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Annals of Occupational Hygiene Advance Access originally published online on February 9, 2005
Annals of Occupational Hygiene 2005 49(5):393-400; doi:10.1093/annhyg/meh108
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© 2005 British Occupational Hygiene Society Published by Oxford University Press


Original Article

Influence of Seasons and Sampling Strategy on Assessment of Bioaerosols in Sewage Treatment Plants in Switzerland

ANNE OPPLIGER1,*, SILVIA HILFIKER2 and TRINH VU DUC1

1 Institute of Occupational Health Sciences, Rue du Bugnon 19, CH-1015 Lausanne, Switzerland; 2 Abteilung für Arbeits- und Umweltmedizin, Institut für Sozial- und Präventivmedizin und medizinische Poliklinik, Sumatrastrasse 30, CH-8006 Zürich, Switzerland

* Author to whom Correspondence should be addressed. Tel: +41 21 314 74 16; fax: +41 21 314 74 30; e-mail: Anne.Oppliger{at}hospvd.ch


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
An assessment of sewage workers' exposure to airborne cultivable bacteria, fungi and inhaled endotoxins was performed at 11 sewage treatment plants. We sampled the enclosed and unenclosed treatment areas in each plant and evaluated the influence of seasons (summer and winter) on bioaerosol levels. We also measured personal exposure to endotoxins of workers during special operation where a higher risk of bioaerosol inhalation was assumed. Results show that only fungi are present in significantly higher concentrations in summer than in winter (2331 ± 858 versus 329 ± 95 CFU m–3). We also found that there are significantly more bacteria in the enclosed area, near the particle grids for incoming water, than in the unenclosed area near the aeration basins (9455 ± 2661 versus 2435 ± 985 CFU m–3 in summer and 11 081 ± 2299 versus 2002 ± 839 CFU m–3 in winter). All bioaerosols were frequently above the recommended values of occupational exposure. Workers carrying out special tasks such as cleaning tanks were exposed to very high levels of endotoxins (up to 500 EU m–3) compared to routine work. The species composition and concentration of airborne Gram-negative bacteria were also studied. A broad spectrum of different species within the Pseudomonadaceae and the Enterobacteriaceae families were predominant in nearly all plants investigated.

Keywords: airborne bacteria • airborne fungi • endotoxin • occupational health • wastewater


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Sewage treatment plants present a potentially high risk of occupational diseases since contaminated wastewater is recognized as an important vehicle for transmission of several viral, fungal and bacterial diseases. Wastewater contains a lot of pathogenic Gram-negative bacteria from humans and animals, which can become aerosolized. Endotoxins, produced by Gram-negative bacteria, are known to result in numerous health problems. Numerous studies, in different industries (animal confinement houses, cotton processing industry, grain processing), have reported associations between airborne endotoxin exposure and respiratory symptoms or pulmonary function decline (Schwartz et al., 1994Go, 1995Go; Donham et al., 2000Go; Rylander, 2002Go; Douwes et al., 2003Go). Results of these different investigations are not necessarily applicable to sewage workers since the composition of organic dust and other volatile components are very different from one occupation to another. Several investigations of waste water treatment workers have shown that certain work-related symptoms were more frequent among employees of sewage treatment plants than among control groups (Nethercott and Holness, 1988Go; Rylander, 1999Go, 2002Go; Thorn et al. 2002bGo). The frequent symptoms reported involve the respiratory and gastrointestinal tracts, but fatigue and headaches are also very commonly reported (Thorn and Kerekes, 2001Go, review). Few studies have reported data concerning the airborne bacterial and fungal loads or the concentration of endotoxins (Laitinen et al., 1994Go; Rylander, 1999Go; Thorn et al., 2002aGo; Prazmo et al., 2003Go). Seasonal influence was rarely investigated (Kiekhaefer et al., 1995Go; Moller-Nielsen et al., 1997Go; Duchaine et al., 2000Go) and the influence of sampling strategy, i.e. personal versus stationary sampling, was reported in only one study (Thorn et al., 2002aGo). The purpose of this work was to collect a comprehensive dataset for bioaerosols concentration (endotoxins, bacteria and fungi) in different seasons, for different worksites (indoors/outdoors) and during special tasks. The composition of the community of Gram-negative bacteria was also investigated. This study was carried out as a part of a prospective cohort study of which some preliminary results have already been presented (Jeggli et al., 2003Go, 2004Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Plant selection
Eleven out of the 79 wastewater treatment plants from the Canton of Zürich, Switzerland, were selected for study to represent a broad array of plant size (2–117 workers) and work conditions (apparently good work conditions or occupational history with symptoms suggesting exposure to endotoxin). Work-related symptoms were defined as those brought about by a specific task, occurring during a specific task in co-workers as well, and without other cause. Fever, chills, fatigue, diarrhoea and headache were defined a priori as general symptoms possibly due to toxic pneumonitis (Rylander, 1999Go). Arthralgias were not included, because they may also result from ergonomic factors (see details in Steiner et al., in pressGo). Procedures for collecting clinical data (check list, coding, quality control) have been described elsewhere (Jeggli et al., 2004Go). All these plants were municipal plants treating household wastewater only. As a first step, stationary sampling was carried out in summer and winter. In the second step, endotoxin exposure was measured by personal sampling in the same 11 plants during 1–2 tasks suspected to represent peak exposures to endotoxin. A control sample was collected in the same plant on the same day (except in one plant where the measures had to be staggered over 1 month) during activities assumed not to involve peak exposures to endotoxin. Peak exposure to endotoxin was assumed for the following tasks: spray removal from basins, tank walls, grids or rakes where the workers were associated with work-related symptoms as defined above (Steiner et al., in pressGo). All the selected plants participated. Measurements were carried out between July 2002 and October 2003.

