Environmental Health Perspectives Volume 107, Supplement 3, June 1999
Microfungal Contamination of Damp Buildings--Examples of Risk Constructions and Risk Materials
Suzanne Gravesen,1 Peter A. Nielsen,1 Randi Iversen,1 and Kristian Fog Nielsen1,2
1Energy and Indoor Climate Division, Danish Building Research Institute, Hørsholm, Denmark; 2Department of Biotechnology, Technical University of Denmark, Lyngby, Denmark
Abstract
To elucidate problems with microfungal infestation in indoor environments, a multidisciplinary collaborative pilot study, supported by a grant from the Danish Ministry of Housing and Urban Affairs, was performed on 72 mold-infected building materials from 23 buildings. Water leakage through roofs, rising damp, and defective plumbing installations were the main reasons for water damage with subsequent infestation of molds. From a score system assessing the bioavailability of the building materials, products most vulnerable to mold attacks were water damaged, aged organic materials containing cellulose, such as wooden materials, jute, wallpaper, and cardboard. The microfungal genera most frequently encountered were
Penicillium (68%),
Aspergillus (56%),
Chaetomium (22%),
Ulocladium, (21%),
Stachybotrys (19%) and
Cladosporium (15%).
Penicillium chrysogenum, Aspergillus versicolor, and
Stachybotrys chartarum were the most frequently occurring species. Under field conditions, several trichothecenes were detected in each of three commonly used building materials, heavily contaminated with
S. chartarum. Under experimental conditions, four out of five isolates of
S. chartarum produced satratoxin H and G when growing on new and old, very humid gypsum boards.
A. versicolor produced the carcinogenic mycotoxin sterigmatocystin and 5-methoxysterigmatocystin under the same conditions.
Key words: allergy,
Aspergillus versicolor, building materials, mold, mycotoxins,
Penicillium chrysogenum, Stachybotrys chartarum. --
Environ Health Perspect 107(suppl 3):505-508 (1999).
http://ehpnet1.niehs.nih.gov/docs/1999/suppl-3/505-508gravesen/abstract.html
This article is based on a presentation at the International Conference on Indoor Mold and Children held 21-24 April 1998 in Alexandria, Virginia.
Address correspondence to S. Gravesen, Energy and Indoor Climate Division, Danish Building Research Institute, Dr. Neergaards Vej 15, DK-2970 Hørsholm, Denmark. Telephone: 45 45 86 55 33. Fax: 45 45 86 75 35. E-mail: sug@sbi.dk
Received 3 September 1998; accepted 13 November 1998.
The correlation between dampness and mold growth, house dust mites, and airway problems has been demonstrated and known for several years (1-5). Previous studies on indoor molds such as Alternaria alternata, Aspergillus fumigatus, and Cladosporium herbarum have been aimed mainly at their allergenic effects and characterization of allergens (6). Newer investigations have, however, dealt with the health implication of exposure to both the allergens and the metabolic products derived from the molds (7,8). Recent studies have revealed that molds growing on building materials produce and liberate several biologically active nonallergenic compounds. Some of these studies demonstrate mold growth on materials with subsequent detection of mycotoxins, e.g., alternariols, chaetoglobosin, mycophenolic acid, satratoxin, and sterigmatocystins--mycotoxins with potential dermatoxic, immunosuppressive and carcinogenic effects (9-11). From documented cases of mold allergies and the recent identification of mycotoxins, it can be concluded that prolonged presence in water-damaged buildings with extended mold growth may result in unwanted health effects (8,12,13).
Consequently, a multidisciplinary pilot study granted by the Danish Ministry of Housing and Urban Affairs was conducted with the purposes of identifying constructions and building materials critical for mold contamination, the most frequently encountered molds found on infested building material, and the possible harmful fungal metabolites produced on the these materials.
The susceptibility of the buildings to humidification was identified by registration of the state of maintenance of 23 public buildings consisting mainly of schools, kindergartens, and other nonindustrial public buildings. A chart with relevant physical, chemical, and building parameters was filled in during the visual inspection of the buildings registered as water damaged. The chart was expanded to collect background information necessary to identify the types of constructions and materials at risk for water ingress, water leakage, humidification, and subsequent microbial growth.
Collection of infected building materials was done during the visual inspection of the buildings and 72 samples, approximately 10 
20 cm, were collected and placed in aroma-tight bags for later chemical and mycological analyses.
For identification of factors regarded as critical for the establishment and subsequent growth of microfungi on the materials, i.e., the bioavailability of the material, a score system was set up with and an index describing the different conditions for the collected materials. The chart described the following parameters:
- surface texture of a material
- state of maintenance
- age
- load (wear and tear on the material)
- availability for cleaning
- cleanliness
Each parameter scored 0 or 1.
