Microfungal Contamination of Damp Buildings--Examples of Risk Constructions and

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Microfungal Contamination of Damp Buildings--Examples of Risk

Constructions and Risk Materials

by Environmental Health Perspectives

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Environmental Health Perspectives Volume 107, Supplement 3, June

1999

Suzanne Gravesen,1 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

Materials and Methods

Results

Discussion

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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

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This article is based on a presentation at the International

Conference on Indoor Mold and Children held 21-24 April 1998 in

andria, 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@...

Received 3 September 1998; accepted 13 November 1998.

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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.

Materials and Methods 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).

Results 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.

Discussion 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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