Mycotoxins in Crude Building Materials

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Mycotoxins in Crude Building Materials from Water-Damaged Buildings

by AEM

Tapani Tuomi,1,* Kari Reijula,1 Tom sson,1 Kaisa Hemminki,2 Eeva-

Liisa Hintikka,1 Outi Lindroos,1 Seija Kalso,3 Pirkko Koukila-

Kähkölä,4 Helena Mussalo-Rauhamaa,5 and Tari Haahtela5

Finnish Institute of Occupational Health (FIOH), Uusimaa Regional

Institute, FIN-00370 Helsinki,1 City of Vantaa Environment Center,

FIN-01300 Vantaa,2 City of Helsinki Environment Center, FIN-00530

Helsinki,3 and HUCH Diagnostics, Mycological Laboratory,4 and

Department of Dermatology and Allergic Diseases,5 Helsinki University

Central Hospital, FIN-00250 Helsinki, Finland

ABSTRACT

We analyzed 79 bulk samples of moldy interior finishes from Finnish

buildings with moisture problems for 17 mycotoxins, aswell as for

fungi that could be isolated using one medium andone set of growth

conditions. We found the aflatoxin precursor,sterigmatocystin, in 24%

of the samples and trichothecenes in19% of the samples.

Trichothecenes found included satratoxin Gor H in five samples;

diacetoxyscirpenol in five samples; and3-acetyl-deoxynivalenol,

deoxynivalenol, verrucarol, or T-2-tetraolin an additional five

samples. Citrinine was found in three samples. Aspergillus

versicolor was present in most sterigmatocystin-containingsamples,

and Stachybotrys spp. were present in the samples wheresatratoxins

were found. In many cases, however, the presence offungi thought to

produce the mycotoxins was not correlated withthe presence of the

expected compounds. However, when mycotoxins were found, some

toxigenic fungi usually were present, even ifthe species originally

responsible for producing the mycotoxinwas not isolated. We conclude

that the identification and enumerationof fungal species present in

bulk materials are important to verifythe severity of mold damage but

that chemical analyses are necessaryif the goal is to establish the

presence of mycotoxins in moldymaterials.

INTRODUCTION

Mycotoxins are " natural products produced by fungi that evoke a toxic

response when introduced in low concentrations to highervertebrates

by a natural route " (J. W. , Editorial, Mycopathologia100:3-5,

1987). These compounds can cause a wide range of acuteand chronic

systemic effects in humans and animals that cannotbe attributed to

fungal growth within the host or allergic reactionsto foreign

proteins. The over 400 known mycotoxins are all complex organic

compounds, most with molecular masses between200 and 800 kDa, that

are not volatile at ambient temperatures.Inhalant exposure to

mycotoxins can occur by inhaling airborneparticulates containing

mycotoxins, including dust and fungalcomponents. In agricultural

settings, mycotoxicoses in both farmanimals and humans can result

from oral, dermal, or inhalant exposureof mycotoxin-contaminated

grain or dust (for reviews, see references. In laboratory

mammals,symptoms can be induced by systemic, oral, dermal,

subcutaneous,or inhalant exposure, with inhalant exposure in

somecases being several orders of magnitude more toxic than dermalor

even systemic administration.

Toxigenic fungi have been isolated from building materials and air

samples in buildings with moisture problems, where the residents have

suffered from nonspecific symptoms possibly relatedto mycotoxin

production, such as cough; irritation of eyes, skin,and respiratory

tract; joint ache; headache; and fatigue. In some cases involving

Stachybotryschartarum (Ehrenberg ex Link) , exposure has

resulted inpulmonary hemorrhage, and S. chartarum isolates from

suchsites have been shown to produce a number of mycotoxins,

includingsatratoxins. Very few studies have, however, establisheda

causal relationship between mycotoxin exposure and building-related

illnesses.

All known mycotoxins are fungal secondary metabolites, which means

that mycotoxin production need not be correlated with thegrowth and

proliferation of the producing species and that factorssuch as

induction, end product inhibition, catabolite repression,and

phosphate regulation will determine production.Therefore, even though

some fungi can grow on almost any naturalor synthetic construction

material, mycotoxin production occurspreferentially on materials that

both allow these fungi to growand provide the conditions for

mycotoxin production. From themany studies of the production of

mycotoxins by fungal isolatesderived from agricultural environments,

a great deal is known about the fungal species that are capable of

producing known mycotoxinsand about the growth media and conditions

that induce production. It is known that some species includestrains

that produce mycotoxins and others that lack this ability. It also

has been established that many of the knownmycotoxin producers are

frequent colonizers in indoor environments. Less is known, however,

about the presenceof mycotoxins in indoor environments, and it is

only in recent years that the presence of some mycotoxins has been

verified incrude building materials.In fact, most mycotoxins have yet

to be extracted from eitherair samples or bulk material derived from

indoorenvironments.