Because knowledge is limited regarding a detailed evaluation of the parameters listed in the chart, the materials were given an index from 0 to 6, with 0 representing a low risk and 6 a high risk for microfungal growth. For example, an old (score 1) wooden floor with cracks and scratches (score 1), a worn-out coat of lacquer (score 1), poor maintenance (score 1), clean floor (score 0), and good cleaning availability (score 0) would give an index of (1+1+1+1+0+0) = 4.
For detection and identification of microfungi, samples from building materials with heavy microbial growth, visible to the naked eye, were taken by means of 5-cm contact plates with V8 agar as growth medium (14).
The plates were inspected after 3 days and again after 1 week. The colonies were counted and identified, if possible to the species level after current taxonomical standard criteria (15). Species from taxonomically difficult genera such as Aspergillus and Penicillium were further cultivated on Czapek yeast autolysate agar (CYA), yeast extract sucrose agar, (YES), malt extract agar (MEA), oat meal agar (OAT), and creatine sucrose agar (CREA) (15) for morphologic and chemotaxonomic criteria (16).
For direct identification, to genus level, of fungi growing on the materials, the tape method was applied. A piece of transparent adhesive tape was gently pressed on the infected material and then stained with lacto-fuchsin before phase contrast microscopy (10,17).
Detection of mycotoxins was performed partly as laboratory experiments on artificially inoculated materials and partly on field samples as described by Nielsen et al. (10,11).
For the artificially inoculated materials (11), the following building materials were used: new gypsum boards, new plywood, old gypsum boards, pieces of old pine, acoustic ceiling boards consisting of mineral wool with glass-fiber wall paper and wallpapered gypsum boards. Materials were artificially infested with pure cultures of Aspergillus versicolor (five isolates), Stachybotrys chartarum (five isolates), and Trichoderma spp. (eight isolates), isolated from infested materials used in Danish buildings.
Mycotoxin Analyses
Trichothecenes were hydrolyzed to their parent alcohols and derivatizated to the heptafluorobutyrlated ester. Extracts from Trichoderma were also analyzed without the hydrolysis step. The hepatafluorobuturylated derivatives were detected using gas chromatography ion trap mass spectrometry and negative ion chemical ionization. Standards of T-2 toxin, HT-2 toxin, diacetoxysrcipenol, fusarenon-X, deoxynivalenol, nivalenol, verrucarol, and trichodermol were available, as described by Nielsen et al. (10,11).
Sterigmatocystins were detected using high-performance liquid chromatography diode array detection on a C18 column with a water-acetonitrile gradient system (10). Extracts were also analyzed for sterigmatocystin by thin-layer chromatography spraying with AlCl3 staining (10).
Identification of Construction Types and Materials Susceptible to Humidification
For the majority of the 72 material samples investigated, the period of humidification was more than 6 months. For 37 of the materials, the main reason for water damage with subsequent infestation of molds was water leakage through roofs. In one-third of the buildings investigated, the water ingress resulted from various defects in flat roofs. Half the roofs suffered from water ingress because of different types of defective junction details and construction failures in incomplete junctions between roofs that had been built together. Rising damp (12 samples) and defective plumbing installations (9 samples) were further causes of mold growth, although with minor impact compared with the defective roof constructions. Other minor reasons for humidification of materials (14 samples) were penetration of cleaning water and condensation.
The susceptibility to fungal intestation of the materials was expressed in the index showing a score from 4 to 6 for 62 of the samples, which indicates a poor resistance to mold infestations (9). The humidification of materials had lasted more than 6 months altogether, indicating that the water ingress was not a result of a single accident but was caused by long-term lack of remediation.
Description and Evaluation of Materials and Constructions at Risk for Mold Infestation
Table 1 indicates the degree of bioavailability in the different materials groups. In this study, building materials most vulnerable to mold attacks were water-damaged, aged organic materials containing cellulose, such as wooden materials, jute, wallpaper, and cardboard. Other groups of materials having a high degree of bioavailability were linoleum and insulation materials for plumbing installations (canvas). The deposition of dust and dirt, together with long-lasting penetration of water, may also lead to fungal growth on inorganic material such as mineral wool. Generally, it can be concluded that leaking roofs were responsible for mold growth in all of the material groups examined.
Identification of the Isolated Molds
Table 2 shows that Penicillium, Aspergillus, and Chaetomium are the genera most frequently isolated from the building materials. S. chartarum is the species most often identified on the material samples, followed by A. versicolor. Penicillium chrysogenum is number three. A substantial number of isolated Penicillia are, however, not identified to the species level, making P. chrysogenum a possible candidate for ranking as number one on the list of species.
As seen in Table 3, trichothecenes were detected on all four building materials naturally infested with Stachybotrys. On the artificially infested materials, four of five cultures of S. chartarum were capable of producing trichothecene mycotoxins on damp building materials from the three heavily contaminated materials investigated. Furthermore, trichodermol was detected, probably originating from a low toxigenic strain of S. chartarum.
