staff, which can be related to the presence of Helicobacter pylori bacteria in the work environment [15] . A disease unit described as 'Sewage Worker's Syndrome' is accompanied by such symptoms as general discomfort, weakness, acute rhinitis and fever [17] . The issue of harmful biological factors hazards in a WWTP's work environment is known and broadly investigated. However, there are no comprehensive tests that would include the presence of cultivable and non-cultivable microorganisms and metabolites of microbiological origin in the air and settled dust in such facilities. No cytotoxicity analyses have been carried out so far for the dust in wastewater treatment plants. That is why this paper was aimed to carry out a comprehensive evaluation of microbiological and toxicological hazards in a sewage treatment plant. It presents the quantity, diversity and profile of chemical compounds of biological origin (including endotoxins and secondary metabolites) occurring in the air and settled dust in a sewage treatment plant. The cytotoxicity was evaluated of settled dust from workstations in the sewage treatment plant against a human lung epithelial cell line and tests were carried out using UHPLC-Q-ToF-UHRMS (ultra-high-performance liquid chromatography-quadrupole time-of-flight ultrahigh-resolution mass spectrometry) to determine why dust from the sewage treatment plant was highly cytotoxic. Such evaluation can be used to define provisions to protect workers from risks to their health and safety, including the prevention of such risks arising or likely to arise from exposure to biological agents at workstations at WWTPs. The results can be utilised by health and safety professionals to recognise activities in which workers may be potentially exposed to biological agents due to their work and thus undertake preventive measures, including the use of appropriate personal protective equipment. The microclimate parameters at the workstations in the sewage treatment plant are summarised in Table 1 . The air temperature in the tested places ranged from 17.4 • C (Workstation 2) to 26.4 • C (Workstation 6). The highest (73.1%) and the lowest (69.8%) relative air humidity were reported for the same workstations, respectively. The airflow rate ranged between 0.03 m s −1 (Workstation 4) and 0.87 m s −1 (Workstation 1). The microclimate conditions at different workstations were highly diversified. Statistically, significant differences were revealed for the measurements carried out on consecutive days and for workstations located close to one another in an open space during measurements performed on the same day. In all cases, the relative humidity at the workstations was high (>57%), which can provide suitable conditions for microorganisms development in the air and their transmission to greater distances, considering the temperature of ca. 25 • C and the reported airflow rate values. The total dust concentration in the sewage plant was very low and ranged from 0.030 mg m −3 (external background) to 0.044 mg m −3 (screenings storage; Table 2 ). At all measurement points, the aerodynamic diameter of the dominant suspended dust fraction was less than 1 µm (Table 2 ). Its share in the total quantity of the measured dust ranged from 73.9% in the sludge thickening building to 92.3% in the sewage treatment plant's environment (screenings storage). The particles with diameters ranging from 1 to 4 µm (1.7-7.5%) constituted the smallest part of the total dust amount. The dust concentration values do not exceed the permissible threshold values at the workstations, and so according to domestic law, there is no need to use respiratory protection against particles. The use of respiratory protection is then voluntary, and the adequate equipment is selected depending on the user's work comfort needs. The possible exposure to the SARS-COV-2 virus is also taken into account. The bacteria count in sewage treatment plant air ranged from 3.05 × 10 2 CFU m −3 (Workstation 3) to 3.71 × 10 3 CFU m −3 (Workstation 4). Actinomycetes were detected only at Workstation 2 (15.0 CFU m −3 ) and the same number was detected in the control atmospheric air (0.5 km away from the sewage treatment plant). The count of mannitol-positive Staphylococci ranged from 2.5 CFU m −3 to 65.0 CFU m −3 . The concentration of Enterobac- teriaceae