presentation air pollution conference - pdf

8/9/2019 Presentation Air Pollution Conference - PDF

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The role of fungi, mycotoxins and volatile organiccompounds in indoor air pollution

V. Polizzi 1, B. Delmulle 2, A. Adams 1, A. Moretti 3, Y. Rosseel 4, E. Adriaens 5, C. Van Peteghem 2, S. De Saeger 2

and N. De Kimpe 1

1Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University, Belgium.2Laboratory of Food Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Belgium.

3Institute of Sciences of Food Production, National Research Council, Bari, Italy 4Department of Data Analysis Faculty of Psychology and Educational Sciences, Ghent University, Belgium

5Laboratory of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Ghent University, Belgium

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Overview

1. Sick building syndrome (SBS) and its relation to fungalgrowth and metabolites

2. Description of the procedure optimized for samplings inmould environments

3. Results of indoor samplings

4. Conclusions and recommendations

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The Sick Building Syndrome (SBS) is described as a set of non-specific symptoms experienced by the occupants of a building withpoor indoor air quality (IAQ).

SBS and its relation to fungal growth and MVOCs

Eye IrritationDry ThroatRhinitis

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Runny NoseHeadacheFatigueSkin IrritationShortness of BreathCoughDizzinessNauseaMental Confusion


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Airborne fungal metabolites as a concern for humanhealth

Allergens

Mycotoxins

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Microbial volatile

organic compounds(MVOCs)


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Some (M)VOCs have adverse effect onrespiratory system, blood vesselsystem, nerve system and may becarcinogenic (Yu et al., Int J Refrigerat , 2008) .

(M)VOC odours are believed to have

SBS and its relation to MVOCs

an n uence on e per ormance opeople, causing mental and cognitivedistraction (Wolkoff et al., Indoor Air , 2006) .

(M)VOCs may be indicative of fungalgrowth and/or mycotoxin production(Larsen and Frisvad, Mycol Res, 1995) .

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Overview





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Optimized procedure for samplings in buildings

Airborne Sample Analysis Platform(ASAP): for bioaerosol present in

indoor airCyclone surface sampler: for dust

Scalpel: for contaminated building

Surface sampling through sterileswab and inoculation on 2 media

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Solid Phase MicroExtraction (SPME) coupled with GC-MS analysisDynamic headspace with thermal desorption of tenax and GC-MS analysis

Thermohygrometer for measurement of indoor temperature and relative humidity

stick-to-it Exposition of Petridish to indoor airLC-MS/MS


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Overview





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Mycotoxin analysis

Mycotoxin Number of positive

substratessamples

Concentrationrange in

substrates(ng/cm 3)

Number of positive air

samples

Concentrationrange in air

(ng/cm 3)

Sterigmatocystin 28 0.063-778.4 3 0.0034-1.7674

Roquefortin C 16 0.106-7.2 1 0.009-4

Chaetoglobosin A 13 1.16-13830 3 0.0067-3.4205

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Aflatoxin B 2 7 0.019-0.253 4 0.0003-0.0211

Aflatoxin B 1 5 0.111-0.332 5 0.0024-0.1463

Roridin E 0 0 3 0.0031-0.082

Ochratoxin A 7 0.008-0.773 3 0.0115-0.228

62 out of 99 samples showed presence of one or more mycotoxins afterLC-MS/MS analysis

Polizzi et al., J Environ Monit , 2009


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Mycotoxin analysis

Mycotoxin Number of positive

substratessamples

Concentrationrange in

substrates(ng/cm 3)

Number of positive air

samples

Concentrationrange in air

(ng/cm 3)

Sterigmatocystin 28 0.063-778.4 3 0.0034-1.7674

Roquefortin C 16 0.106-7.2 1 0.009-4

Chaetoglobosin A 13 1.16-13830 3 0.0067-3.4205

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Aflatoxin B 2 7 0.019-0.253 4 0.0003-0.0211

Aflatoxin B 1 5 0.111-0.332 5 0.0024-0.1463

Roridin E 0 0 3 0.0031-0.082

Ochratoxin A 7 0.008-0.773 3 0.0115-0.228

62 out of 99 samples showed presence of one or more mycotoxins afterLC-MS/MS analysis



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Observed counts Expected countsAir 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

