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ISSN 1464-0325

www.rsc.org/jem Volume 11 | Number 10 | October 2009 | Pages 1709–1888

De Kimpe, De Saeger et al.Fungi in mouldy interiors

Sun and ZhengOil spill response strategies

Baun et al.Limits for nanoparticles in EU waters

Krivit and MarwanLow-energy nuclear reaction research

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View Article Online / Journal Homepage / Table of Contents for this issue

PAPER www.rsc.org/jem | Journal of Environmental Monitoring

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JEM Spotlight: Fungi, mycotoxins and microbial volatile organic compoundsin mouldy interiors from water-damaged buildings

Viviana Polizzi,ab Barbara Delmulle,a An Adams,b Antonio Moretti,c Antonia Susca,c Anna Maria Picco,d

Yves Rosseel,e Ruben’t Kindt,f Jan Van Bocxlaer,f Norbert De Kimpe,*b Carlos Van Peteghema

and Sarah De Saeger*a

Received 7th April 2009, Accepted 2nd July 2009

First published as an Advance Article on the web 20th July 2009

DOI: 10.1039/b906856b

Concerns have been raised about exposure to mycotoxin producing fungi and the microbial volatile

organic compounds (MVOCs) they produce in indoor environments. Therefore, the presence of

fungi and mycotoxins was investigated in 99 samples (air, dust, wallpaper, mycelium or

silicone) collected in the mouldy interiors of seven water-damaged buildings. In addition, volatile

organic compounds (VOCs) were sampled. The mycotoxins were analysed by liquid chromatography-

tandem mass spectrometry (LC-MS/MS) (20 target mycotoxins) and quadrupole time-of-flight

mass spectrometry (LC-Q-TOF-MS). Morphological and molecular identifications of fungi were

performed. Of the 99 samples analysed, the presence of one or more mycotoxins was shown in

62 samples by means of LC-MS/MS analysis. The mycotoxins found were mainly roquefortine C,

chaetoglobosin A and sterigmatocystin but also roridin E, ochratoxin A, aflatoxin B1 and aflatoxin

B2 were detected. Q-TOF-MS analysis elucidated the possible occurrence of another 42 different

fungal metabolites. In general, the fungi identified matched well with the mycotoxins detected. The

most common fungal species found were Penicillium chrysogenum, Aspergillus versicolor (group),

Chaetomium spp. and Cladosporium spp. In addition, one hundred and seventeen (M)VOCs

were identified, especially linear alkanes (C9–C17), aldehydes, aromatic compounds and monoterpenes.

Introduction

From the early 1970s, reports on illnesses associated with

poor indoor air quality have appeared in the medical and

aGhent University, Faculty of Pharmaceutical Sciences, Laboratory ofFood Analysis, Harelbekestraat 72, B-9000 Ghent, Belgium. E-mail:Sarah.Desaeger@UGent.be; Fax: +32 9 264 81 99; Tel: +32 9 264 81 34bGhent University, Faculty of Bioscience Engineering, Department ofOrganic Chemistry, Coupure links 653, B-9000 Ghent, Belgium. E-mail:norbert.dekimpe@ugent.be; Fax: +32 9 264 62 43; Tel: +32 9 264 59 51cInstitute of Sciences of Food Production, National Research Council, ViaAmendola 122/o, I-70126 Bari, ItalydPavia University, Faculty of Sciences, Department of Territorial Ecologyand Environment, Viale Taramelli 24, 27100 Pavia, ItalyeGhent University, Faculty of Psychology and Educational Sciences,Department of Data Analysis, Henri Dunantlaan 1, B-9000 Ghent, BelgiumfGhent University, Faculty of Pharmaceutical Sciences, Laboratory ofMedical Biochemistry and Clinical Analysis, Harelbekestraat 72, B-9000Ghent, Belgium

Environmental impact

Fungi and their toxic metabolites have been indicated as one of the

associated with poor indoor air quality. This is the first study wher

presence of 20 mycotoxins, volatile organic compounds and fungi. S

sample collection procedures including different techniques and fun

one or more mycotoxins were found and related to fungal growth.

human health in water-damaged houses, especially for those myco

contribute to a better understanding of the SBS.

This journal is ª The Royal Society of Chemistry 2009

scientific literature. In 2004, Bornehag et al.1 stated that

people living and working in mouldy buildings exhibit more

respiratory symptoms and diseases than people in non-

problem buildings and thus provided evidence for a true

association between dampness and health. A common core

group of six symptoms, including nasal, eye and mucous

membrane irritation, headache, dry skin, and lethargy, was

present in nearly all reports. These symptoms, without a clear

indication of the causative agent(s), have been coined as ‘Sick

Building Syndrome’ (SBS).2

The first case in which a toxigenic fungus was more clearly

correlated with human illness was reported in 1999.3 In this

study, Stachybotrys chartarum was isolated from the bron-

choalveolar lavage fluid of a child with pulmonary hemorrhage.

The same Stachybotrys was also recovered from his water-

damaged house. The patient recovered completely after removal

from the environment and subsequent cleaning of the house. In

another study, building materials visibly contaminated with

possible causes of the Sick Building Syndrome (SBS), an illness

e mouldy environments were simultaneously monitored for the

ubstantial efforts were devoted to the establishment of optimal

gal identification protocols. In more than 60% of the samples,

Thus, mycotoxin production may be a possible risk factor for

toxins with a higher incidence in air and dust. These findings

J. Environ. Monit., 2009, 11, 1849–1858 | 1849

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moisture-related fungi were screened for cytotoxicity. In all cases

in which building occupants had reported verifiable skin, mucous

membrane, respiratory, central nervous system or neuro-

psychological abnormalities, cytotoxicity was found.4

Concerning human inhalational exposure, only fungal

metabolites that can become airborne are of interest. In a study

of Smith et al.,5 47% of the fungal isolates from the air flora of

a damp building displayed spore-associated cytotoxic properties.

This fact emphasises the potential health hazards of the contin-

uous inhalation of toxigenic fungal propagules by occupants of

mouldy houses.