family bacteria ranged from 2.5 CFU m −3 (Workstation 1) to 1.98 × 10 2 CFU m −3 (Workstation 5) ( Table 3 ). The count of Pseudomonas fluorescens bacteria was also low and amounted to 7.5 CFU m −3 -40.0 CFU m −3 . Haemolytic Staphylococci were most numerous at Workstation 5 (3.25 × 10 2 CFU m −3 ), while their lowest counts were observed at Workstation 3 and 5 (50.0 CFU m −3 ). The fungi count at the tested workstations ranged from 1.91 × 10 3 CFU m −3 (Workstation 4) to 3.23 × 10 4 CFU m −3 (Workstation 5), while for xerophilic fungi, the values ranged from 1.13 × 10 3 CFU m −3 (Workstation 3) to 2.39 × 10 4 CFU m −3 (Workstation 6) ( Table 3) . The results show that the highest microbiological contamination occurred at Workstation 4 (sludge thickening building) and 5 (screenings storage). However, a statistical analysis of the results comparing the microbiological contamination in the air from the tested workstations and control atmospheric air revealed only a few statistically significant differences. It suggests a comparable microbiological condition of the air in and outside the sewage treatment plant premises. The microorganisms count in the sewage treatment plant air was tested in many previous papers, but the characteristic levels have not been determined, so there is still a need to conduct microbiological analyses in such facilities. Li et al. detected the highest count of bacteria (1.7 × 10 3 CFU m −3 ) and fungi (9.3 × 10 2 CFU m −3 ) in a sewage treatment plant in China at sludge thickening stations [9] . The microorganisms count reported by other authors for sewage treatment plant air is highly diversified and ranges from 10 2 to 10 5 CFU m −3 [2, 8] . Moreover, bacteria concentrations in bioaerosol in such work environments can be twice higher and fungi concentrations 8-10 times higher in spring than winter [13] . The volume of microorganisms emission to the air in a sewage treatment plant was proven to vary and depend on the season but also the facility size, quantity and nature of the treated sewage and the sewage treatment process, i.e., sewage treatment plant operation and applied technology [2, 18] . Settled dust at the tested sewage treatment plant workstations occurred only in the sludge thickening building, while other workstations were located in an open space, where settled dust did not accumulate. Enterobacteriaceae family bacteria (1.14 × 10 6 CFU g −1 ) and mannitol-positive Staphylococci (4.03 × 10 5 CFU g −1 ) dominated the dust samples collected from the sludge thickening building, while the total bacteria count was 2.73 × 10 5 CFU g −1 . A similar contamination level was reported for fungi and xerophilic fungi (2.00 × 10 5 CFU g −1 ). In the tested dust, Actinomycetes (1.74 × 10 3 CFU g −1 ) and haemolytic Staphylococci (2.08 × 10 3 CFU g −1 ) ( Figure 1 ) were the least numerous. Enterobacteriaceae family bacteria (1.14 × 10 6 CFU g −1 ) and mannitol-positive Staphylococci (4.03 × 10 5 CFU g −1 ) dominated the dust samples collected from the sludge thickening building, while the total bacteria count was 2.73 × 10 5 CFU g −1 . A similar contamination level was reported for fungi and xerophilic fungi (2.00 × 10 5 CFU g −1 ). In the tested dust, Actinomycetes (1.74 × 10 3 CFU g −1 ) and haemolytic Staphylococci (2.08 × 10 3 CFU g −1 ) ( Figure 1 ) were the least numerous. The literature provides no data on the microorganisms count in the settled dust from workstations in a sewage treatment plant. Only settled dust from the agricultural work environment (poultry breeding rooms, cereal dust) was microbiologically characterised. A higher count of bacteria (3.33 × 10 7 CFU g −1 -1.57 × 10 8 CFU g −1 ) and P. fluorescens bacteria (1.00 × 10 5 CFU g −1 -5.73 × 10 5 CFU g −1 ) was reported. The haemolytic Staphylococci count was higher for poultry farm dust (8.50 × 10 5 CFU g −1 ) and similar for cereal dust (2.98 × 10 3 CFU g −1 ) [19, 20] . The counts of Actinomycetes (≤2.05 × 10 3 CFU g −1 ) and xerophilic fungi (3.35 × 10 5 CFU g −1 -4.45 × 10 5 CFU g −1 ) were on a similar level. The lowest counts were in turn reported for mannitol-positive Staphylococci (1.20 × 10 4 CFU g −1 -3.49 × 10 4 CFU g −1 ), Enterobacteriaceae family bacteria (≤2.30 × 10 4 CFU g −1 ) and fungi (7.48 × 10 4 CFU g −1 -8.75 × 10 5 CFU g −1 ). High-throughput sequencing of the DNA from the air and settled dust collected at Workstation 4 in the sewage treatment plant (sludge thickening building) revealed a high phylogenetic diversity of the microorganisms (Figure 2a,b) . One hundred and seventy (170) genera of bacteria/archeons and 475 genera of fungi, representing 19 and 9 types, SD:1.00 × 10 1 SD:5.00 × 10 0 SD:0.00 × 10 0 SD:0.00 SD:1.00 × 10 1 SD:1.08 × 10 4 SD:1.09 × 10 4 No.