Applied and Environmental Microbiology, February 2000, p. 627-631, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Department of Environmental Sciences, Environmental and Occupational Health Group, Wageningen University, Wageningen, The Netherlands,1 and Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, Iowa 52242-50002
Received 30 July 1999/Accepted 10 November 1999
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ABSTRACT |
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As part of environmental management policies in Europe, separate
collection of organic household waste and nonorganic household waste
has become increasingly common. As waste is often stored indoors, this
policy might increase microbial exposure in the home environment. In
this study we evaluated the association between indoor storage of
organic waste and levels of microbial agents in house dust. The levels
of bacterial endotoxins, mold
(1
3)-glucans, and fungal
extracullar polysaccharides (EPS) of Aspergillus and Penicillium species were determined in house dust extracts
as markers of microbial exposure. House dust samples were collected in
99 homes in The Netherlands selected on the basis of whether separated
organic waste was present in the house. In homes in which separated
organic waste was stored indoors for 1 week or more the levels of
endotoxin, EPS, and glucan were 3.2-, 7.6-, and 4.6-fold higher,
respectively (all P < 0.05), on both living room and
kitchen floors than the levels in homes in which only nonorganic
residual waste was stored indoors. Increased levels of endotoxin and
EPS were observed, 2.6- and 2.1-fold (P < 0.1), respectively, when separated organic waste was stored indoors for 1 week or less, whereas storage of nonseparated waste indoors had no
effect on microbial agent levels (P > 0.2). The
presence of textile floor covering was another major determinant of
microbial levels (P < 0.05). Our results indicate
that increased microbial contaminant levels in homes are associated
with indoor storage of separated organic waste. These increased levels
might increase the risk of bioaerosol-related respiratory symptoms in
susceptible people.
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INTRODUCTION |
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Separate collection of organic household waste and nonorganic household waste has become increasingly common in many European countries as part of national or local environmental management policies. This often involves indoor storage of separated organic waste, including fruits, vegetables, and food remains in the home. Indoor storage of waste is especially common in apartment buildings in densely populated areas. As microbial decomposition of organic waste starts and possibly proceeds to a substantial extent inside an organic waste bin, the bin might become an important source of bacteria and mold spores inside the house.
Although the specific role of microbial exposure in the development of
noninfectious respiratory disease is still not clear, there is strong
evidence that microbial contaminants and allergens from house dust
mites are related to the prevalence and severity of respiratory
symptoms, particularly in damp houses (1, 3, 10, 17, 24,
26). Exposure to bacteria, especially exposure to bacterial
endotoxins, is known to be associated with respiratory symptoms
(2, 14, 16, 19). It is thought that molds initiate both
allergic and nonallergic inflammatory reactions (1, 10, 17,
24). The latter could be related to
(1
3)-glucans, which are
cell wall components of most fungi (20-22).
In the present study we investigated the influence of indoor storage of
organic waste on microbial levels in the home environment as the first
step in a health risk evaluation. The effect of indoor storage of
household waste on concentrations of biological contaminants indoors
has not been reported previously. We measured the levels of bacterial
endotoxins and fungal
(1
3)-glucans, which are known or probable
inducers of airway inflammation, and the levels of extracellular
polysaccharides (EPS) of Aspergillus and
Penicillium species, which were used as markers for fungal
exposure. In addition, the levels of house dust mite and cat
allergens, which are two well-recognized indoor air allergens
associated with housing characteristics, were measured to provide
external validation of the results.
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MATERIALS AND METHODS |
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Study population. In the summer of 1997 samples of settled house dust were collected in 99 households in The Netherlands. Approximately one-half of the houses (53 houses) were selected from a large birth cohort study on the development of asthma and mite allergy in The Netherlands. Most of these houses (>80%) were single-family houses. In addition, we studied 46 households in apartment buildings in a small city of approximately 30,000 inhabitants in The Netherlands. A stratified sampling strategy was devised for both populations such that one-half of the households had a 5- to 30-liter organic waste bin in the kitchen. Data concerning the frequency at which the organic bin was emptied and variables such as the type of floor covering and the presence of pets were collected by questionnaire. Members of all households agreed to participate in the study by signing an informed consent document.