Investigations have focused primarily on intact airborne

fungal spores assuming that spores are the main airborne parts of

fungi. However, different studies, all concerning Stachybotrys

chartarum, have demonstrated that the number of fungal frag-

ments released from contaminated surfaces was always higher

than the number of intact spores.6,7 These fungal fragments,

separated from conidia by means of filters with decreasing pore

size, contained macrocyclic trichothecenes.8

Therefore, the high number of fungal fragments released in

combination with their potential to deliver harmful antigens and

mycotoxins to the lung suggests that future exposure measure-

ments need to include measurements of fungal fragments as well.

This would provide more realistic exposure profiles.

Several field studies, however, have shown that airborne

fungal concentrations in problematic buildings are similar to

those observed in non-problematic buildings.6,9 It is, therefore,

important to investigate other potentially hazardous factors as

well, such as mycotoxins and microbial volatile organic

compounds (MVOCs).

Concerning mycotoxins, the majority of studies to date have

focused on trichothecene mycotoxins. However, it has been

shown that, while fungal isolates from water-damaged buildings

produce highly cytotoxic trichothecenes (e.g. verrucarins, ror-

idins, verrucarols, T-2 toxin, diacetoxyscirpenol, neosolaniol,

satratoxins, nivalenol and deoxynivalenol), they may also

produce other mycotoxins, including ochratoxin A, zearalenone,

aflatoxins and sterigmatocystin,10–15 which may induce health

problems upon inhalation. In fact, aflatoxins are classified by the

International Agency for Research on Cancer (IARC) as carci-

nogenic to humans (group 1), while ochratoxin A and ster-

igmatocystin are classified in group 2B ‘possibly carcinogenic to

humans’.16 Adverse effects have also been reported for other

mycotoxins. Chaetoglobosin A has a cytotoxic effect on various

cell lines,17 roquefortine C has a neurotoxic effect on cockerels18

and zearalenone is a cytotoxic potent estrogen having a negative

effect on the reproductive system.19

The Sick Building Syndrome has also been associated with the

presence of (M)VOCs.20 (M)VOCs can have adverse effects on

the respiratory systems, blood vessel systems and nerve systems,

may be carcinogenic21 and can cause mental and cognitive

distraction of the exposed subjects, which results in reduced

performance, especially if the odor is perceived as unpleasant or

unrecognizable.22 In addition, the detection of MVOCs may

indicate fungal growth. Certain species specific production

profiles may enable the identification of fungi based on the

detected fungal volatiles.23 In specific cases, certain marker

molecules, such as trichodiene, can be detected as indicators of

ongoing mycotoxin production.24

1850 | J. Environ. Monit., 2009, 11, 1849–1858

To date, mouldy interiors have not been monitored simulta-

neously for mycotoxins, MVOCs and fungal growth. The aim of

this study was to analyse MVOCs as possible indicators of fungal

growth and to reveal the presence of fungi and mycotoxins in

water-damaged buildings. These findings could contribute to

a better understanding of the SBS. Therefore, a broad range of

mycotoxins (20 compounds) was monitored using a multi-

mycotoxin liquid chromatography tandem mass spectrometry

(LC-MS/MS) method (based on Delmulle et al.25). This is in

contrast with earlier indoor reports, which concentrated on

a more limited range of mycotoxins. The samples were further

analysed with a quadrupole time-of-flight (Q-TOF) MS, to

identify less studied fungal metabolites. Since several authors

reported many errors in literature concerning fungal identifica-

tions and hence the connection between mycotoxins and the

source of the toxins,14,26 substantial efforts were devoted to the

establishment of optimal sample collection procedures and

fungal identification protocols.

Materials and methods

Reagents

Methanol (VWR International, Zaventem, Belgium), acetonitrile

(Biosolve BV, Valkenswaard, The Netherlands), stabilized

dichloromethane and ethyl acetate 99.5+% (Acros Organics,

Geel, Belgium) were high-performance liquid chromatography

grade. Water was obtained from a Milli-Q Gradient System

(Millipore, Brussels, Belgium) and formic acid from Acros

Organics. Ammonium acetate was supplied by Grauwmeer

(Leuven, Belgium) and anhydrous sodium acetate by Merck

(Darmstadt, Germany). Standards of nivalenol (NIV), deoxy-

nivalenol (DON), T-2 toxin (T-2), verrucarin A (VERA), ver-

rucarol (VERO), ochratoxin A (OTA), aflatoxin B1 (AFB1),

aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2),

zearalenone (ZEA), sterigmatocystin (STERO) and roridin A

(RORA) were provided by Sigma (Bornem, Belgium). Diac-

etoxyscirpenol (DAS), neosolaniol (NEO) and deepoxy-deoxy-

nivalenol (DOM) were obtained from Coring System Diagnostix

GmbH (Gernsheim, Germany). Satratoxin G (SATG), satra-

toxin H (SATH) and roridin E (RORE) were purchased from the

New-Zealand Government Research Organisation (Wellington).

Chaetoglobosin A (CHAETO) was provided by Alexis

Biochemicals (Lausen, Switzerland) and roquefortine C (ROQC)

by IRIS biotech GmbH (Marktredwitz, Germany). Stock solu-

tions were prepared in methanol and stored at �20 �C.

Sampling procedures

The sampling procedures were approved by the Ethical

Committee of the Ghent University Hospital (B6702006019,

17/07/2006). Samples were taken in mouldy interiors from 7

water-damaged buildings throughout Belgium (House n� 1 to 7).

Criteria of choice were: visible fungal growth for at least 1 m2 of

surface area and presence of malodour. In some cases, namely in

houses 1, 3 and 4, at least one of the inhabitants reported health

problems, mainly respiratory problems, starting after mould

growth. Instead, in building 2 (a library), where different stored

books were heavily contaminated by fungi, headache and dizzi-

ness were experienced both by the people who moved the books

This journal is ª The Royal Society of Chemistry 2009

Table 1 Summary of the samples collected from 7 water-damaged buildings (positive sample ¼ containing 1 or more mycotoxins)

Sampled place N� of samples taken Type of sample N� of positive samples per house Type of positive sample

House n� 1 16 2 Air 5 5 Wallpaper8 Dust6 Wallpaper

House n� 2 21 3 Air 12 1 Air3 Dust 11 Mycelium15 Mycelium

House n� 3 14 2 Air 10 10 Wallpaper2 Dust10 Wallpaper

House n� 4 11 2 Air 9 2 Air7 Dust 7 Dust2 Mycelium

House n� 5 13 3 Air 9 3 Air2 Dust 2 Dust4 Wallpaper 1 Wallpaper2 Mycelium 2 Mycelium2 Silicone 1 Silicone

House n� 6 12 4 Air 8 4 Air3 Dust 2 Dust5 Wallpaper 2 Wallpaper

House n� 7 12 4 Air 9 4 Air2 Dust 2 Dust6 Mycelium 3 Mycelium

Total number of samples 99 62

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in the sampled room and by both persons carrying out the

sampling only during the stay in the room. In addition, one

recently built house (1991) was chosen as a control because it was

a clean, smoke-free environment with no building-related

complaints by the inhabitants. Moreover, sampled house n� 4

was re-sampled only for VOCs after long ventilation and clean-

ing, since it was still sensory unpleasant. In all cases except for

house n� 1, relative humidity (RH) and temperature values were

recorded with a thermohygrometer (Lufft, Fellbach, Germany).

