THE DUSTFALL COLLECTOR - A SIMPLE WAY TO MEASURE EXPOSURE TO MICROORGANISMS

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1 THE DUSTFALL COLLECTOR - A SIMPLE WAY TO MEASURE EXPOSURE TO MICROORGANISMS H Würtz 1 National Institute of Occupational Health, Lersø Parkallé 105, DK-2100 Copenhagen ABSTRACT A new developed dustfall collector has been investigated which collects airborne dust by sedimentation. The dustfall collector was placed at least 1.5 m above the floor. It was made of cardboard and the collecting surface was covered with aluminium foil; a chicken wire covered the box. After 140 days the dust was collected using the VacuMark sampler. Bioaerosols were sampled three times during a school day with one week intervals using IOM samplers. Airborne total particulate mater (TPM) was sampled on closed faced Millipore cassettes. Floor dust was sampled at the end of a school day using an HVS-3-sampler. Dust collected by the three sampling methods was weighed and analysed for culturable fungi. Comparing the levels of culturable fungi of airborne dust or collected from the dustfall collector in mechanically ventilated classrooms indicates an association between the two ways of measuring the exposure to culturable fungi. INDEX TERMS Schools, Fungi, Exposure assessment, Dustfall collector, Bioaerosols, Floor dust, TPM INTRODUCTION Many epidemiological studies have stated a relationship between mould/moisture and health outcomes but no dose response have been established yet (Bornehag et al, 2001). The reason for that could be that exposure assessment is not good enough (Husman, 1996). For quantification of exposure personal exposure measurements or stationary measurements of airborne dust usually have to be used. These measurement may be poor predictors of long term avarage exposures because bioaerosol levels vary widely due to e.g. the activity level and degree of ventilation see e.g. (Würtz et al, 1999). Some researchers have used floor dust as a surrogate for exposure measurements because this dust is considered to be less influenced by indoor activities and ventilation and therefore represents more of a long term pattern than air sampling (Wickman et al, 1992) and some analysis require bigger amounts of dust than possible to get using air sampling. Using floor dust is not without problems. It is not known how well the dust reflects the exposure, the age of the dust is often not known and the mass and concentration of e.g. of microorganisms may vary depending of the level of dust and dirt from the outside environments. Usually only one floor dust sample is taken from each site but studies of the reproducibility shows that the content of fungi may vary from one period to each other (Verhoeff et al, 1994). The purpose of this study was to investigate if a new sampling method (the dustfall collector) could overcome these problems. The dustfall collector collects dust by sedimentation from the air during long sampling periods e.g. 5 months or more. 1 Contact author: hw@ami.dk 425

2 MATERIALS AND METHODS Design: 6 schools (a total of 35 classrooms) were investigated in the winter 1999/2000. Airborne dust: bioaerosols were sampled by stationary sampling during a school day three times in each classroom with a least one week intervals. IOM inhalable dustsamplers operated at an airflow of 2 L/min mounted with polycarbonate filters (25 mm; 0.4 µm; Nuclepore) were used for collecting inhalable dust which was analysed for culturable fungi. In parallel airborne TPM (total particulate matter) was sampled onto teflon filters (25 mm; 1µm; Fluopore; Millipore) mounted in closed faced Millipore cassettes operated at an airflow of 1.9 L/min (1.25 m/s inlet velocity). The samplers were placed 1.5 m above the floor in the rear end of a classroom. The dustfall collector: the dust was collected in boxes made of cardboard (51 cm x 35.5 cm; INCO) covered with aluminium foil (25 µm) upward facing side. To prevent collection other than airborne dust chicken wire was used to cover the boxes. The boxes were placed on cardholders, on shelves or cupboards at least 1.5 m above the floor in the same rooms as the airborne measurements. The heights above floor of the dust collectors in the present study had a mean of 2.2 m and a standard deviation of 0.33 m. The sedimented dust was collected using VacuMark samplers mounted with polycarbonate filters (76mm; 1 µm; Nuclepore). The VacuMark casette was equilibrated at constant temperature and relative humidity and weighed before and after sampling the dust. When using the VacuMark sampler a special designed metal frame were used to hold the aluminium foil. The total vacuumed area was 0.13 m 2. Airborne dust was sampled for 140 days in the period January until April Floor dust: dust from the class rooms floors was collected using a modified HVS-3 sampler (ASTM Designation: D ) connected to a vacuum cleaner (Nilfisk). The pressure drop through the HVS-3 was set at 12 in. water and the nozzle pressure drop was set approx. at 8 in. water by using a variotransformer (Lübcke). The dust was separated from the air by a cyclone and collected in a 250 ml glass jar. Floors were vacuumed without moving the furnitures until approximately 2 cm level of dust was seen in the jar. A bicycle computer (cyclocomputer, Cateye vectra, model cc-7000) measured the vacuumed distance. The floors were mopped every day morning or late afternoon and therefore the dust from the floors was considered to be at most one day old. Other measurements: The teachers recorded how many windows were opened for how long time in the sampling period. A parameter IAER was defined as an 'indicator of air exchange rate' for a sampling period. IAER was estimated as N*T/A where N is number of windows opened, T is time the windows were open and A is the sampling period. Analysis of dust and bioaerosols: filter samples for cultivation were kept at room temperature for no more than 24 hours and then resuspended in the filter cassettes by adding 10 ml sterile 0.05% Tween 80. The cassettes were vigorously shaken on a shaking table for 15 min (500 rpm) at room temperature. The floor dust samples were kept at room temperature for no more than 24 hours before cultivation and the dust from the dustfall collector was kept at constant temperature and humidity and handled within 3 weeks. This method is validated by Macher (2001), who found that the concentration of culturable fungi and bacteria in dust from floors kept at room temperature, did not change over a period of 25 days. Both types of dust are suspended in 0.05% Tween20 by orbital shaking at 250 rpm for one hour. The dustfall collector samples were kept in 25% glycerol and then frozen at -80 C until cultivation. The mass of TPM was 426