-tested workstation number; EB-external background (atmospheric air sampled 0.25 km from the sorting plant); M-mean; SD-standard deviation; A, B, C-the means marked with the same letter in the same column do not differ in a statistically significant way (Tukey's HSD test; p < 0.05). The literature provides no data on the microorganisms count in the settled dust from workstations in a sewage treatment plant. Only settled dust from the agricultural work environment (poultry breeding rooms, cereal dust) was microbiologically characterised. A higher count of bacteria (3.33 × 10 7 CFU g −1 -1.57 × 10 8 CFU g −1 ) and P. fluorescens bacteria (1.00 × 10 5 CFU g −1 -5.73 × 10 5 CFU g −1 ) was reported. The haemolytic Staphylococci count was higher for poultry farm dust (8.50 × 10 5 CFU g −1 ) and similar for cereal dust (2.98 × 10 3 CFU g −1 ) [19, 20] . The counts of Actinomycetes (≤2.05 × 10 3 CFU g −1 ) and xerophilic fungi (3.35 × 10 5 CFU g −1 -4.45 × 10 5 CFU g −1 ) were on a similar level. The lowest counts were in turn reported for mannitol-positive Staphylococci (1.20 × 10 4 CFU g −1 -3.49 × 10 4 CFU g −1 ), Enterobacteriaceae family bacteria (≤2.30 × 10 4 CFU g −1 ) and fungi (7.48 × 10 4 CFU g −1 -8.75 × 10 5 CFU g −1 ). High-throughput sequencing of the DNA from the air and settled dust collected at Workstation 4 in the sewage treatment plant (sludge thickening building) revealed a high phylogenetic diversity of the microorganisms (Figure 2a ,b). One hundred and seventy (170) genera of bacteria/archeons and 475 genera of fungi, representing 19 and 9 types, respectively, were detected in the dust. The number of bacteria/archeons genera identified in the air amounted to 166 (19 types), and the number of fungal genera amounted to 475 (7 types). Both the dust and the air were mainly contaminated by Actinobacteria (68.0 and 21.0%), Proteobacteria (28.0 and 36.0%) and Firmicutes (2.0 and 23.0%). Moreover, a significant share was discovered of the bacteria genus and Bacteroidetes (11.0%) in the settled dust in the sewage treatment plant. Other bacteria types constituted less than 1% OTU Both the dust and the air were mainly contaminated by Actinobacteria (68.0 and 21.0%), Proteobacteria (28.0 and 36.0%) and Firmicutes (2.0 and 23.0%). Moreover, a significant share was discovered of the bacteria genus and Bacteroidetes (11.0%) in the settled dust in the sewage treatment plant. Other bacteria types constituted less than 1% OTU (operational taxonomic unit) in the tested air and settled dust (Figure 2a) . The bacteria genera observed most often among Actinobacteria in the dust included Bifidobacterium (5.0%), Gordonia (3.0%), Corynebacterium (1.0%), Terracoccus (1.0%), Candidatus Microthrix (0.94%) and Cellulosimicrobium (0.71%). The Proteobacteria were dominated by Sphingomonas (4.0%), Acinetobacter (4.0%), Psychrobacter (4.0%), Pseudomonas (4.0%), Serratia (2.0%) and Stenotrophomonas (1.04%) genera. The Firmicutes identified most often in the sewage treatment plant's dust include Turicibacter (1.0%). The Chryseobacterium (10.0%) genus representing Bacteroidetes occurred in the dust with high OTU values. The air collected from the sewage treatment plant was dominated by such Actinobacteria genera as Cellulosimicrobium (59.0%) and Bifidobacterium (6.0%). Pseudomonas (12.0%) and Stenotrophomonas (8.0%) were the most common Proteobacteria. The presence of 44 bacteria genera with the OTU < 1% (Table S1 ) was confirmed among the Firmicutes. Ascomycota and Basidiomycota were the most common fungi in both sample types (dust and air) (Figure 2b ). In the settled dust, Mycosphaerella (12.0%), Cladosporium (6.0%), Neoascochyta (5.0%), Alternaria (4.0%), Didymella (4.0%), Chalastospora (3.0%), Botrytis (2.0%) and Citeromyces (2.0%) had the highest OTU among Ascomycota (59.0%). Basidiomycota were dominated by Cryptococcus (3.0%), Cystobasidium (3.0%), Rhodotorula (1.0%) and Sporobolomyces (1.0%). The Aspergillus and Penicillium genera fungi quantities were similar (2.0-3.0%) in the dust and air samples. Moreover, a high percentage of Mortierella genera representing Mortierellomycota was observed in the dust. For fungi in the air samples, the most common DNA sequences related to Ascomycota included Cladosporium (14.0%), Mycosphaerella (7.0%), Alternaria (5.0%), Didymella (2.0%), Candida (2.0%) and Dissoconium (1.0%) genera. The highest OUT of Basidiomycota in the air was identified for Sporobolomyces (4.0%), Hypholoma (3.0%), Udeniomyces (3.0%), Coprinellus (1.0%), Bjerkandera (1.0%) and Malassezia (1.0%) (Table S2) . Tests presenting in such a detailed way the bacteria and fungi diversity in the sewage treatment plant's air and settled dust were carried for the first time under this study. Previously, many authors were interested in the qualitative and quantitative composition of microorganisms in sludge and sewage. The bacteria genera most often isolated from the air in mechanical and biological sewage treatment plants included Citobacter, Enterobacter, Klebsiella, Serratia and Pantoea, whereas pathogenic bacteria such as Salmonella, Escherichia and Shigella were rarely isolated [21, 22] . Absidia, Actinomucor, Alternaria, Aspergillus, Cladosporium, Fusarium, Geotrichum, Mucor and Penicillium mould and Candida, Cryptococcus and Rhodotorula yeast were most often described as fungi characteristic for sewage treatment plant environment [21, 23] . In the present study, a high-throughput DNA sequencing from the samples collected in the sludge thickening building revealed a broader spectrum of microorganisms occurring in the sewage treatment plant air and dust, including potentially pathogenic microorganisms that can threaten human life. Moreover, Candidatus Microthrix bacteria representing filiform bacteria often occurring in the air in sewage treatment plants using activated sludge technology were detected in the tested samples. The presence of such bacteria can be related to sludge sedimentation problems, i.e., "swelling" [24, 25] . Settled dust in the tested sewage treatment station occurred only in the sludge thickening building (Workstation 4). The endotoxin concentration in the dust samples amounted to 0.214 nmol LPS mg −1 on average (Figure 3 ). diversified and ranges from 0.6 to 370 ng m −3 in the air [28, 29] . The highest endotoxin concentrations were confirmed for the sewage sludge treatment areas. Interestingly, endotoxins from different bacteria can differ in their molecular structures [30, 31] . Moreover, bacteria can adopt to an unfavourable environment to ensure viability. It is known that bacteria are able to modify their primary LPS structure under certain growth conditions. Thereby they can reinforce the external membrane to assure optimal protection against the environment [32, 33] . The literature examples demonstrate that exposure to bacterial endotoxin through inhalation may result in respiratory tract inflammation and toxic pneumonia caused by the non-specific activation of alveolar macrophages that release inflammation mediators. Endotoxins may also cause fever, chills, cough and flu symptoms [34, 35] . In the sludge thickening building of the sewage treatment plant, three chemical compounds were detected in the air and 16 in the settled dust (Table 4 ). Metabolites characteristic for Aspergillus (3-Nitropropionic acid) and Penicillium (Flavoglaucin) genera mould were detected in the air, next to metabolites not related to any specific mould genus (non-specific)-asperphenamate. Compounds produced by Penicillium spp., (quinolactacin A, citreohybridinol, flavoglaucin, pentoxifylline), Fusarium spp. (beauvericin, enniatin A1, enniatin B, enniatin B1) and Aspergillus (3-Nitropropionic acid) dominated in the air. Lichen (lecanoric acid, usnic acid) and plant (prunasin) metabolites, and non-specific compounds (asperglaucide, asperphenamate, cyclo(L-Pro-L-Tyr, emodin) ( Table 4) were also detected. It is worth pointing out that the concentrations of lichen and plant metabolites were higher (14-123 ng g −1 ) than those of metabolites of microbiological origin (concentration range: 0.24-18.1 ng g −1 ). Previously, the literature published no such detailed analysis of mycotoxins content in the air and settled dust from a sewage treatment plant. The