Satratoxins belong to the macrocyclic trichothecene class of

mycotoxins. Over 100 trichothecenes with irritatory and immuno

suppressive effects are known. Most trichothecenes were

originallyisolated from species of Fusarium, but they also may be

producedby other fungi, such as species of Stachybotrys,

Trichothecium,Cylindrocarpon, Myrothecium, Trichoderma,

Vertinosporum, and Acremonium. Other mycotoxins potentially presentin

indoor environments include the carcinogenic aflatoxins andtheir

precursor, sterigmatocystin, which has immuno suppressive and

carcinogenic properties. Fumonisins, ochratoxins (nephrotoxicand

carcinogenic), zearalenone (estrogenic), gliotoxin

(immunosuppressive),patulin (carcinogenic and neurotoxic), and

citrinine (nephrotoxic)also may be present (reviewed in reference).

Penicilliumand Aspergillus species also may produce mycotoxins,

commonlyfound in association with indoor air problems).

Aspergillusochraceus Wilhelm (ochratoxin A), Aspergillus fumigatus

Fresenius(fumitremorgins, gliotoxin, and verrucologen), Aspergillus

versicolor(Vuillemin) Tiraboschi (sterigmatocystin), Aspergillus

flavusLink (aflatoxins), Aspergillus parasiticus Speare

(aflatoxins),and Penicillium citrinum Thom (citrinine) are among

those of particularconcern. Sterigmatocystin also may be produced

byA. flavus, Aspergillus nidulans, Aspergillus rugulosus,

Aspergillusunguis, Bipolaris spp., and Chaetomium spp., while

Penicilliumverrucosum and Penicillium viridicatum may produce

citrinine.

In the present study, over a period of 4 months, we collected samples

for mycotoxin analysis from four major environmental laboratories in

southern Finland that are collectively responsiblefor over 90% of the

mycological analyses performed on moisture problem sites in this

area. Samples were selected based on mycologicalanalyses down to

genus level. A group of 17 mycotoxins likelyto be encountered in

indoor environments were analyzed, including 4 macrocyclic

trichothecenes, 10 nonmacrocyclic trichothecenes,citrinine,

sterigmatocystin, and ochratoxin A. As only one setof growth

conditions was used to isolate fungi growing on oneparticular medium,

we did not attempt to identify the fungi responsible for producing

the mycotoxins in each case. Rather, our objectives were to establish

(i) whether these mycotoxins occur in moisture problem sites, (ii) in

what materials individual mycotoxins occur, and (iii) which fungal

species are associated with mycotoxin-containing samples.

MATERIALS AND METHODS

Sample composition. We analyzed 79 bulk samples of moldy interior

finishes, including samples of wallpaper, cardboard, wood, plywood,

plasterboard, paper-covered gypsum board, mineral wool, plaster,

sand, soil, linoleum, polyurethane insulation, pipe insulation, and

paint.The samples were collected from buildings where a moisture

problem had been detected either by a municipal inspector or by an

occupational hygienist. Additionally, in all these buildings, the

examining inspector, hygienist, or physician had recorded the

presence of symptomatic individuals, or possibly a mold-induced

disease. The79-sample subset was selected from a larger group based

on two criteria: (i) selected samples were usually covered with

visible fungal growth, and (ii) one or more of the following species

dominated in CFU measurements: Fusarium spp., Stachybotrys spp.,

Trichotheciumspp., Cylindrocarpon spp., Myrothecium spp., Trichoderma

spp.,Verticinosporum spp., Acremonium spp., Bipolaris spp.,

Chaetomiumspp., A. fumigatus, A. ochraceus, A. nidulans, A. flavus,

A. unguis,A. versicolor, A. rugulosus, P. verrucosum, P. citrinum,

and P. viridicatum. Samples were collected over a period of 4 months

by health inspectors, occupational hygienists, or environmental

inspectors and made available to us by the City of Helsinki

Environment Center, Helsinki, Finland; the City of Vantaa Environment

Center,Vantaa, Finland; the Finnish Institute of Occupational Health

(FIOH), Uusimaa Regional Institute, Helsinki, Finland; HUCH

Diagnostics,Mycological Laboratory, Helsinki University Central

Hospital, Helsinki, Finland; and the Department of Dermatology and

AllergicDiseases, Helsinki University CentralHospital.