Dust sampling and extraction.
House dust samples were
collected by vacuuming 1-m2 portions of living room and
kitchen floors by using an internationally standardized protocol, as
described previously (7, 18). Samples were taken from smooth
and wall-to-wall textile floor coverings. When a rug with a surface
area of at least 1 m2 was present, the sample was taken
from the rug. Filters were weighed before and after dust samples were
collected by using an analytical balance in a preconditioned room at
approximately 20°C and 50% relative humidity. Dust samples were
stored at
20°C until extraction was performed. Endotoxins, EPS, and
allergens were extracted from the dust as described by Douwes et al.
(7), and afterwards
(1
3)-glucans were extracted
(6). Briefly, the dust samples were suspended in 2.5 to 20 ml (depending on the weight of the dust) of 0.05% (vol/vol) Tween 20 in pyrogen-free water (<0.3 g, 3 ml; 0.3 to 0.5 g, 5 ml; 0.5 to
1.0 g, 10 to 15 ml; >1.0 g, 20 to 50 ml). The suspensions were
rocked vigorously for 2 h at room temperature, and after
centrifugation each supernatant was stored at
20°C until it was
analyzed. After extraction at room temperature, extraction at 120°C
was used to dissolve
(1
3)-glucans (6). Each pellet was
resuspended in the same volume of 0.05% Tween 20 in pyrogen-free
water, vigorously shaken for 15 min at room temperature, autoclaved at
120°C and 105 Pa for 1 h, and then shaken for 15 min. The samples were centrifuged, and each supernatant was collected
and stored at
20°C until it was analyzed.
Microbial and allergen analyses. The concentrations of endotoxins, EPS antigens, house dust mite allergens, and cat allergens were determined by using the dust extracts prepared at room temperature. Bacterial endotoxin levels were measured by a quantitative kinetic chromogenic Limulus amebocyte lysate assay (Kinetic-QCL no. 50-650 U; Bio Whittaker, Walkersville, Md.) as described previously (7). Levels of fungal EPS antigens of Penicillium and Aspergillus spp. were measured with a sandwich enzyme immunoassay (EIA) essentially as described by Douwes et. al. (9). Briefly, microplates were coated overnight at 4°C with isolated anti-Aspergillus/Penicillium-EPS immunoglobulin G antibodies (10 µg/ml) which had been raised in rabbits and isolated from their serum as previously described (11). The specificity of these antibodies for EPS of Aspergillus and Penicillium spp. has been shown by Kamphuis et. al. (11) and Douwes et al. (9), who used EPS of species belonging to other genera and crude fungal allergen extracts, respectively. Then the plates were washed with phosphate-buffered saline containing 0.05% Tween 20 (PBT), and free sites on the well surfaces were blocked by incubation for 0.5 h at 37°C with PBT containing 0.1% milk proteins (Protifar; NV Nutricia, Zoetermeer, The Netherlands) (PBTM). Dust extracts were diluted 1/5, 1/10, and 1/20 with PBTM and incubated for 1 h at 37°C in the microwells. After plates were washed with PBT, the amount of bound EPS was measured by incubating the plates for 1 h at 37°C with peroxidase-labelled rabbit anti-Aspergillus/Penicillium-EPS immunoglobulin G diluted 1/2,000 with PBTM, followed (after another wash with PBT) by incubation for 0.5 h at room temperature with o-phenylenediamine (2 mg/ml in 0.05 M citrate-phosphate buffer; pH 5.5) containing 0.015% H2O2. The reaction was stopped with HCl, and the results were read spectrophotometrically at 492 nm.