To sample visible fungal growth on surfaces, mycelium was

scraped off with a sterile disposable scalpel (Swann-Morton,

Sheffield, United Kingdom) and if not possible, mouldy wall-

paper or silicone from window sealings were cut away. For

fungal identification, mycelium was sampled by ‘‘stick-to-it’’ lift

tape (Procare, Groningen, The Netherlands). The tape was

pressed gently to the mycelium and pulled back.

Dust was collected with a Cyclone Surface Sampler from

Burkard Manufacturing Company Limited (Hertfordshire,

United Kingdom). The area of surface sampled was always

measured (cm2).

Airborne mycotoxins were collected in all houses with an

Airborne Sample Analysis Platform (ASAP) system model 2800

Bioaerosol sampler (Thermo, New York, USA). The instrument

was operated at a flow rate of 200 litres per min. Ambient

particles between 1 and 10 micron in diameter were collected on

polyurethane foam (PUF) (Thermo Scientific, USA) during

a period of 3.5 h, resembling the breathing of an average man

(moderate activity, 80 kg) during 24 h (assuming breathing of

28 L/min). These high flow rates enabled the collection of suffi-

cient mycotoxin concentrations in a relatively short time frame,

which was preferable for practical reasons (sampling in private

homes). The PUF substrate was enclosed in a self-sealing

collection module called Intergrated Bio Aerosol Smart Sample

(iBASS� cartridge) (Thermo, New York, USA), in which two

This journal is ª The Royal Society of Chemistry 2009

PUF strips are sampled simultaneously. All samples described

(Table 1) were extracted for mycotoxin analysis with LC-MS/MS

and, afterwards, 23 of the samples were additionally analysed

with LC-Q-TOF-MS.

The (M)VOCs in the houses were sampled by dynamic head-

space absorption on Tenax and by solid-phase microextraction

(SPME) followed by gas chromatography-mass spectrometry

analysis. The 50/30 mm divinylbenzene/Carboxen/poly-

dimethylsiloxane (DVB/Car/PDMS) SPME fibre was purchased

from Supelco (Bornem, Belgium). The SPME fibre was exposed

to the mouldy environment for the longest time permitted, i.e.

30 minutes, 3.5 h or 24 h depending on the possibility to leave the

equipment in the buildings. The same sampling times were

applied to the dynamic headspace technique performed by means

of a hand-made pump to which a Tenax adsorption tube (length

18 cm, i.d. 4 mm, o.d. 6 mm) was attached, filled with 250 mg of

Tenax TA 60/80 mesh (Supelco, Bellefonte PA, USA).

Analytical procedures

Fungal identification. Mycelium was eluted from stick-to-it lift

tape by using sterile water that was dropped in peptone penta-

chloronitrobenzene agar and potato dextrose agar (PDA) con-

taining Petri dishes (three replicates for each sample and each

substrate). All growing colonies were purified and transferred on

malt extract agar, potato dextrose agar, water agar, yeast extract

agar, or Czapek agar substrates according to a previous genus

identification of the growing colonies, and then morphologically

identified at the species level.27

Further, fungal strains were cultured for 48 hours with

shaking (125 rpm) at 25 �C in 100 mL of Wikerham’s medium

(4% D-glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt

extract). The mycelium was vacuum-filtered on Whatman n� 4

filter paper and washed with distilled water, frozen (�20 �C) and

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lyophilized. Lyophilized mycelium powder was used for genomic

DNA isolation, according to the E.Z.N.A. Fungal DNA

Miniprep Kit (Omega Bio-tek, Doraville, GA) instructions.

Extracted DNA was dissolved in 100 mL of sterile water and

stored at �20 �C. DNA concentrations were estimated using

a DNA ladder and electrophoresis.

Sequence analysis was performed in order to confirm the

identity of the strains established by morphological analysis. The

species identification of the strains was obtained by sequencing

a region of the b-tubulin gene by using primers Bt2a28 and T2.29

Glass and Donaldson28 and O’Donnell and Cigelnik29 developed

a series of primers for amplifying b-tubulin from filamentous

fungi, able to amplify different fungal genera, and therefore

suitable for identification of a large spectrum of fungi. Poly-

merase chain reaction (PCR) was performed using the following

conditions: denaturation at 94 �C for 10 min; 35 cycles of

denaturation at 94 �C for 50 s, annealing at 53 �C for 50 s,

extension at 72 �C for 1 min; final extension at 72 �C for 7 min,

followed by cooling at 4 �C until recovery of the samples.

Amplification of a 400 bp product was verified via gel electro-

phoresis on a 1.5% agar gel. Once verified, PCR products were

purified by filtration through Sephadex G-50 medium (Sigma)

prior to DNA sequencing. Sequence analysis was performed with

the Big Dye Terminator Cycle Sequencing Ready Reaction Kit

for both strands (Applied Biosystems). The resulting sequences of

all the isolates and reference strains were aligned by the Clustal

method with DNAMAN (version 5.2.9, Lynnon Biosoft) or

Bioedit (version 7.0.5, Ibis Therapeutics Carlsband, CA 92008)

software. The b-tubulin homology sequences were searched per-

forming a pairwise method in DDBJ/EMBL/GenBank Interna-

tional Nucleotide databases using Basic Local Alignments Search

Tool (BLAST) at http://www.ncbi.nlm.nih.gov/blast/.