Isolation and identification of fungal species. Fungal propagules

were isolated from a suspension of 10 g of material in 90 ml of

buffer solution (0.3 mM KH2PO4, 2.1 mM MgSO4,2 mM NaOH, 0.02% Tween

80). Dilutions from 102 to 105 were spread on 2% malt extract agar

(Difco, Detroit, Mich.).Plates were incubated, in the dark, at 25°C

for 7 days prior to enumeration and identification. Fungi were

identified morphologically to species or genus level.

Preparation and analysis of mycotoxin samples. Mycotoxins were

extracted with aqueous 95% methanol, purified by a hexane wash and

solid-phase extraction, separated by reverse-phasehigh-pressure

liquid chromatography (HPLC), identified by tandemmass spectrometry,

and quantified using electrospray ionization(ESI) on a quadrupole ion

trap mass analyzer, as described previously.

The analytes were introduced to the mass spectrometry detector by

injecting 10 µl of sample through an HPLC system consisting of an

Alliance 2690 separations module (Waters Associated, Milford,Mass.)

connected to a Lichrocart 250-3 Purospher RP18 column

(Merck,Darmstadt, Germany) online with a four-by-four Purospher

precolumn(Merck), both operated at 30°C ( chromatography column

oven model 7981, HPLC Technology Company Ltd.). A methanol-

aqueousbuffer (10 mM ammonium acetate) solvent system was used.

Sodiumacetate (20 µM) was added to solvents for enhancement of

cationizationin ESI-mass spectrometry. An initial methanol

concentration of20% was held for 4 min, after which the concentration

of methanol was raised linearly to 70% at 8 min. This concentration

was held for 11.5 min, after which the concentration was raised

linearly within 1 min to 90%. The final concentration was held for

15.5 min. The flow rate was 400 µl/min. Between samples, 10 µl of

puremethanol was injected into the column and the column was eluted

for 4 min with 90% methanol before lowering the methanol

concentrationto 20% in 1 min and conditioning for 4 min with this

solvent.This protocol minimized cross contamination of samples.

Mass spectral analysis was performed on a Finnigan LCQ (Finnigan

Corp., San , Calif.) fitted with an ESI probe. The operating

conditions were optimized using T2 toxin, roridin A (RDRA), andT2-

tetraol. These conditions were as follows. The ESI probe was operated

in the positive ion mode and set at a voltage of 1.10 kV. Pressurized

nitrogen (690 kPa) was used as auxiliary and sheath gas with a flow

rate of 2.5 and 47 dm3/min, respectively. Helium was used for

collision-induced dissociation at a pressure of 275 kPa. Capillary

temperature was 260°C, and capillary voltage was 46 V with a tube

lens offset of 55 V. Thesystem includes two octapole ion guides with

an interoctapolelens in between. The first octapole direct current

offset potential was 3.24 V, and the second was 6.5 V, with the

interoctapolelens voltage set at 16 V and the octapole RF amplitude

at 400. The electron multiplier voltage was set to 800 V. For

collision-induceddissociation experiments, the relative collision

intensity in the ion trap varied from 12.6 (verrucarol) to 25.0

(satratoxinH [sATH] and RDRA). Maximum injection time was 200 ms, and

totalmicroscans were set to 3. Samples were not analyzed in

replicates.To each sample, 2 µg of the alkaloid reserpine was added

as aninternal standard prior to the extraction procedure. Each

sampleseries of six samples contained one or more blank samples to

exclude the possibility of false positives. Blank samples were

analyzed prior to injecting the actual samples and once more after

thelast sample had been analyzed. The ion trap, particularly whenused

as a tandem mass spectrometric device as in the present study,is

qualitatively reliable. However, the accuracy of the quantitative

analysis was limited by the characteristics of the ion trap, which is

a semiquantitative rather than a precise quantitative instrument.

Yields of the extraction and purification procedure ranged from 7 to

92%, and detection limits ranged from 0.02 to 200 ng. Irrespective of

the compound, the intensity of atleast two major fragments was used

for quantitation purposes.