The incidence of the detected mycotoxins on eachspecific type of sample

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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 0.2

Absent 9 20 14 6 1 10 17 12 11 0.8

observed counts against the expected counts assuming the null hypothesis(there is no association between mycotoxin incidence and type of sample)


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Assessment of the correlation between the incidenceof a specific mycotoxin and the type of sample

P-value Pearson residuals

Air Wallpaper Mycelium Dust SiliconeSTERO 0.00629 -1.29 1.45 -1.2 0.88 -0.72

CHAETO < 0.0001 -0.11 -1.44 3.76 -1.86 -0.52ROQC 0.102 -1.3 0.5 1.52 -0.88 -0.53

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. . - . - . . - .

AFB1 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

Positive values for Pearson residuals indicate that moreincidences are observed than expected


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Assessment of the correlation between the incidenceof a specific mycotoxin and the type of sample

P-value Pearson residuals

Air Wallpaper Mycelium Dust SiliconeSTERO 0.00629 -1.29 1.45 -1.2 0.88 -0.72

CHAETO < 0.0001 -0.11 -1.44 3.76 -1.86 -0.52ROQC 0.102 -1.3 0.5 1.52 -0.88 -0.53

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. . - . - . . - .

AFB1 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

Positive values for Pearson residuals indicate that moreincidences are observed than expected


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No risk assessmenthas been erformedNo risk assessment

has been erformed

Mycotoxins can be 10 times more toxicthrough inhalation than by oral intake (Creasia etal., Fundam Appl Toxicol , 1987)

for human inhalationexposure tomycotoxins

for human inhalationexposure tomycotoxins

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Studies concerning inhalation toxicity shouldaddress first aflatoxin B1 and B2, ochratoxin Aand roridin E


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To date, little is knownTo date, little is known

Synergistic cytotoxic and inflammatoryproperties of amoebe and Stachybotryscalifornicus or Penicillium spinolosum spores has

on synergistic effects of simultaneous exposure

to mycotoxins

on synergistic effects of simultaneous exposure

to mycotoxins

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een s own Y i-Piri a et a ., Environ Toxico , 2007

Synergistic cytotoxicity and apoptosismechanisms of spore from co-coltures of S.chartarum and Aspergillus versicolor has been

reported (Bloom et al., Appl Enviorn Microbiol , 2007)


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Fungal identification

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Chaetomium spp.Cladosporium spp.Penicillium spp.

Aspergillus spp.

Trichoderma harzianum Stachybotrys chartarum



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(M)VOCs117 (M)VOCs were detected in the indoor air of 11 buildings

Common (M)VOCs in mouldy buildings:

OH

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Common (M)VOCs found also in the control house:

o uene e y enzene xy enes s yrene , - - er - u y - -me y p eno

O

O

O

limonene

-pinene 1,3,5-trimethylbenzene lilial

nonanal

decanal

5-13

linear alkanes (C9-C17)

H

H

H



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(M)VOCs117 (M)VOCs were detected in the indoor air of 11 buildings

Common (M)VOCs in mouldy buildings:

OH

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Common (M)VOCs found also in the control house:

o uene e y enzene xy enes s yrene , - - er - u y - -me y p eno

O

O

O

limonene

-pinene 1,3,5-trimethylbenzene lilial

nonanal

decanal

5-13

linear alkanes (C9-C17)

H

H

H



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Mould-specific volatile compounds detected indoor

O

OH

OMe

OAc

OMe

5-methyl-3-heptanone

valencene

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Fungal species isolated from the contaminated building materials arecurrently under screening for VOC production

MVOCs may be present in concentrations too low to be collected or tobe identified by standard analytical techniques (Wolkoff et al., Indoor Air ,2006)

eugeno eugeno acetate


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(M)VOCs Health effectsMonoterpenes, benzyl acetate, bornylacetate, benzyl alcohol

React with ozone and NO x to form secondary sensorypollutants which cause eye and respiratory tract

irritation2-Hexylcinnamaldehyde, ionone derivatives,

lilial, limonene, linaloolSuspected allergens causing skin irritation: they

contain chemical groups considered responsible for

binding with skin proteins

1,4-Dichlorobenzene

Short-term exposure gives irritation to the eyes andthe respiratory tract

Long-term exposure regards liver, kidneys and blood

Styrene

, ,

Classified as a possible human carcinogen by theInternational Agency for Research on Cancer (IARC)

