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Applied and Environmental Microbiology, September 2008, p. 5621-5627, Vol. 74, No. 18
0099-2240/08/$08.00+0 doi:10.1128/AEM.00619-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands,1 Environmental Health Sciences Research Center, University of Iowa, Iowa City, Iowa2
Received 14 March 2008/ Accepted 21 July 2008
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Several studies have used airborne dust sampling methods, such as active airborne dust sampling with an ion charge device (2, 13) and passive airborne sampling with a petri dish (8, 9, 21) or dust fall collector (6, 24). Of these, the active airborne dust sampling and ion charge device methods have high logistic and equipment costs and have as yet been applied only in short-term and small experimental studies, e.g., to compare with other dust sampling methods (3, 6, 11, 21). The petri dish method is simple and inexpensive but collects dust on a small surface and therefore needs long sampling times (8, 9, 21). The aim of this project was therefore to develop a rigorous, low-cost passive sampling technique for airborne dust in order to assess exposure to airborne microbial and allergenic components in the home environment. Würtz et al. (24) developed a passive sampling technique, the dust fall collector, using an aluminum foil-covered inner surface of a pizza box to collect settling dust in school classrooms. For the measurement of endotoxin on surfaces, Thorne et al. (19) used electrostatic wiping cloths (cloths), which may also be used to collect settled dust from horizontal surface areas. Both methods yielded sufficient dust for endotoxin measurement, but the dust fall collector cannot easily be sent by regular mail before and after sampling, and both methods are rather difficult to handle carefully, without causing contamination, by home residents. We therefore designed a new electrostatic dust fall collector (EDC) by combining several of their features. The EDC consists of a custom-fabricated polypropylene sampler that has electrostatic cloths attached to it to provide a sampling surface. Airborne dust settles on this surface and is captured by the electrostatic properties of the cloth. The feasibility of this method for endotoxin measurements was evaluated in a validation study in urban and farm homes. The comparability of the new method to active airborne measurements as well as to floor dust sampling was investigated, and reproducibility within homes and between two sampling periods was studied.
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Sampling procedures. (i) Floor dust sampling.
Floor dust samples were collected by home residents in their living rooms as described previously (17) using a vacuum cleaner equipped with a 25-µm mesh nylon sample sock (Allied Filter Fabrics, Sydney, Australia). The sampling area was 1 m2 if there was a carpet or rug with a >4-m2 surface. In homes with a smooth floor or small rug, a 4-m2 surface area of the smooth floor was sampled. The sampling time was always 2 min. The whole procedure was repeated in the second period, and thus four living room floor samples were collected per home.
(ii) Active airborne dust sampling.
Active airborne dust samples were collected over 6 or 8 days. Harvard impactors (Air Diagnostics and Engineering, Inc., Naples, ME) were placed by a field worker at an approximately 1.5-m height, and the pump was run at a flow of 10 liters/min. The flow was calibrated before and after sampling, and no significant drop in the flow rate was noted for any of the measurements. Dust was collected on 37-mm Teflon filters (Anderson, Smyrna, GA) with a 2-µm pore size in three or four Harvard impactors, with a sample changer switching between the impactors after each 24-h period. Each filter was thus loaded with dust from two 24-h sampling periods to prevent possible overloading of the filters and to average out day-to-day variations. The whole procedure was repeated in the second period. In total, 6 or 8 samples were collected per home.
(iii) Electrostatic dust fall collector.
Passive airborne dust samples were collected in each home and in seven stables with the newly developed EDC. In this evaluation study, prototype cardboard samplers were used that were covered with a plastic film and four electrostatic cloths, each with an area of 0.032 m2 (Zeeman, Utrecht, The Netherlands), attached to their surfaces by aluminum-foil-covered frames. The cloths were rendered pyrogen free before use by heating overnight at 200°C. Each EDC contained four cloths. The EDC was opened horizontally to expose the cloths to the air, allowing the collection of settling dust for 14 days. Two EDCs were placed on top of a bookshelf or a like surface at least 1.50 m above the floor by the field worker visiting the home on day 0 (Fig. 1). At each of the farms, one EDC was placed in the stable, at a site where the animals could not reach it and where it was exposed to as little air disturbance as possible, such as away from doors or windows. The home residents were instructed to close the EDC carefully without touching the cloths after the sampling period. The EDCs were returned to the laboratory in a preaddressed envelope. The total number of cloths was 16 per home—2 sampling periods x 2 EDCs x 4 cloths per EDC—and 8 per stable.