The procedure used to calibrate the assay was modified from the previously described procedure as the specific activities of standard preparations containing isolated EPS appeared to differ considerably when the EIA was used and as dose-response curves of many house dust extracts were not parallel to the dose-response curve of the standard. Therefore, we calibrated the EIA in the present study by including in each test plate serial dilutions (1/5 to 1/640) of a sieved bulk house dust extract to which an arbitrary value of 5,000 EPS units per ml was given. When this was done, the obtained results obtained with different dilutions of the same test sample usually varied less than 20%. The limit of detection of the assay was 19 EPS units/ml. The levels of house dust mite allergen Der p 1 (13) and cat allergen Fel d 1 (4) were measured with monoclonal antibody-based EIA kits (catalog no. 5H8/4C1 and 6F9/3E4; Indoor Biotechnologies, Chester, United Kingdom) by using the protocol of the supplier with some slight modifications. The limits of detection of these tests were 5.36 ng/ml for Der p 1 and 1.47 ng/ml for Fel d 1. The levels of
(1
3)-glucans in the heated dust extracts were
measured with an inhibition EIA (6). The detection limit of
the assay was 80 ng/ml. The blank paper filters used for dust sampling
contained
(1
3)-glucan (mean, 300 µg per filter). Therefore, a
correction for this amount of
(1
3)-glucan was used as described previously.
Statistical analysis. A statistical analysis was performed by using SAS statistical software (version 6.12; SAS Institute, Cary, N.C.). Indoor microbial and allergen levels were expressed per square meter and per gram of dust, and a sample with undetectable microbial or allergen levels was given a value of two-thirds of the lowest observed amount per gram of dust or per square meter for the specific component determined. Except for Der p 1, the indoor microbial and allergen levels were normally distributed after natural log transformation. Therefore, data were summarized as geometric means (exponent of the average of natural log-transformed concentrations) and geometric standard deviations (exponent of the standard deviation of natural log-transformed concentrations). First, crude unadjusted analyses of the differences in allergen and microbial contaminant levels between houses with organic waste bins and houses without organic waste bins were performed. Endotoxin, glucan, Penicillium/Aspergillus- EPS, and cat allergen levels were evaluated by performing Student's t tests with natural log-transformed concentrations, and Der p 1 levels were evaluated by performing nonparametric Wilcoxon tests because of the nonnormality of Der p 1 levels. Subsequently, natural log-transformed concentrations of microbial contaminants determined per square meter or per gram of dust were used as the response variables in an analysis of covariance. Homes with organic waste bins indoors were compared to homes without organic waste bins indoors. By including potentially confounding variables in the regression model, the corrected effect of organic waste bins indoors compared to no organic waste bins indoors could be determined (12). The possible confounding factors included type of floor covering, population sample, and the presence of pets.
In the final analysis, households with only residual nonorganic waste indoors (n = 26) were compared with households with nonseparated organic and residual waste indoors (n = 25), households with organic waste bins that were emptied at least twice per week (n = 32), and households with organic waste bins that were emptied at most once per week (n = 17), corrected for type of floor covering and population sample, by using the following model: Ln(microbial agent concentration) =
where Ln(microbial agent concentration) is the natural
log-transformed concentration of microbial agents; Int is the
intercept; MW is the presence of mixed waste indoors (organic and
nonorganic waste not separated); OBA is the presence of an
indoor organic waste bin that is emptied twice a week or more;
OBB is the presence of an indoor organic waste bin that is
emptied once a week or less; FC is the type of floor covering (textile
or smooth); PS is the population sample (birth cohort study or
apartment); and
1 through
5 are the
regression coefficients for the respective effects.
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RESULTS |
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The distributions of some relevant variables in the whole study
population and both population samples are shown in Table 1. By design, one-half of the households
had an organic waste bin indoors. However, people who lived in
apartments emptied their organic waste bins less frequently than
participants in the birth cohort study emptied theirs. Furthermore,
when no organic waste bin was present indoors, more households in
apartment buildings than households in the birth cohort study did not
separate their waste. Textile floor coverings (rugs or wall-to-wall
carpets) were also more common in apartment houses than in the birth
cohort study households. The distributions of textile floor coverings were the same for groups of houses defined on the basis of
different waste collection characteristics.