Under experimental conditions, each of five isolates of A. versicolor produced sterigmatocystin (Table 3). Two of the isolates produced sterigmatocystins, constituting about 1% of the biomass scraped off the material.
Table 4 lists the number of findings of the different genera on each material group. S. chartarum was most often identified on wood, insulation materials for installations (canvas), and gypsum boards. A. versicolor was not connected to any specific group of materials but had a broad range of favorite substrates such as damp floor joists, wet, dirty mineral wool, and moist wallpaper.
Studies from the most recent years have demonstrated that microfungi produce a substantial number of biologically very active substances other than allergens (10,18-21). A new paradigm for the significance of exposure to microfungi in the indoor climate has been developed since mycotoxins of the trichothecene type have been detected from airborne spores, dust, and infected buildings (20,22,23). Furthermore, it has been experimentally documented that the toxic effects of mycotoxin T-2 toxin were 10 to 20 times stronger by inhalation than by ingestion (24).
As the buildings investigated in this study were specially selected and known to have problems, they do not provide sufficient data to recommend certain building materials to inhibit or avoid mold infestation in case of leakage and humidification for a prolonged period of time. Such recommendations will be published as one of the outcomes of the Danish Mold Programme, 1998-2001.
The main reasons for water ingress, which caused damage to the buildings, were leakage through flat roofs, rising damp, and defective plumbing installations.
The most important factor for mold growth was water activity (aw). Water activity of 0.96 corresponding to a relative humidity of 96% (at steady state) yielded significantly poorer growth of S. chartarum compared with an aw = 0.98% (9). This indicates the importance of using dry gypsum boards in a new building and keeping the boards dry to prevent microbial growth.
Compared to the main reason for the dampness of materials (leaking roofs), rising damp and defective plumbing installations were minor causes of mold growth in the cases investigated.
The susceptibility to fungal growth or the potential for microbial growth is an expression of the interaction between the material itself and the different influences of the environment. The evaluation of the materials examined in this study demonstrated a high degree of susceptibility to fungal growth, indicating a low resistance to mold infestation. Because the surface of a material damaged and exposed to various degradation processes as it ages, the actual age of a material plays an important role for microbial infestation.
In addition to the parameters included in the evaluation chart, the content of biologically degradable components is essential. A material can be classified as organic or inorganic depending on the amount of biodegradable components; cellulose is an important component.
Concerning possible adverse health reactions, the investigated building materials infested with S. chartarum and A. versicolor consistently demonstrated the presence of the trichothecene mycotoxins and the carcinogenic mycotoxin sterigmatocystin, respectively (10,11,20,22,23).
A building-associated fungal flora (funga) was identified, if possible to species level. The microfungal genera most frequently isolated from the 72 samples of building materials were Penicillium (68%), Aspergillus (56%), Chaetomium (22%), Ulocladium (21%), Stachybotrys (19%), Cladosporium (15%), Acremonium (14%), Mucor (14%), Paecilomyces (10%), Alternaria (8%),Verticillium (8%), and Trichoderma (7%). These are all known to cause different types of inhalation allergy (25). The species most frequently encountered were S. chartarum, P. chrysogenum, and A. versicolor.
A. alternata, C. herbarum, and Ulocladium chartarum are important inhalation allergens also frequently recorded outdoors (6,25). Heavy exposure to spores from A. fumigatus and Paecilomyces variotii may cause allergic alveolitis (hypersensitivity pneumonitis) (6,25).
Furthermore, it is experimentally documented that other microfungi frequently found in this study such as Alternaria spp., Chaetomium spp., Penicillium brevicompactum, Penicillium polonicum, and Aspergillus niger produce mycotoxins such as alternariol, alternariol-monomethyl ether, chaetoglobosins A and C, verrucosidin, verrucofortine, mycophenolic acid, naphtho-
-pyrones, and many unknown secondary metabolites (26,27).
Under field conditions the toxic macrocyclic trichothecenes and trichodermol were detected by scraping fungal material from each of four materials investigated, which were heavily contaminated with S. chartarum. This is the first time that trichothcenes have been detected in Danish buildings. Materials infested with A. versicolor produced the cancerogenic mycotoxin sterigmatocystin, and 5-methoxy-sterigmatocystin, constituting a potential health hazard. This is the first time this mycotoxin has been detected from building materials. The production of sterigmatocystin from two of the isolates was 1% of the total biomass removed, which is considered a large amount.
For the assessment of the condition of a mold-infested building, to establish standards for remediation, including choice of safe materials and constructions, and to recommend methods for removal and prevention of mold growth, more medical, biologic, and technical knowledge must be gained. Such recommendations will, however, be published as part of the outcomes of the Danish Mold Programme 1998-2001.
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Last Updated: May 18, 1999