Mycotoxin analysis by means of LC-MS. Extraction of PUF

(air) and silicone samples was performed in a Gosselin tube

(Nunc, Wiesbaden, Germany) by adding 15 mL of methanol:

water (90:10 v/v) to the sample. After shaking for 15 min on

a horizontal Mini Shaker S03 (Orbital, Stuart Scientific, Surrey,

England), the extract was removed and kept separate, followed by

addition of 15 mL of dichloromethane:ethyl acetate (50:50 v/v) to

the sample and shaking for 15 minutes. The dichlor-

omethane:ethyl acetate layer was combined with the methanol:

water layer and filtered through a folded paper filter, sort 15,

provided by VWR International (Zaventem, Belgium). After

Table 2 Optimized ESI+ MS/MS conditions for the 5 mycotoxins added tmycelium and filter samples

Compound Precursorion (m/z)

Conevoltage (V)

Productions (m/z)

Satratoxin G 562.3 15 545.1[M + NH4]+ 249.2

Satratoxin H 546.3 18 529.3[M + NH4]+ 511.2

Chaetoglobosin A 529.2 25 130.2[M + H]+ 511.2

Roquefortine C 390.0 40 193.1[M + H]+ 322.1

Roridin E 532.2 23 361.1[M + H]+ 113.0

1852 | J. Environ. Monit., 2009, 11, 1849–1858

washing the filter with 2 mL of methanol:water (90:10 v/v) and

dichloromethane:ethyl acetate (50:50 v/v), the filtrate was evap-

orated to dryness under nitrogen in a hot water bath (60 �C)

(Grant Instruments JB2, VWR International Zaventem,

Belgium) and redissolved in 200 mL of methanol:water (30:70 v/v).

The 200 mL extract was transferred to a microvial (Waters,

Belgium) and injected into the LC-MS-MS system.

Dust samples were extracted twice with 1.5 mL of meth-

anol:water (90:10 v/v) in a microcentrifuge tube from Greiner

Bio-one (Wemmel, Belgium). After shaking for 15 min, the

extract was filtered and washed with 2 mL of methanol:water

(90:10 v/v). Afterwards, it was evaporated to dryness under

nitrogen and redissolved in 200 mL of methanol:water (30:70 v/v).

The 200 mL extract was transferred to a microvial and analysed

by means of LC-MS/MS. For wallpaper samples the same

procedure was applied, without filtering, and with an extraction

volume of twice 15 mL.

Mycelium samples were extracted in microcentrifuge tubes

with 1.5 mL of ethyl acetate. After shaking for 15 min and

centrifugation for 5 min (823 g) in a Multicentrifuge 4371 from

Heraeus (Brussels, Belgium), the ethyl acetate layer was removed

and kept separate, followed by addition of 1.5 mL of dichloro-

methane to the sample and shaking for 15 min. The dichloro-

methane layer was combined with the ethyl acetate layer and

filtered through a folded paper filter. After washing the filter with

2 mL of ethyl acetate and 2 mL of dichloromethane, the filtrate

was evaporated to dryness under nitrogen and redissolved in

200 mL of methanol:water (30:70 v/v). The 200 mL extract was

transferred to a microvial and injected for target mycotoxin

analysis into the LC-MS-MS system.

LC-MS-MS analysis was based on a method previously pub-

lished25 using a Waters Alliance 2695 XE HPLC system coupled

to a Micromass Quattro Micro triple quadrupole MS (Waters,

Milford, MA, USA). This system was equipped with Masslynx

4.0 software for data processing. Sample extracts were injected

on a C18, 3.5 mm, 150 � 3.0 mm, reversed-phase SunFire

analytical column (Waters, Zellik, Belgium). A mobile phase

consisting of variable mixtures of ammonium acetate (10 mM)

and sodium acetate (20 mM) in water (solvent A) and in methanol

(solvent B) separated the 20 target mycotoxins. The mass spec-

trometer was operated in the positive electrospray ionization

(ESI+) mode using multiple reaction monitoring (MRM).

Detection limits, product and fragment ions of the different

mycotoxins were described before.25 This method was extended

o the existing LC-MS/MS method and their detection limit (LOD) for

Collisionenergy (eV)

LOD (pg/ml)for fungal samples

LOD (pg/ml) forfilter samples

10 2.0 51510 2.1 501050 1.4 9.51025 4.9 2.82015 1.5 0.920

This journal is ª The Royal Society of Chemistry 2009

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with five additional mycotoxins, the optimized ESI+ MS/MS

conditions and detection limits of which are shown in Table 2.

During the analysis, identification criteria according to the

‘Commission Decision 2002/657/EC’ were used, i.e. the presence

of the two selected fragment ions, relative retention time of the

analyte and relative ion intensities.30

Twenty-three of the 99 sample extracts were additionally

analysed by means of liquid chromatography quadrupole time-

of-flight mass spectrometry (LC-Q-TOF-MS). An Alliance 2690

(LC) system was coupled to a micro quadrupole orthogonal

acceleration TOF-MS (Waters, Milford, MA, USA) equipped

with an orthogonal electrospray source (Z-spray�). An Atlantis

dC18 column, 3 mm, 150� 2.1 mm (Waters, Milford, MA, USA),

was used to analyse the same samples in scan mode. A mobile

phase of variable mixtures of formic acid (10 mM) in water

(solvent A) and formic acid (10 mM) in acetonitrile (solvent B)

was used. Compounds with m/z between 100 and 800 were

recorded in ESI + scan mode at a scan accumulation time of

2.0 sec and an interscan time of 0.1 sec. First, a standard mix was

analysed to check the masses of known mycotoxins. Afterwards,

the samples were analysed and the accurate masses of the

compounds present were measured in comparison with the

standard mycotoxin DOM, used as the internal lock mass. These

masses were manually annotated using accurate mass listings.14,31

(M)VOC analysis by means of GC-MS. The SPME desorption

was performed for 2 min in the inlet of an Agilent 6890 Gas

Chromatograph (GC) Plus (Agilent Technologies, Diegem,

Belgium) coupled with a quadrupole mass spectrometer HP 5973

Mass Selective Detector (MSD), equipped with a programmed

temperature vaporizer (CIS-4 PTV) injector (Gerstel, M€ulheim

a/d Ruhr, Germany), and a HP5-MS capillary column (30 m �0.25 mm i.d.; coating thickness 0.25 mm). Working conditions