RESULTS

Thirty-four of the 79 samples analyzed (43%) contained one or more of

the mycotoxins. Mycotoxins were found inmost of the material

categories tested, with most (82%) of themycotoxin-positive samples

containing cellulosic matter, suchas paper, board, wood, or paper-

covered gypsum board.

Fifteen samples (19%) contained trichothecenes, 5 containing the

macrocyclic trichothecene satratoxin G or SATH,and 10 containing one

of the nonmacrocyclic trichothecenes, diacetoxyscirpenol(DAS),

deoxynivalenol (DON), 3-acetyl-DON (3-Ace-DON), T2-tetraol,or

verrucarol. The most prevalent toxin was sterigmatocystin,which was

detected in 19 samples (24%), while three samples (4%)contained

citrinine (Table 2).

Fungi associated with mycotoxin-containing samples. Eighteen of 63

samples contaminated with Aspergillus spp. contained

sterigmatocystin, with A. versicolor occurringmost frequently (13

samples). Three sterigmatocystin-containing samples did not yield any

Aspergillus isolates. Species of Penicilliumwere isolated in two of

the three cases where sterigmatocystinwas found in the absence of

Aspergillus spp. In addition to 14 samples containing

sterigmatocystin, toxin-containing samples contaminated with

Penicillium spp. included two of the three citrinine-

containingsamples. The majority of the 56 samples that contained

Penicilliumspp., however, were negative for both citrinine and

sterigmatocystin. Species of Fusarium were detected in 12

samples,only two of which were associated with the production of

nonmacrocyclictrichothecenes characteristic of Fusarium spp.

Satratoxins, with one exception, were found only in associationwith

Stachybotrys species.

Some species were more frequently associated with mycotoxin-

containing materials, even when the toxins found were not

characteristicof these species. For example, A. ochraceus was foundon

eight occasions, all of which were associated with the production of

mycotoxin. Yet, ochratoxin A, which is characteristic of this

species, was not detected in any of the analyzed samples. On the

other hand, all six samples containing Aspergillus niger werefree

from mycotoxin.

DISCUSSION

The present samples are a subset selected from a large pool of

buildings with moisture problems and were biased in their

microbiologyas examined on one particular universal growth medium.

Therefore,we cannot draw any conclusions regarding the fungal

frequencyon moisture-damaged building materials in general. One in

fivesamples of material from which species of Aspergillus were

recovered contained detectable levels of sterigmatocystin, making it

the single most prevalent toxin in this study and, perhaps,

indicatingthat sterigmatocystin is more ubiquitous than previously

thought.As in previous studies, most sterigmatocystin-

producingstrains appeared to be A. versicolor, but it also is

possiblethat this toxin may have been produced by Penicilliumspp.

Spread plating on malt extract agar favors the growth of rapidly

growing species of Aspergillus, Penicillium, and Alternariaat the

expense of the generally slower-growing species of

Stachybotrys,Acremonium, and Fusarium. The isolation of Fusarium

spp.might require direct plating on medium specific for this purpose.

In the present study, 15% of materials were contaminatedwith Fusarium

species, but 10 of 12 samples containing nonmacrocyclictrichothecenes

characteristic of Fusarium spp. yielded no Fusariumcultures.

Verrucarol has been reported in Stachybotrys spp.,but judging from

extensive reviews of the mycotoxins characteristicof different

species of Fusarium and other fungi, it is highlyunlikely that the

other nonmacrocyclic trichothecenes present(DAS, DON, 3-Ace-DON, and

T2-tetraol) originated from fungi otherthan Fusarium spp. It seems

that the procedure used to isolate the fungi left most of the

Fusarium spp.undetected.

The mycology of the building materials did not correlate well with

the toxin contents, although when a mycotoxin was found in a sample,

representatives of a fungal genus known to containtoxigenic species

were present. It is possible for toxigenic specieswith different

growth requirements to be present in the same sample,as they may have

proliferated during different stages of the waterdamage. For example,

a surface may be overgrown by S. chartarum,which prefers cellulosic

matter with a high water content, withnitrogen deficiency promoting

satratoxin production, but at an earlier stage of the water damage,

at a lower relative humidity,A. versicolor could havedominated.