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Unadjusted analyses of microbial agent and allergen concentrations in
homes with and without organic waste bins indoors (Table 2) showed that bacterial endotoxin and
fungal Aspergillus/Penicillium-EPS concentrations both in
the living room and in the kitchen were significantly higher in homes
with organic waste bins (endotoxin, 1.8- to 3.4-fold [P
0.05]; EPS, 1.8- to 2.4-fold [P
0.1 and P
0.05]). We observed a trend toward increased
(1
3)-glucan levels expressed per square meter (1.5- and 1.7-fold
[P > 0.1]). In contrast, neither Der p 1 nor Fel d 1 allergen levels were associated with the type of
waste container inside the house. Similar results were obtained after
we adjusted for possible confounding factors, such as the type of floor
covering, the sample population, and the presence of pets (data not
shown). The presence of a textile floor covering was strongly
associated with both microbial and allergen concentrations in house
dust (2- to 100-fold higher levels [P < 0.05]),
while the population sample was not significantly associated with
measured concentrations in house dust. The presence of pets, especially
cats, was a major determinant for cat allergens but not for house dust
mite allergens or microbial agents.
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In subsequent regression analyses we made a further distinction between
unseparated organic and residual waste (mixed waste) and organic waste
stored in a separate bin, and we included the effect of emptying
frequency. The data showed that the greatest explained variance
occurred with a model in which the effect of organic waste itself, the
emptying frequency of the organic waste bin, and two confounding
factors, type of floor covering and population sample, were taken into
account. The predicted values for microbial contaminant amounts
per square meter in the four different groups of homes as derived
from this regression model are shown in Fig. 1. The predicted concentrations in living
room floor dust in houses with residual waste and smooth floors were
225 endotoxin units/m2 for endotoxin, 23 µg/m2 for
(1
3)-glucans, and 123 EPS
units/m2 for EPS. The presence of separated organic
waste stored indoors and a low bin-emptying frequency led to
significantly increased microbial agent levels (endotoxin, 3.2-fold
[P < 0.05]; EPS, 7.6-fold [P < 0.05]; glucan, 4.6-fold [P < 0.05]). The
presence of a textile floor covering increased the concentrations 20- to 100-fold (P < 0.05). The combined effect of textile
floor covering and the presence of an organic waste bin resulted in 25- to 840-fold increases in microbial agent levels. An organic waste bin
indoors that was emptied at least twice a week was associated with
moderately enhanced microbial agent levels (endotoxin, 2.6-fold
[P < 0.05]; EPS, 2.1-fold [P < 0.1]; glucan, 1.6-fold [P > 0.2]), while
storage of unseparated waste had no effect on microbial concentrations
(endotoxin, 0.9-fold [P > 0.2]; EPS, 1.3-fold
[P > 0.2]; glucan, 1.7-fold [P > 0.2]). Similar results were obtained for kitchen floors; for
example, there were 3.6-, 1.5-, and 6.1-fold increases in the
endotoxin, glucan, and EPS concentrations, respectively, due the
presence of an organic waste bin that was emptied once a week or less
frequently (data not shown). Analyses of microbial agent concentrations
expressed per gram of dust produced the same results, although the data were less marked.
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DISCUSSION |
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Indoor storage of organic waste was associated with significantly increased concentrations of bacterial endotoxins and fungal antigens in dust from both kitchen and living room floors. The type of floor covering was another important and independent determinant of microbial contamination in the home environment. To confirm that increased levels of microbial agents were specifically associated with indoor storage of organic waste and not due to other differences between homes with and without organic waste containers (for instance, the overall hygiene status of the homes), we measured the levels of cat and house dust mite allergens in the dust samples. These two allergens are well-known indoor allergens that would probably be present at higher levels in dirtier homes (for example, homes that are cleaned less frequently) but should not be specifically associated with indoor storage of organic waste. The presence of an organic waste bin indoors was not associated with cat and house dust mite allergen levels in the home environment, indicating that there was a specific association between indoor storage of organic waste and biocontaminant concentrations. The lack of an association between house dust mite allergen levels and waste characteristics might be explained by the fact that most of the house dust samples had undetectable levels of house dust mite allergens. The cat allergen levels were also relatively low compared to the levels in previous studies (23, 25). However, we found that there was a strong association between both of these allergens and textile floor coverings, a well-known determinant of indoor allergen exposure.