were as follows: injector temperature 250 �C, transfer line

temperature 250 �C, oven temperature: start 35 �C, hold 5 min,

programmed from 35–120 �C at 5 �C min�1, from 120–280 �C at

20 �C min�1, hold 3 min; carrier gas (He) 1.2 mL min�1; splitless

Table 3 Summary of mycotoxin containing samples (‘positive samples’ ¼ sa

Air Conc. rangein air (ng/m3) Dust

Wallp

Housenumber

Number ofpositivesamples

Housenumber

Number ofpositivesamples

Housnumb

ROQC 4 1 0.009–4 6 2 16

STERO 5 3 0.0034–1.7674 4 3 15 2 36 2 57 2 6

CHAETO 5 3 0.0067–3.4205 n.d. 6

AFB2 4 2 0.0003–0.0211 4 75 2

AFB1 4 2 0.0024–0.1463 1 15 3

RORE 2 1 0.0031–0.082 n.d.5 2

OTA 4 1 0.0115–0.228 4 75 12 1

This journal is ª The Royal Society of Chemistry 2009

mode; Electron Impact (70 eV); acquisition parameters: scanned

m/z: 40–200 (2–10 min), 40–300 (>10 min).

Thermal desorption of the Tenax tubes was carried out in

a Thermodesorption System TDS2 from Gerstel coupled with

the GC-MS system. The TDS2 was operated as follows: start

25 �C and end at 260 �C at a rate of 60 �C/s, hold 7 min; purging

gas helium; transfer line temperature to the injector 275 �C.

During thermal desorption the CIS-4 PTV inlet was held at

�100 �C by means of liquid N2 for 10 min to cryofocus the

compounds. After complete desorption the inlet was heated to

260 �C at 12 �C/s. The GC-MS parameters were the same as for

the SPME analysis. Fungal metabolites were identified by

comparison of the obtained mass spectrum with mass spectral

libraries32 (Nist 98; Wiley 6th) and by comparison of the calcu-

lated linear retention index with literature values.32,33

Results and discussion

Mycotoxin analysis

Mycotoxins are not volatile but can be found on fungal spores

and particles.34,35 As a consequence, airborne fungal spores and

particles are of particular interest in view of human inhalation

exposure to mycotoxins. Therefore, besides different substrates

and dust, the indoor air of the mouldy buildings was sampled as

well.

Of the 99 samples analysed, 62 showed the presence of one or

more mycotoxins after LC-MS-MS analysis. An overview of the

type of samples and the presence of mycotoxins is shown in

Table 1. Mainly sterigmatocystin, roquefortine C and chaeto-

globosin A were found, in 31, 17 and 16 samples, respectively,

but also roridin E (3), ochratoxin A (10), aflatoxin B2 (11) and

aflatoxin B1 (10) were detected at concentration ranges reported

in Table 3. There was a large spread on the concentrations

measured for different mycotoxins and also for different

substrates containing the same mycotoxin.

mples containing the specific mycotoxin)

aper Mycelium Silicone Conc. rangeon thedifferentsubstrates(ng/cm2)

eer

Number ofpositivesamples

Housenumber

Number ofpositivesamples

Housenumber

Number ofpositivesamples

5 2 7 n.d. 0.106–7.223 2 2 n.d. 0.063–778.4

10 5 2112 2 9 n.d. 1.16–13 830

5 2n.d. n.d. n.d. 0.019–0.253

n.d. 7 3 5 1 0.111–0.332

n.d. n.d. n.d.

n.d. n.d. n.d. 0.008–0.773

J. Environ. Monit., 2009, 11, 1849–1858 | 1853

Table 4 Overview of the 2 � 5 contingency tables for all mycotoxins (observed counts) against the expected counts assuming the null hypothesis

Observed counts Expected counts

Air Wallpaper Mycelium Dust Silicone Air Wallpaper Mycelium Dust Silicone

STERO Present 3 15 4 9 0 6 10 7 7 0.5Absent 9 5 10 4 1 6 10 7 6 0.5

CHAETO Present 3 2 11 0 0 3 5 4 3.5 0.3Absent 9 18 3 13 1 9 15 10 9.5 0.7

ROQC Present 1 7 7 2 0 3 6 4 3 0.3Absent 11 13 7 11 1 9 14 10 10 0.8

AFB2 Present 4 0 0 7 0 2 4 3 2 0.2Absent 8 20 14 6 1 10 16 11 11 0.8

AFB1 Present 5 0 3 1 1 2 3 2 2 0.2Absent 7 20 11 12 0 10 17 12 11 0.8

RORE Present 3 0 0 0 0 1 1 1 1 0.1Absent 9 20 14 13 1 11 19 13 12 0.9

OTA Present 3 0 0 7 0 2 3 2 2 0.2Absent 9 20 14 6 1 10 17 12 11 0.8

Table 5 Mycotoxin incidence for a specific sample (mycelium, wallpaper, air, dust or silicone)

P-value Pearson residualsAir Wallpaper Mycelium Dust Silicone

STERO 0.00629 �1.29 1.45 �1.2 0.88 �0.72CHAETO <0.0001 �0.11 �1.44 3.76 �1.86 �0.52ROQC 0.102 �1.3 0.5 1.52 �0.88 �0.53AFB2 <0.0001 1.2 �1.9 �1.6 2.99 �0.42AFB1 0.0016 2.12 �1.82 0.43 �0.79 2RORE 0.0336 3.10 �1.00 �0.84 �0.81 �0.22OTA <0.0001 0.71 �1.83 �1.53 3.28 �0.41

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Furthermore, the incidence of the detected mycotoxins for

each specific kind of substrate was investigated. For each

mycotoxin a 2 � 5 contingency table was constructed (Table 4).

To assess whether there was a significant association between the

incidence of the mycotoxin and the type of sample (air, wall-

paper, mycelium, dust or silicone), Fisher’s exact test was applied

using a network algorithm36 (Table 5). If the p-value was lower

than 0.05, the null hypothesis (‘‘there is no association between

mycotoxin incidence and type of sample’’) was rejected. In this

case, it is informative to inspect the Pearson residuals (Table 5).