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FIG. 1. The EDC. The sampler consists of four electrostatic cloths mounted in a 40- by 30-cm plastic folder that is left for 14 days in the horizontal position with the cloths exposed to the air. The folder is kept closed before and after sampling and during transport and storage.
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Analysis of samples.
Endotoxin analysis was performed in a suitable dilution in pyrogen-free water with the quantitative kinetic chromogenic Limulus amoebocyte lysate (LAL) assay (Lonza; LAL-Lysate lot EL004V; standard Escherichia coli O55:B5 lot 4L3560; reference standard endotoxin/control standard endotoxin ratio, 18 EU/ng). Each single filter or cloth extract from a home was measured in a 1/50 dilution and each floor dust sample at 1/5,000. The cloth extracts from the stable EDCs were tested in a 1/1,000 dilution and the crude stable dust extracts in a 1/20,000 dilution. The samples were retested at a more appropriate dilution when necessary due to too high or low values at the outer ends of the calibration line. No sample was diluted lower than 1/25. In the whole series of sample extracts, only seven (three from filters and four from cloths) had an endotoxin concentration below the limit of detection (LOD; 0.25 EU/ml for a 1/25 dilution) and were assigned the value of the LOD.
Statistical analyses.
First, arithmetic mean endotoxin loads per period per home were computed for all methods. For the EDC, this included the average of values measured in the four cloths per EDC and the average of the two EDCs used in the same period per home. In subsequent analyses, correlations were calculated between endotoxin levels in cloths from the same EDC, between mean values for duplicate EDCs used in parallel in the same period, between different sampling periods, and between the various sampling methods. Since data showed a log-normal distribution, Pearson correlation coefficients (r) and geometric means (GM) and geometric standard deviations (GSD) were calculated based on ln-transformed values.
We then explored the variability over time and between homes in endotoxin exposures by performing mixed-effect regression analyses. The home ID was included as a random factor to correct for correlations between repeated measurements. A factor identifying farm/nonfarm homes was introduced as a fixed effect. The analyses were conducted with the ln-transformed arithmetic mean endotoxin load per period and per home for all sampling methods (active airborne, n = 3 or 4 filters; passive airborne, n = 8 cloths; floor dust, n = 2 sample socks). Statistical analyses were performed with SAS statistical software (version 9.1; SAS Institute, Cary, NC).
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Endotoxin measurement.
Endotoxin could be measured in all but 4 of the 256 electrostatic dust fall sampling cloths from farm and nonfarm living rooms and in 127 of the 130 filter extracts. Thus, airborne dust endotoxin could be readily detected and quantified after recovery, not only from filters but also from EDC cloths exposed to the air for 14 days. All EDC cloths from stables, all crude stable dust, and all living-room floor dust samples yielded detectable endotoxin levels. Field control cloths, treated similarly as the sample cloths (heated to 200°C and attached to an EDC for 14 days but without exposure to the air), were all negative. The LOD was 213 EU/m2 for the EDC sampling and 0.05 EU/m3 for the active airborne dust sampling. For the floor dust sampling, the LOD varied—depending on the sampling area and extraction volume—from 62.5 to 2,000 EU/m2.
Table 1 presents the geometric mean endotoxin levels for farm and nonfarm homes separately, as measured with all three sampling methods. The mean endotoxin levels measured in the different types of samples were all far above the respective detection limits. The levels in the samples from farms were consistently higher than those for nonfarms, with GM ratios between the farm and nonfarm being lowest for the floor dust samples (2.5), slightly higher for active airborne samples (2.9), and highest for the EDC samples (3.3).
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TABLE 1. Indoor home endotoxin geometric means (GSDs) measured with different dust sampling methodsa
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Correlation between sampling periods.
The mean endotoxin levels measured over two 14-day periods in the same home correlated very well for the EDC (r = 0.85) as shown in Fig. 2. The correlation coefficient was very similar for the floor dust sampling (r = 0.79) but weaker for the active airborne dust sampling (r = 0.57). The lower correlation for active airborne dust sampling was completely due to a much lower correlation for the samples from farms (r = 0.19) than for the nonfarm samples (r = 0.92). No such differences between farms and nonfarms were seen in the EDC or floor dust samples.