latest studies included assessing four metabolite concentrations: aflatoxin B1, gliotoxin, ochratoxin A and sterigmatocystin. The authors detected in the air only aflatoxin B1 and sterigmatoxin at concentrations lower than ng m −3 [36] . [26] . The bacterial endotoxin concentrations obtained in the present study are higher than the values described in the literature for household rooms of different functions (kitchen, bedroom, living room-from 0.092 nmol LPS mg −1 to 0.155 nmol LPS mg −1 ) [27] . Based on studies carried out in many sewage treatment plants in Finland and Norway, the endotoxin concentration in such work environments is diversified and ranges from 0.6 to 370 ng m −3 in the air [28, 29] . The highest endotoxin concentrations were confirmed for the sewage sludge treatment areas. Interestingly, endotoxins from different bacteria can differ in their molecular structures [30, 31] . Moreover, bacteria can adopt to an unfavourable environment to ensure viability. It is known that bacteria are able to modify their primary LPS structure under certain growth conditions. Thereby they can reinforce the external membrane to assure optimal protection against the environment [32, 33] . The literature examples demonstrate that exposure to bacterial endotoxin through inhalation may result in respiratory tract inflammation and toxic pneumonia caused by the non-specific activation of alveolar macrophages that release inflammation mediators. Endotoxins may also cause fever, chills, cough and flu symptoms [34, 35] . In the sludge thickening building of the sewage treatment plant, three chemical compounds were detected in the air and 16 in the settled dust (Table 4 ). Metabolites characteristic for Aspergillus (3-Nitropropionic acid) and Penicillium (Flavoglaucin) genera mould were detected in the air, next to metabolites not related to any specific mould genus (non-specific)-asperphenamate. Compounds produced by Penicillium spp., (quinolactacin A, citreohybridinol, flavoglaucin, pentoxifylline), Fusarium spp. (beauvericin, enniatin A1, enniatin B, enniatin B1) and Aspergillus (3-Nitropropionic acid) dominated in the air. Lichen (lecanoric acid, usnic acid) and plant (prunasin) metabolites, and non-specific compounds (asperglaucide, asperphenamate, cyclo(L-Pro-L-Tyr, emodin) ( Table 4 ) were also detected. It is worth pointing out that the concentrations of lichen and plant metabolites were higher (14-123 ng g −1 ) than those of metabolites of microbiological origin (concentration range: 0.24-18.1 ng g −1 ). Previously, the literature published no such detailed analysis of mycotoxins content in the air and settled dust from a sewage treatment plant. The latest studies included assessing four metabolite concentrations: aflatoxin B1, gliotoxin, ochratoxin A and sterigmatocystin. The authors detected in the air only aflatoxin B1 and sterigmatoxin at concentrations lower than ng m −3 [36] . This study is the first to show the cytotoxic effect of the settled dust samples from a sewage treatment plant. Human lung epithelial cells A-549 (Photo 1) were exposed to 0.3 to 100 mg mL −1 concentrations of water-soluble fractions of dust for 48 and 72 h. The curve representing the test dust's cytotoxicity is shown in Figure 4 . High cytotoxicity of the tested dust was observed. Concentrations of 20 mg mL −1 and more caused 100% cytotoxicity for the tested cells after 48 h and 72 h of exposure. Low IC 50 values (6.98-7.72%, depending on the exposure duration) confirm the high cytotoxicity of the tested dust, which inhibits the test cell population growth by 50%. High cytotoxicity of the dust settled in the sludge thickening buildings suggests the presence of non-biological inhalation hazards at the tested workstation, related to the process carried out at the workstation. 500 mycotoxins and other secondary metabolites in food crops: Challenges and solutions Assessment of microbiological indoor air quality in cattle breeding farms Test No. 442D: In Vitro Skin Sensitisation Are-nrf2 Luciferase Test Method GESTIS: International Occupational Exposure Limit Values for Chemical Agents Applied Statistics for Behavioral Sciences Informed Consent Statement: Not applicable. The data presented in this study are available on request from the corresponding author. The authors declare no conflict of interest.