Positive values indicate that more incidences are observed than

expected. For sterigmatocystin, for example, significant associ-

ations with the five types of samples were found. As can be seen

from the Pearson residuals, this was mostly due to its high inci-

dence in wallpaper and the low incidence in mycelium. On the

other hand, chaetoglobosin A showed a significant association

with mycelium. For roquefortine C, no association was observed.

Aflatoxin B2 was found in air and dust and was absent in wall-

paper and mycelium. Aflatoxin B1 as well was mainly present in

air. Since aflatoxins are classified by the International Agency for

Research on Cancer (IARC) as group 1 carcinogens, their pres-

ence in the indoor air of water-damaged interiors, is of serious

concern. Roridin E belongs to the toxic trichothecene group and

was again mostly found in air. Ochratoxin A is classified by

IARC in group 2B ‘possibly carcinogenic to humans’ and was in

our study mostly observed in respirable dust.

These results demonstrate the presence of mycotoxins in air,

settled dust and mycelium in indoor environments, thus

1854 | J. Environ. Monit., 2009, 11, 1849–1858

involving a potential health hazard for inhabitants of mouldy

water-damaged houses. However, no risk assessment has been

performed so far for human inhalational exposure to myco-

toxins. Nevertheless, health problems caused by inhaled myco-

toxins tend to be more serious than those caused by mycotoxins

absorbed via the digestive tract and the skin.7,37–39 Creasia et al.40

showed that mycotoxins can be 10 times more toxic through

inhalation than by oral intake. The mechanism of toxicity was

not elucidated, but a higher resorption rate of the toxins in the

lungs as compared to in the bowels and a first pass effect in the

liver after enteric resorption were discussed.37,40

In several cases, different mycotoxins were present in one

single sample. To date little is known on synergistic or antago-

nistic effects of simultaneous exposure to mycotoxins.37,39 Several

authors suggested the synergistic interaction between different

exposure agents as a biologically plausible theory that could

explain adverse health effects experienced at fairly low indoor

airborne microbial concentrations.38,41 Nielsen et al.42 suggested

synergistic effects of mycotoxins together with other biologically

active metabolites (e.g. endotoxins, bacteria, mites). Recent

reports have shown synergistic effects in cytotoxicity and

apoptosis mechanisms in mouse macrophages challenged by

spore extracts from co-cultures of S. chartarum and Aspergillus

versicolor.43

As LC-MS-MS only allows screening for target compounds

that are determined in advance, 23 of the 99 sample extracts were

additionally analysed by means of LC-Q-TOF-MS. In these

samples, 42 fungal metabolites with a molecular mass ranging

This journal is ª The Royal Society of Chemistry 2009

Table 6 Possible annotation of fungal metabolites in various samples bymeans of LC-Q-TOF-MS analysis

Mass (range) Fungal Metabolite

170.060 Penicillic acid182.060 Carolic acid234.105 Cyclo(Phe-Ser)238.148 Agroclavine I238.191 Culmorin250.121 Vertinolide252.139 Frequentin254.152 Elymoclavine

PalitantinEpoxyagroclavine I

266.141 Ascochitine280.120 Cyclopeptin280.135 Phaseic acid282.186 Fumagillol294.115 Italinic acid294.108 Cyclopenin296.121–296.126 Deoxynivalenol308.129 50-Hydroxyasperentin314.079 Aflatoxin B2

318.110 Epi-dechlorogriseofulvinDechlorogriseofulvin

318.225 3,4-Epoxy-6-hydroxy-dolabella-7,12-dienone

320.129 PR-toxinMycophenolic acid

326.040 Gliotoxin330.074 Aflatoxin B2a

Aflatoxin G2

Bis-dechlorogeodin338.136 3-Acetyldeoxynivalenol365.174 Brevianamide A373.227 Equisetin384.339 Desmosterol388.189 Desacetylpebrolide392.143 Vermiculin423.295 Dihydroxyaflavinine458.231 Fumagillin508.236 Acetyl-T2 toxin511.232 Verruculogen528.262 Chaetoglobosin A532.115 Dethiosecoemestrin544.193 Satratoxin G572.289 SMTP-6 (triprenyl phenol

derivative from Stachybotrysmicrospora)

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from 170.06 to 685.41 were tentatively identified by LC-Q-TOF-

MS in one or several samples (Table 6). These preliminary

results confirm that fungal metabolites, other than those usually

analysed by targeted techniques, can be present in mouldy inte-

riors. It is clear that more data need to be generated on the

occurrence of different fungal metabolites and mycotoxins in

indoor environments. Since reference standards are non-existent

for most of these fungal metabolites, identification through

accurate mass measurements will become the method of choice,

giving the possibility to screen for a much wider range of possible

metabolites in water-damaged buildings. This will give new

insights into relevant mycotoxins that should be further investi-

gated in target analysis of indoor environments. Important to

mention is that Q-TOF analysis is characterized by a ca. 5 ppm

error range. Therefore, more sophisticated instruments such

as Fourier transform MS could result in a more accurate

identification.

This journal is ª The Royal Society of Chemistry 2009

Fungal identification

Visible fungal growth on surfaces was sampled because this can

give an indication of the fungal population in the house and to

correlate fungal growth with the mycotoxins detected. Molecular

fungal identification was only carried out for a limited number of

samples (houses n� 1 to 3), since it was practically impossible to

do PCR-analysis on all 99 samples. Mostly, correlations between

the presence of fungi and mycotoxins in the same sample were

found. The results are gathered in Table 7. Values for relative

humidity (RH) and temperature were recorded as well. In

general, the highest RH values were measured in those houses

with the most visually extended mould growth, namely houses 3,

4 and 7. However, it was not possible to correlate the relative

humidity and temperature values with mycotoxin concentrations

since no quantitative data on the fungal load in the houses were

available.

For house n� 1, the detection of roquefortine C and ster-

igmatocystin could be matched with the presence of Penicillium

chrysogenum44 and Aspergillus versicolor,45 respectively.

For house n� 2, 11 mycelium samples were molecularly iden-

tified. Roquefortin C detection in mycelium samples M1, M5,

M6, M10 and M11 could be explained by the presence of

P. chrysogenum, P. expansum45,46 and P. carneum47 while in

samples M2, M7 and M8 any Roquefortine C producing fungal

strain was absent. Chaetoglobosin A was detected in most

mycelium samples of house 2. However, possible producers were

isolated only from mycelium samples M4, M5 and M9 e.g.