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FIG. 2. Comparison of the endotoxin levels measured in the same homes over two sampling periods by electrostatic dust fall sampling (A), active airborne dust sampling (B), and floor dust sampling (C).
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FIG. 3. Comparison of the endotoxin levels measured by different dust sampling methods: active airborne dust sampling versus EDC sampling (A), floor dust sampling versus EDC sampling (B), and active airborne dust sampling versus floor dust sampling (C).
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TABLE 2. Within- and between-home variance components of ln-transformed endotoxin levels measured in two sampling periods with a 3-week time interval (model 1), and effect of being a farm on measured endotoxin levels (eβ) relative to reference nonfarm homes (intercept e ) (model 2)
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The endotoxin levels measured with the EDC or with floor dust sampling showed a four- to fivefold larger between- than within-home variance (Table 2, model 1), whereas for active airborne dust sampling, the within-home variance was larger than the between-home variance. When the farm/nonfarm factor was taken into account, the between-home variance decreased for all three methods, as part of the between-home difference is due to whether the home is a farm or not (Table 2, model 2). The between/within-home variance ratio for active airborne sampling decreased from 0.82 to 0.46 and for the EDC sampling from 4.64 to 2.94. This ratio was least affected for the floor dust sampling, changing from 3.95 to 3.77. Apparently, for the floor dust sampling, the farm/nonfarm environment factor did not influence the between-home variance as much as for both the airborne dust sampling methods. The factor difference for the farm/nonfarm environment from the mixed-model regression analysis confirmed the conclusion from the crude data analysis (Table 1) that farm homes have approximately three times higher endotoxin levels than nonfarm homes. Additional analyses were performed, including other home characteristics—pets, smoking in the home, reported mold growth, and type of floor cover from which the sample was taken—separately or in combination in the statistical models. In all cases, the difference between farms and nonfarms remained essentially unchanged—e.g., the farm/nonfarm factor for endotoxin measured by the EDC varied from 2.6 to 3.5 in the adjusted models (not shown). Thus, confounding by the other home characteristics could be excluded.
Endotoxin in EDC cloths from stables.
The EDC also proved to be suitable for airborne dust collection and endotoxin measurements in stables (Fig. 4). As expected, high levels for endotoxin were found (GM, 1,030,000 EU/m2). The crude settled dust samples from the stables showed similar endotoxin levels (GM, 1,300,000 EU/m2). These levels are approximately 100 times higher than the levels measured with the EDC in the farm living rooms (10,000 EU/m2; Table 1).
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FIG. 4. Endotoxin in stable dust samples from seven farms. (A) EDC; (B) crude dust.
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The estimated endotoxin levels in stables also showed a moderate to high correlation in time, with a correlation of 0.58 for both the EDC and the crude settled dust samples.
The endotoxin levels of the two types of stable dust samples correlated moderately (r = 0.52). Finally, when comparing stable dust endotoxin levels with the EDC results from the homes corresponding to the stables, we found a moderate correlation (r = 0.55) for the crude settled stable dust and a remarkably high (r = 0.82) correlation between the EDC endotoxin levels measured in the stables and in the living rooms of the same farms.
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The EDC endotoxin levels measured in living rooms as well as in stables showed good reproducibility between two different sampling periods. This indicates that the method may give a reliable proxy for long-term exposure assessment. Previous reproducibility studies have shown that floor and mattress dust sampling with a vacuum cleaner may result in reproducible proxies for exposures during a period of up to 1 year (1, 5). For the EDC, this needs further investigation since the interval between the sampling periods was only 3 weeks in the present validation study. From the correlation between the results of the two periods for all sampling methods, it seemed that endotoxin levels assessed by active airborne sampling in the farm environment were more susceptible to variation over time compared to the other two methods. Since the active airborne dust sampling selectively captured PM10, this suggests that the PM10 fraction may be more variable in time, but the numbers of observations in this study are too small to draw definite conclusions.