3 determined by weighing the Teflon filter before and after sampling. Before weighing, the filters were equilibrated at a constant air temperature and humidity for at least one night. Culturable counts Ten-fold dilutions of the suspension fluid were spread onto the media. Mesophilic fungi were cultivated on Dichloran Glycerol Agar (Oxoid CM729) supplemented with penicillin (30mg/L) and streptomycin (30 mg/l) at 25 C. The number of colony forming units (cfu) from the air samples was calculated as cfu/m 3 and cfu/g airborne TPM The minimum detectable concentration (LOD) of the air samples was 10 cfu/filter which was equivalent to approx. 30 cfu/m 3 depending on the volume of the sampled air. The number of cfu of fungi in the dustfall collectors was calculated as cfu/g sedimented dust or as the flux (cfu/m 2 /day). LOD of the boxes was 40 cfu/ml, which was equivalent to approx. 2 cfu/mg depending on the weight of the sedimented dust or LOD is 2 cfu/m 2 /day. The amount of cfu in the floor dust was calculated as cfu/g floordust and cfu/m 2. LOD of the floor dust was approx. 1 cfu/mg depending on the weight of the sedimented dust og approx. 80 cfu/m 2 depending on the sampled area of the floor. Statistical analysis: the Wilcoxon signed rank test or Student T-test (SAS software, PROC UNIVARIATE) was used to test (at the 0.05 level) if there was a difference between the results of culturable fungi using two different measurement methods. The data was tested altogether and also when divided into mechanically and naturally ventilated class rooms. RESULTS Figure 1 shows the estimated degree of ventilation (IAER) in the 35 classrooms investigated in the present study. 2.0 Indication of air exchange rate L;(15) M;(18) Q1;(6) D;(18) Q2;(12) A;(12) P;(15) Figure 1: Indication of air exchange rate (IAER) in 6 schools. Black symbols illustrates class rooms with mechanical ventilation and white symbols illustrate class rooms with natural ventilation. In each class room IAER was registered three times with at least one week intervals. Schools are labelled with letters and in brackets are numbers of IAER. In figure 2-5 the y-axis is illustrated using base 10 logarithms. In figure 2 and 3 the level of airborne culturable fungi collected over a period of 140 days are plotted against 3 repeated airborne measurements. Data were only included if >1 cfu/agarplate had been found and therefore only 40% of the paired results from the class rooms are included. The statistical tests did not show a significant difference between the median of culturable fungi in the dust of 3 427

4 airborne measurements and dust collected in the dustfall collector for mechanically ventilated rooms (figure 2: p=0.06 and figure 3:p=0.95). However, for the total and naturally ventilated rooms there was a difference when testing data in figure 2 (p= and p=0.008 respectively) but did not differ significantly when testing data in figure 3 (p=0.7 and p=0.7 respectively). 1e+5 cfu/mg inhalable dust 1e+4 1e+3 1e cfu/mg sedimented dust; 140 days old Figure 2: culturable fungi in 5 schools (13 class rooms) where airborne measurements have been repeated three times with at least one week interval (n=39)(1 is missing) compared with dust collected in dustfall collectors (n=13) for 140 days. Black symbols illustrate class rooms with mechanical ventilation and white symbols illustrate class rooms with natural ventilation. Different symbols and colours indicate different schools except the white and black circle, which are from the same school where both naturally and mechanically ventilated classrooms occurred cfu/m 3 of inhalable air cfu/m 2 /day Figure 3: culturable fungi in 6 schools (14 class rooms) where airborne measurements (n=42) have been repeated with 1 week intervals compared with dust collected in dust collectors (n=14) for 140 days. Symbols as in figure