P. expansum48 and P. discolor.45 Samples M7 and M8 contained

sterigmatocystin but no fungal strain that could be the producer

was detected. Moreover, roridin E was found in the air sample of

house 2.

In house n� 3, wallpaper samples (W7 to W10) from the same

surface contained sterigmatocystin while A. versicolor was only

found in W9 and W10. The same was observed in wallpaper W11

to W16 with W15 containing A. versicolor.

For the rest of the samples (houses n� 4 to 7), only morpho-

logical identification was assessed. In house n� 4, the detection of

the toxins aflatoxin B2, ochratoxin A and sterigmatocystin in the

dust samples D1–D7 could be due to the presence of strains

belonging to the genus Penicillium and Aspergillus. For example,

A. flavus and A. parasiticus are known producers of AFB2,49

while either Penicillium verrucosum, P. nordicum or A. ochraceous

might be responsible for OTA production.45 Sterigmatocystin,

which was also detected, is a precursor of the aflatoxins,49 but is

also produced by some non-aflatoxigenic strains such as A. ver-

sicolor or A. nidulans.45 The dust sample D2 was collected from

the same corner as D1 but only 2 Cladosporium strains could be

detected. The same toxins plus aflatoxin B1 and roquefortin C

were recovered after air sampling.

In the silicon sample of house n� 5 the fungal strain identified

as Aspergillus flavus probably is responsible for the production of

the detected aflatoxin B1.45 Sterigmatocystin, found in mycelium

sample M12, M13 and M14 and in the air and dust (D8–D9) of

house n� 5, can originate from either the Aspergillus sydowii or

the Chaetomium spp. strain. Also chaetoglobosin A, detected in

samples M13 and M14, is a known metabolite of Chaetomium

spp. Besides sterigmatocystin and chaetoglobosin, also aflatoxin

B1 and B2, ochratoxin A and roridin E were detected in the air of

J. Environ. Monit., 2009, 11, 1849–1858 | 1855

Table 7 Summary of detected mycotoxins and morphologically identified fungi in different samples, namely W ¼ wallpaper samples, M ¼ myceliumsamples, D ¼ dust samples. Fungal samples of houses 1–3 were molecularly identified as well. Values of relative humidity (RH, %) and temperature (T,�C) during sampling are given for each house, except house n� 1

Sampling place Samples Mycotoxins Fungi

House n� 1 W1 ROQC Penicillium chrysogenum, Scopulariopsis fusca, Ulocladium chartarumW2 ROQC, STERO P. chrysogenum, S. fusca, Aspergillus versicolorW3 ROQC, STERO P. chrysogenum, S. fusca, A. versicolor, P. communeW4 ROQC P. chrysogenum, S. fuscaW5 ROQC, STERO P. chrysogenum, S. fusca, A. versicolorW6 ROQC P. chrysogenum, S. fusca, A. versicolor

House n� 2 M1 CHAETO Trichoderma atroviride, P. chrysogenumRH ¼ 56% M2 CHAETO, ROQC T. atroviride, T. harzianumT ¼ 21.4 �C M3 CHAETO T. atroviride, P. chrysogenum, A. flavus, A. fumigatus, T. harzianum

M4 CHAETO T. atroviride, P. expansumM5 CHAETO, ROQC T. atroviride, A. flavus, P. expansum, P. purpurogenum, P. vulpinumM6 CHAETO, ROQC T. atroviride, P. chrysogenumM7 ROQC, STERO, CHAETO T. atroviride, T. harzianumM8 ROQC, STERO, CHAETO T. atroviride, A. fumigatusM9 CHAETO T. atroviride, P. discolorM10 ROQC T. atroviride, P. carneumM11 ROQC T. atroviride, P. carneum

House n� 3 W7 STERO T. atrovirideRH ¼ 72% W8 STERO T. atrovirideT ¼ 17.4 �C W9 STERO P. verrucosum, P. chrysogenum, Stachybotrys chartarum, A. versicolor

W10 STERO P. verrucosum, P. chrysogenum, A. versicolor, A. nigerW11 STERO T. atroviride, P. italicum, P. corrylophilumW12 STERO T. atroviride, P. crustosumW13 STERO P. verrucosum, P. italicum, P. chrysogenum, P. olsonii, P. glabrum,

P. carneumW14 STERO T. atroviride, P. chrysogenum, P. verrucosum, P. olsoniiW15 STERO P. chrysogenum, A. versicolorW16 STERO P. chrysogenum, S. fusca

House n�4 D1 AFB2, OTA, STERO Alternaria alternata, Penicillium spp., Aspergillus spp.RH ¼ 68% D2 AFB2, OTA, STERO Cladosporium spp.T ¼ 18 �C D3-D7 AFB2, OTA, STERO Cladosporium spp., Penicillium spp., Aspergillus spp.House n� 5 Silicon AFB1 Penicillium spp., Cladosporium spp., Wallemia sebi, Aspergillus flavus groupRH ¼ 53.8% M12 STERO Trichoderma spp., Penicillium spp., A. sydowii, FusariumT ¼ 22.4 �C M13 STERO, CHAETO Chaetomium spp., Penicillium spp., Acremonium strictum

M14 STERO, CHAETO A. sydowii, Chaetomium spp., A. strictumD8-D9 STERO Cladosporium spp., Penicillium spp., Aspergillus spp., A. ochraceous

House n� 6 W17 STERO, CHAETO, ROQ C S. chartarum, Chaetomium spp., A. ustusRH ¼ 41.5% M15 CHAETO, ROQ C S. chartarum, Chaetomium spp., Penicillium spp.T ¼ 20.6 �C D10-D11 STERO, ROQC S. chartarum, Penicillium spp.House n� 7 M16 AFB1 Aspergillus spp.RH ¼ 94% M17 AFB1 A. strictumT ¼ 21 �C M18 AFB1 A. versicolor group, Chaetomium spp., Aspergillus spp.

D12-D13 STERO Chaetomium spp., Aspergillus spp., Rhizopus spp., Acremonium spp.,Penicillium spp., unidentified Dematiaceae

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house n� 5, possibly produced by the isolated fungal strains

Aspergillus spp., A. ochraceous (in D8 and D9) and Fusarium spp.