The levels of endotoxin measured with EDC sampling correlated reasonably well with the endotoxin levels measured with active airborne or with floor dust sampling, while the active airborne and floor dust sampling showed only low correlation with each other (r = 0.3). A similar low correlation (r = 0.21) for endotoxin measurement by active airborne and floor dust sampling was reported by Park et al. (12). The results of our study suggest that characteristics of the dust collected with the EDC in homes are somewhere in between the characteristics of the dust collected with the other two methods. Active airborne dust sampling was conducted with PM10 selective filter heads, while the EDC as well as the floor dust sampling are not size-selective methods. Particles captured by the EDC must be airborne to be captured at 1.5-m height and on average must be smaller than floor dust particles; nevertheless, they may be larger than the PM10 particles. Thus, it can be assumed that the EDC collects particles that may be missed by the active airborne dust sampling. Such intermediate dust properties may explain the good correlation of the EDC with both other methods and the much weaker correlation of the active airborne dust sampling with the floor dust sampling. The high correlation between the EDC endotoxin levels measured in stables and in living rooms from the corresponding farms indicates that the levels we found in those homes may be influenced by the 100-times-higher levels found in the stable. It has been described previously that particles may be transported from the workplace into the homes for bakery allergens (22) and animal allergens (10). The results from this small validation study suggest this may also be true for endotoxin. The high correlation to the active airborne dust measurements and the good repeatability over time supports our conclusion that the EDC can serve as a good proxy of airborne exposure to endotoxins.
Cloths within the same EDC or in two EDCs used in parallel in a home gave very similar results. Thus, differences in endotoxin yields between cloths—either due to a different dust-capturing efficacy, a different release of lipopolysaccharides from cloths, and/or analytical errors in the LAL assay—appeared to be small compared to the range of airborne endotoxin levels measured with the EDC. This means that a single cloth from an EDC should be sufficient to characterize a home's airborne endotoxin level. Since with one EDC we collect airborne dust on four parallel cloths, the four cloths may be used separately to measure diverse agents for which different extraction and analysis procedures after sampling are needed. Preliminary studies showed that it is possible to measure β-1,3-glucans or culture-viable molds from the cloths (data not shown). Future work will be directed toward measuring other agents, like fungal extracellular polysaccharide antigens, peptidoglycans, or allergens.
Whether a home was situated on a farm or not determined the level of exposure for all methods. Levels of endotoxins found in farm homes were approximately three times higher than those found in nonfarm homes. Similar differences for the floor dust sampling between farm and nonfarm homes have been reported previously (6, 16, 23).
Very little information is available on airborne endotoxin levels in homes, due to the low levels of airborne endotoxin found in these environments and the high logistic and equipment costs to sample with active airborne sampling methods. Park et al. (11, 12) reported GMs of 0.64 to 0.77 EU/m3 for urban homes, and Thorne et al. (20) performed 24-h inhalable dust sampling in the homes of rural asthmatic children and reported a GM of 6 EU/m3 and a GSD of 2.8. Both studies found higher levels than we found in nonfarm or farm homes. Hyvarinen et al. (6) reported for their active airborne dust sampling endotoxin levels comparable to what we found. In that study, endotoxin was measured with a "pizza box"-like sampler, but the results were only reported in EU/mg (24) and not in EU/m2. Since we could not accurately assess the net dust weight captured on the EDC cloths, we cannot compare our results with that study. Moreover, the sampling period used by Hyvarinen et al. was up to several months, much longer than in our study. This further complicates any comparison of measured endotoxin levels. For other passive or settling airborne dust samples, endotoxin levels have not been reported as yet. The petri dish sampling method (8, 9, 21) collects dust on a much smaller surface; thus, a longer sampling duration would be necessary to measure endotoxin with this method. Another disadvantage of both the petri dish sampling and the dust fall collector (24) is that captured dust must be transferred before extraction and analysis, which might lead to the loss of dust and therefore a loss of precision. In addition, it is not possible to send a petri dish or a dust fall collector after sampling back to the laboratory by regular mail. With the new EDC, this is more than a theoretical option, as shown by our first experiences in the European GABRIEL study. In that multicenter population study, we have distributed several thousand EDCs to study participants, who, with the help of a simple photoillustrated instruction, deploy the EDC sampler themselves and return the sampler together with a completed questionnaire by mail in a simple 20- by 40-cm envelope.
In conclusion, the EDC can be used as a tool to assess airborne endotoxin exposures in home and/or work environments. It is easy to use for the participants of a study, can be sent by mail, and is a cheap and reliable method to collect airborne dust. The electrostatic dust fall collector is a potential alternative to or a complement for vacuum dust sampling in large-scale epidemiological studies.
We thank the participants of this validation study for their cooperation and James A. Deddens, University of Cincinnati, for his help with the statistical analysis.
Published ahead of print on 1 August 2008. ![]()
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