5 In figure 4 and 5 the results of the dust from the dustfall collector are substituted by the results from settled dust from the floors. Only results from two measurements of airborne dust are included because the third airsample was taken at the same day as the floor sample, which might result in bias. The statistical tests did show a significant difference between the median of culturable fungi in the dust of 3 airborne measurements and floor dust for both total, natural and mechanically ventilated rooms (figure 4: p=0.03, p= and p< respectively; figure 5: p=0.0002, p=0.005 and p=0.03 respectively). 1e+5 cfu/mg inhalable dust 1e+4 1e+3 1e cfu/mg floor dust Figure 4: culturable fungi in 6 schools (13 class rooms) where airborne measurements have been repeated three times with at least one week interval (n=39) compared with dust collected from floors (n=13), one day old at most. Black symbols illustrate class rooms with mechanical ventilation and white symbols illustrate class rooms with natural ventilation. Different symbols and colours indicate the different schools except the white circle and the black triangle, which are from the same school where both naturally and mechanically ventilated classrooms occurred cfu/m3 inhalable air e+2 1e+3 1e+4 1e+5 1e+6 cfu/m 2 floor dust, 1 day old Figure 5: culturable fungi in 6 schools (12 class rooms) where airborne measurements have been repeated three times with at least one week interval (n=36) compared with dust collected from floors (n=13), one day old at most. Symbols as in figure

6 DISCUSSION Figure 1 and 2 indicate an association between the median of repeated airborne measurements and the dustfall collector results for culturable fungi in mechanically ventilated rooms, both for the concentration pr mg dust and pr m 3 of air. This association was not seen in the naturally ventilated rooms when testing the concentration pr mg dust in figure 2 possibly because of the larger variability in the IAER. In figure 3 there was anyhow not a significant difference in the naturally ventilated rooms, which can be explained by variation in the data. Figure 3 and 4 show that there is a significant difference between the two measurement methods, which means no association between the two measurement methods. It is assumed that the air in classrooms is well mixed due to peoples activity and then the position of the sampler in the class room is of minor importance. This assumption is supported by a study that showed that there was no observable influence on the mass deposition rate in heights (0.3 m and 1.5 m above the floor) (Edwards et al. 1998). With this method more dust for analysing can be collected than the traditional samples. The dustfall collector method needs more extensive testing regarding influence of height and placement in a room and influence of the collection time. CONCLUSION The results indicate that the dustfall collector is a valuable tool for estimating the median exposure to airborne culturable fungi. The method is cost effective and simple to use for collecting airborne dust in the indoor environment. ACKNOWLEDGEMENT The technicians M. Simkus, Gitte H Hansen, D.Narv and S.H.Nielsen are acknowledged for their technical assistance. M.Sc.Thomas Schneider and Ph.D.Erik Holst are acknowledged for helpful discussions. The project is part of the Danish research programme Moulds in buildings. The programme is supported by the Danish government and private companies through the Danish Research agency. REFERENCES Bornehag C-G, Blomquist G, Gyntelberg F et al Dampness in buildings and Health. Nordic interdisciplinary review of the scientific on associations between exposure to ''Dampness'' in buildings and health effects (Norddamp). Indoor Air. (11), pp Edwards RD, Yurkow EJ, and Lioy PJ Seasonal deposition of housedusts onto household surfaces. The Science of the Total Environment. Vol. 224, pp Husman T Health effects of indoor-air microorganisms. Scandinavian Journal of Work, Environment and Health. Vol. 22 (1), pp Macher JM Evaluation of a procedure to isolate culturable microorganisms from carpet dust. Indoor Air. Vol (11), pp Verhoeff AP, van Reenen-Hoekstra ES, Samson RA et al Fungal propagules in house dust. I. Comparison of analytic methods and their value as estimators of potential exposure. Allergy. Vol. 49, pp Wickman M, Gravesen S, Nordvall SL et al Indoor viable dust-bound microfungi in relation to residential characteristics, living habits, and symptoms in atopic and control children. Journal of Allergy and Clinical Immunology. Vol. 89 (3), pp Würtz H Skimmelsvampe i Bygninger. pp Würtz H, Kildesø J, Meyer HWet al A pilotstudy on airborne microorganisms in Danish classrooms. Indoor Air 99, 8 th International Conference on Indoor Air Quality and Climate. Vol. 1, pp