Only in relatively few studies conducted to assess the relationships among mold growth, house dust mites, home dampness, and respiratory symptoms have actual levels of exposure to microbial agents been determined. Endotoxin, glucan, and EPS levels were readily detected in house dust samples, as shown in previous studies. The endotoxin levels determined in this study were, however, 2- to 10-fold lower than the levels in previous studies performed by Michel et al. and Douwes et al. (5, 7, 8, 14, 16). This is in contrast to the glucan levels, which were more comparable to the levels found in previous studies performed in Germany and The Netherlands (5, 6, 8). The presence of textile floor coverings was again strongly associated with increased levels of endotoxin, glucan, and EPS. This should be interpreted as indirect validation of our finding that the presence of an organic waste bin results in 1.6- to 7.6-fold increases in microbial agent concentrations. The microbial contaminant levels that were expressed per square meter were more variable than the levels expressed per gram of dust, indicating that there was an association between the amount of dust sampled and the biocontaminant levels expressed per square meter. However, significantly increased microbial exposure levels expressed per gram of dust were found in households with indoor storage of organic waste, which indicated that the association between microbial levels in dust and waste storage does not depend only on the amount of dust in a home.
This study was the first step in a health risk evaluation of
source-separated organic waste collection. So far, the health implications of elevated microbial exposure are uncertain, as health
effects have not been evaluated directly. In various
epidemiological studies, however, bioaerosol-related respiratory
symptoms and morbidity in moldy and damp houses have been described
(1, 3, 10, 17, 24, 26). More recent studies have
demonstrated that endotoxins are quantitatively associated with the
severity of airflow limitation and respiratory symptoms in asthmatics
(14-16), and epidemiological and toxicological studies have
revealed that endotoxins are causative agents of nonspecific,
nonallergic airway inflammation (19). Similar associations
with respiratory symptoms and nonspecific airway inflammation have also
been suggested for
(1
3)-glucans (20-22) and EPS
(9). In a recent study, the effects of endotoxins and
(1
3)-glucans on the lung function of children were assessed
(5). We found that 25-fold-higher levels of microbial agents
in living room floor dust were associated with a 1.6-fold increase in
peak flow variability in a subgroup of atopic children with asthmatic
symptoms. This implies that there may be similar or even stronger
effects due to the presence of an infrequently emptied organic waste
bin together with the presence of a textile floor covering. Therefore,
health risks associated with indoor storage of organic waste,
particularly in asthmatics and other susceptible individuals, must be
considered a possibility.
In conclusion, we found that increased microbial contaminant concentrations in the home environment were associated with indoor storage of separated organic waste, which might increase the risk of respiratory diseases related to such contaminants.
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ACKNOWLEDGMENTS |
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We are indebted to the participating households. We thank Bart Flipse, Wobbe van der Meulen, Lützen Portengen, Isabella van Schothorst, Jack Spithoven, and Siegfried de Wind for technical assistance with exposure measurements and laboratory analyses.
This study was supported by The Ministry of Housing, Spatial Planning
and the Environment (VROM), by The Netherlands Organization for
Scientific Research (NWO), and by an EC research project (Prevention of
disease caused by waste handling with special reference to endotoxin
and
(1
3)-glucan [BHM4-CT96-0105]).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Environmental Sciences, Environmental and Occupational Health Group, Wageningen University, P.O. Box 238, 6700 AE Wageningen, The Netherlands. Phone: 31 317 482080. Fax: 31 317 485278. E-mail: DICK.HEEDERIK{at}STAFF.EOH.WAU.NL.
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