(in M12), respectively.

Also the mycotoxins detected in house n� 6 can be explained

through the isolation of fungal strains that may be their

producers. In fact, chaetoglobosin A found in wallpaper (W17)

and mycelium (M15), might be produced by the recovered

Chaetomium or, possibly Penicillium strains (in case it would

concern P. aurantiogriseum or P. discolor).45 Many strains

belonging to the genus Penicillium, collected in samples M15,

D10 and D11, can excrete roquefortine C47 while some Chaeto-

mium species45 and P. luteum50 can produce sterigmatocystin.

In house n� 7 the presence of aflatoxin B1, found in samples

M16, M17 and M18, is probably due to the presence of the

unidentified Aspergillus strains, while sterigmatocystin found in

D12 and D13 might derive from the Chaetomium spp.

1856 | J. Environ. Monit., 2009, 11, 1849–1858

In few cases the presence of the mycotoxins could not be

ascribed to a specific fungal strain. Loss of fungal viability might

be a reasonable explanation for the absence of the mycotoxin

producer. Krysinska et al.51 detected deoxynivalenol, nivalenol

and ochratoxin A in grains and grain dust in 100%, 94.4% and

91.7% of the samples, respectively. Afterwards, it was shown that

Fusarium species capable of producing these mycotoxins were

only present in 58.3% of the grain dust samples. Furthermore,

since all mycelium samples were taken from the same surface,

diffusion of the mycotoxins could be another explanation.

Hanssen and Jung52 measured 600 mg/kg aflatoxin B1 even at

a distance of 45 mm from the mycelium zone after inoculating

whole-meal bread with Aspergillus flavus. They also found that

diffusion characteristics of mycotoxins appeared to be linked to

the amount of water available. Dry conditions may restrict

diffusion more effectively. In this study, all samples were taken in

This journal is ª The Royal Society of Chemistry 2009

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water-damaged houses with a high relative humidity, which

makes diffusion of mycotoxins possible. The most common

fungal species isolated from the seven sampled mouldy buildings

were Penicillium chrysogenum, Aspergillus versicolor (group),

Chaetomium spp. and Cladosporium spp.

(M)VOC analysis

In addition to mycotoxins and fungal species, volatile organic

compounds were sampled since it has been suggested that fungal

volatiles may be useful in the early detection of mould growth

and/or mycotoxin production.15,24

117 (M)VOCs were identified in the indoor air of the 9

buildings sampled (7 mouldy interiors, 1 control house and house

n� 4 after cleaning) (data not shown). The sampling technique

that yielded the highest (M)VOC concentrations and the best

peak shape was the dynamic headspace absorption on Tenax.

SPME-GC-MS confirmed the presence of almost all compounds.

When an environment was sampled twice, a longer sampling time

was shown to allow detection of more volatiles.

In the control house, the (M)VOC concentration was higher

than in the mouldy interiors, which can be due to the fact that

a more recent building emits more VOCs than an established

one.22,53 The concentration of M(VOC)s was also higher in house

n� 4 after cleaning than when evident mould growth and high

humidity were present. This is in agreement with some studies

where the sum of VOCs in reported ‘healthy’ buildings appeared

to be higher or equal to that measured in ‘sick’ buildings.54

Common (M)VOCs found in the seven mouldy buildings were

toluene, ethylbenzene, xylenes, 1-ethyl-2-methylbenzene and 2,6-

di-tert-butyl-4-methylphenol. Limonene, a-pinene, nonanal,

decanal, 2-methyl-3-(4-tert-butylphenyl)propanal, 1,3,5-trime-

thylbenzene and linear saturated alkanes (C9–C17) were also

found in the control house. Most of these compounds have been

described in a study in literature, where 2103 dwellings were

sampled for VOCs.55,56 Styrene,57 1,3-dimethylbenzene, trime-

thylbenzene, nonanal,58 2,6-di-tert-butyl-4-methylphenol, long-

ifolene, pinenes, limonene, ethylbenzene, toluene, octane and

nonane59 can be derived from the isolated fungal species. To

study the origin of these compounds, fungal species isolated from

the contaminated building materials are currently under

screening for VOC production. So far however, none of the

compounds detected is surely produced by the isolated fungi or

can be held responsible for the mouldy odour. Organic

compounds that are responsible for this odour may be present in

concentrations too low to be collected and/or identified by

standard analytical techniques.22

Conclusions

This is a first report on the simultaneous analysis of fungi,

mycotoxins and (M)VOCs in mouldy interiors. The occurrence

of mycotoxins in buildings is demonstrated. In general, the

fungal strains identified matched well with the mycotoxins

detected. However, it is not possible to evaluate the significance

of the detected mycotoxin concentrations due to the lack of

knowledge on the toxicity of mycotoxin inhalation. In addition,

due to the absence of quantitative data on fungal samples, only

the mycotoxin and (M)VOC presence and not the concentration

This journal is ª The Royal Society of Chemistry 2009

is discussed. Moreover, research is needed on the toxicity of co-

occurring indoor mycotoxins and other biologically active

agents. Since aflatoxin B1, aflatoxin B2, ochratoxin A and roridin

E found in indoor dust and air, may represent a health hazard,

studies concerning inhalational toxicity should address these

mycotoxins first.

At present, no mould-specific VOC was detected even in those

environments where a strong mouldy odour was present. The

presence of the majority of the (M)VOCs detected was in

agreement with other studies. These compounds were probably

emitted from building materials and cleaning products or were

infiltrations from the outdoor air. Many of them may cause

documented or suspected adverse health effects in humans.

Acknowledgements

This work was financially supported by the Research Founda-

tion – Flanders (FWO-Vlaanderen) (research project G.0034.07

and a postdoctoral fellowship of An Adams) and BOF UGent

(research project 015/102/07 and basic equipment 05B05106).

Special thanks to V. Ricci (ISPA-CNR, Italy) for his valuable

technical assistance. Also thanks to V. Vandecasteele and W.

Dubois (‘‘Vlaamse Overheid, Wonen – Vlaanderen’’, Belgium)

for the provision of water-damaged buildings, to Hamide

Senyuva (TUBITAK-ATAL, Turkey) for her valuable contri-

bution concerning the mycotoxin database.

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