Our findings agree with those of Gravesen et al., in which

sterigmatocystin was detected in 19 of 23 samples of building

materials artificially contaminated with strains of Aspergillussp.

recovered from Danish buildings with moisture problems. Theyalso

found trichothecenes in six of eight natural samples

tested.Previously, in Finnish water-damaged buildings,

trichotheceneswere detected in dust and construction material

samples, as wellas from samples of artificially enriched microbial

media.We hypothesize that sterigmatocystin and trichothecenes

occurfrequently in cellulosic construction materials of problem

houses,where some of the fungi used to select the samples analyzed

inthe present study (A. ochraceus, Stachybotrys, Fusarium,

Trichoderma,and Acremonium) have proliferated as a result of

prolonged exposureto high wateractivities.

Risk assessment of the inhalation of mycotoxins cannot be made from

the analysis of bulk samples of construction materials,even if dose

responses of humans to airborne mycotoxins were known.However, as

many of the fungi that we isolated can elicit allergenicreactions in

addition to being toxic, it seems that care should be exercised when

moisture-damaged sites are torn downor renovated. Sterigmatocystin is

an International Agency forResearch on Cancer class 2B carcinogen and

also has immunotoxicproperties, while satratoxin G and SATH are

probably thechemical agents responsible for stachybotryotoxicosis in

mammals. In a recent study, S. chartarum and A. versicolorwere

implicated as causes of building-associated pulmonary diseasein

workers in three adjacent office buildings. A. versicolor

predominatedin the indoor air, and S. chartarum was isolated from

bulk samplescontaining parts-per-million levels of satratoxins.

Unfortunately, sterigmatocystin could not be isolated in that study,

due to peak interference in UV-HPLC. In addition to the work of

Hodgson etal. (2 to 5 µg/g), satratoxins have previously been found

in building materials by Johanning et al. (16 µg/g), Croftet al. (not

quantified), and et al. (17 µg/g). To our knowledge,

sterigmatocystin has not previously beenextracted from building

materials naturally contaminated by fungi.The levels of satratoxins

in our present study (0.77 µg/g of extracted material) were lower

than those previously found inbuilding materials naturally

contaminated by S. chartarum but as high as those found in building

materials artificially inoculated with S. chartarum and incubated to

enrich toxins and almost as high as those encountered with

Stachybotrys-contaminatedrice or fodder.

Mycological analyses of air and crude building materials are

routinely performed in environmental laboratories to evaluate the

extent and spread of damage in buildings with moisture problems and

to assess the risk to residents. The isolation of toxigenic species

does not substantiate the presence of mycotoxins. However,the present

study demonstrates that when mycotoxins are foundin bulk materials,

some genus known to include toxigenic species usually is present,

even if strains from the fungal species probably responsible for

producing the mycotoxin are not recovered. In this context, we

suggest that the sources of mycotoxic fungal contamination should be

removed and necessary precautions should be taken to prevent exposure

to potentially harmful aerosolized particles during renovation of

buildings with moisture problems.

As the techniques to collect and analyze airborne propagules develop,

mycotoxins can be analyzed from indoor air, enablingan assessment of

the possible health consequences of mycotoxinsfor residents of water-

damaged buildings. In future studies, the ubiquitousness of

mycotoxins in indoor environments can be evaluatedwhen more

mycotoxins are added to the analysis protocol and whenmore moldy

materials are sampled. There are techniques availableto analyze most

fungi present in environmental samples. Identifying the fungi

responsible for producing mycotoxins in building materialswill

require using such techniques in combination with the enrichmentof

pure fungal isolates on building materials and extraction of

mycotoxins from these isolates. The present study underlines the need

for such research. In the largest screen from indoor environments

with respect to the number of mycotoxins and samples analyzed, we

found mycotoxins in more than 40% ofsamples.

ACKNOWLEDGMENTS

This work was supported in part by the Academy ofFinland.

We thank a Vanninen at VERIFIN (Finnish Institute for

Verification of the Chemical Weapons Convention) for furnishing of

mycotoxin standards; Tapio Suorti (Vantaa), Kari Vähämäki(FIOH), and

Marjatta Malmberg (HUCHS) for collecting samples;Taru Järvimaa

(Vantaa) and Tuula Laakso (Helsinki EnvironmentCenter) for

identifying fungal species; Sirkku Kokko (HUCHS),Tuovi Karpeeki

(FIOH), and Marita Airaksinen and Sulasalmi(Vantaa) for

preparing mycological samples; and Hilkka kauppiat FIOH for

performing the mycotoxin samplepretreatments.