Toluene, xylenes, ethylbenzene,trimethylbenzene

Narcotics, irritants, affect the central nervous system

Alkanes Octane, decane and undecane are possible co-

carcinogensSaturated short aldehydes Contact allergens due to the formation of imines withskin proteins

Toluene diisocyanate Sensitizer; probable human carcinogen

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Example of MVOC possibly related with SBS

6-pentyl-2-pyrone

High production of 6-pentyl-2-pyrone by a Trichoderma

harzianum strain isolated froma mouldy environment

6-pentyl-2-pyrone contains chemical groups heldresponsible for triggering skin irritation

the mucosal irritation potency of this compound wastested by means of a slug mucosal irritation assay (SMI)

23Polizzi et al., submitted , 2010


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Irritation and tissue damage caused by 6-pentyl-2-pyroneaccording to the SMI assay

Total MP 1 Protein 1 LDH1 ALP1 Class

(%) (g/ml.g) (U/l.g) n/5 Irritation Damage- PBS1 -3.8 2.8 9 2 - 0/5 No No

+BAC1 1% 26.1 7.6 139 20 7.6 3.8 3/5 Severe Severe

6-pentyl-2-pyrone 1% 14.9 5.0 34 11 0.1 0.1 0/5 Moderate Mild

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6-pentyl-2-pyrone 5% 18.8 3.0 72 46 2.8 2.6 1/5 Severe Moderate

6-pentyl-2-pyrone 10% 16.0 4.9 70 57 2.4 2.5 0/5 Severe Moderate1PBS= phosphate-buffered saline, BAC= benzalkonium chloride, MP=Mucos production, LDH=lactate dehydrogenase,

ALP=alkaline phosphatase activity

A heavy indoor contamination (e.g. 1 m2

on plasterboard at 25C) by Trichodermasp. could produce quantities of 6-pentyl-2-pyrone that fall within theconcentration range classified in the SMI as irritating and damaging the mucosal

membrane (Polizzi et al., submitted , 2010) .


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This is a first report on thesimultaneous analysis of fungi,

mycotoxins and (M)VOCs in

mouldy interiors

Mainly sterigmatocystin,roquefortine C and

chaetoglobosin A were found,in 31, 17 and 16 out of 99

samples, respectively

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en c um c rysogenum , sperg us vers co or group, aetom um spp.and Cladosporium spp. are the most common fungal species recovered

In a sampling of a mouldybuilding, mould-specificvolatile compounds weredetected for the first time

6-Pentyl-2-pyrone is a MVOCpotentially related to SBSsymptoms, as confirmed by

the SMI assay


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Recommendations

Research is needed on the inhalation toxicity of mycotoxinsand of co-occurring indoor mycotoxins.

Studies concerning inhalation toxicity should address firstaflatoxin B1 and B2, ochratoxin A and roridin E.

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There is a need of techniques with lower detection limits fordetection of (M)VOCs in indoor environments.

The screening in vitro of fungal samples is useful foridentification of MVOCs potentially hazardous for humanhealth.


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Slug mucosal irritation assay

Cut-off values for the TotalMucus production are used toclassify the compounds intonon-irritants, mild, moderate &

Daily during 5 successive days

30 Contact Period with100 l of testsubstance

Measure Mucusproduction

Irritation

Calculate Total Mucusproduction

Test procedure (5Test procedure (5- -day)day)

30 CP

n=5

EndpointsEndpoints CalculationsCalculations PredictionPredictionModelModel

severe irritants .

Transfer slug to newpetri dish. Add 1 mlPBS and remove thisafter 1 h.

Transfer slug to newpetri dish. Add 1 mlPBS and remove thisafter 1 h.

Tissue damageMeasure Protein,

LDH & ALPrelease in samples

Calculate MeanProtein, Mean LDHrelease and countnumber of slugsinducing ALP release

Sample 1

Sample 2

A decision tree combining theresults of the Mean Protein,Mean LDH and number of slugsshowing ALP release is used toconvert the results into tissuedamage grades: no, mild,moderate and severe tissue damage

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