Air sampling
All micro-organism samples were collected in duplicate twice: once in the morning and once 4 h later in the afternoon. Endotoxins were sampled continuously for 4 h at a stationary point. In each plant, we sampled at two different work stations: (i) in the enclosed area (called indoors), at the water inlet near the rake that removes big particles from incoming water (grid basin); (ii) in the unenclosed area (called outdoors), above the bubbling aeration basin. All indoor air samples were collected 1.5 m above the floor and the oxygenated tank samples were collected between 50 cm and 1.2 m above the footbridge. For personal sampling, the cassette was placed close to the breathing zone of the worker and samples were collected continuously for at least 22 min, (22–170 min) for special tasks and 4 h for the group of control measurements. Outdoor and indoor temperatures and relative air humidity were measured each sampling with an ECOLOG apparatus (Ecolog TH1, Elpro-Buchs).

Bacteria and fungi
Airborne bacteria and fungi were sampled with a single-stage impactor (MAS-100 Eco, Microbiological Air Sampler MBV; Vevey, Switzerland) operated at a flow rate of 100 l min–1. We sampled 20 l for fungi, 50 l for non-specific bacteria and 100 l for Gram-negative bacteria. These volumes were chosen since higher volumes result in overloading. Samples for total cultivable bacteria were impacted on Tryptone Soya agar plates, Gram-negative bacteria on MacConkey and fungi on Dichloran-glycerol agar base (DG18) plates (all from Oxoïd, Basel, Switzerland). Fungi were incubated at 25°C for 5 days while bacteria were incubated at 30°C for 7 days, since we have previously observed new colonies after 6 days of incubation. All plates were checked daily for colony growth. Results are expressed in colony forming units (CFU) per cubic metre of air. The mean of the two samples collected in the morning and the two samples collected in the afternoon was used for the statistical analysis. The most frequently appearing colony morphology types of Gram-negative bacteria were isolated and subsequently identified using enzymatic test kits (API 20 E and API 20 NE, Biomérieux, France).

Endotoxin analysis
Endotoxins were sampled for 4 h onto polycarbonate filters (37 mm diameter, 0.4 µm pore size) placed into a ready-to-use polystyrene cassette (endofree cassette, Aerotech Laboratories, Inc. Phoenix, AZ, USA). Sampling was carried out with a pocket pump (MSA Escort Elf, Mine Safety Appliance Company, Pittsburgh, PA, USA, or SKC pocket pump 210-1002, SKC Inc., PA, USA) calibrated at 1.5 l min–1. Airflow was calibrated before and after field sampling with a pocket calibrator (DryCal DC-Lite, Bios International, Pompton Plains, NJ, USA). Thus, ~360 l of air was sampled. After sampling, cassettes were transported in a cold box to the laboratory within 3 h where they were stored at –20°C for 1–3 months.

Endotoxins were extracted by shaking the filters at room temperature for 1 h in 10 ml of non-pyrogene water in a 50 ml conical polypropylene tube. Filter extraction solutions were vortexed vigorously prior to drawing a sample for endotoxin analysis. Finally, 0.1 ml of the filter solution was analysed with a quantitative kinetic chromogenic limulus amebocyte lysate (LAL) assay (Cambrex Biowhittaker Europe, Verviers, Belgium) at 37°C with an automated microtitre plate reader. Escherichia coli O55:B5 endotoxin (Biowhittaker) was used as standard endotoxin and a positive product control was included in each sample to monitor product inhibition or enhancement. Results were expressed in endotoxin units (EU) per cubic metre of air. The detection limit of the test was 0.005 EU ml–1 of analysed substrate.

Statistical methods
The normality of the data distribution was tested with the Shapiro-Wilk normality test and non-parametric tests were used if necessary. Hypotheses of the differences between groups of bioaerosols data (summer/winter, outdoors/indoors) were tested by using the paired t-test, Mann–Whitney or Wilcoxon rank–sum test. All statistics were carried out by using Systat software (SYSTAT Software Inc., Canada) or STATA (Stata Corporation, TX, USA). Bonferroni correction for multiple comparisons was used when necessary. The data were generally presented as arithmetic mean values ± standard error (SE) and range (minimum and maximum values).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Effects of seasons
The mean concentration of cultivable fungi per cubic metre of air, both indoors and outdoors, was ~8-fold greater in summer than in winter (Table 1) and this difference was significant (Wilcoxon signed rank test: Z = 3.3, P = 0.001). On the other hand, the mean number of total cultivable bacteria, cultivable Gram-negative bacteria and endotoxins levels were not significantly different in summer than in winter (Table 1) (Wilcoxon signed rank test: Z = –0.29, P = 0.76 for total cultivable bacteria and Z = 0.12, P = 0.90 for Gram-negative and Z = –1.76, P = 0.07 for endotoxins). The climatic parameters are summarized in Table 2.


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Table 1. Mean ± SE and range (minimum–maximum) of different bioaerosols sampled indoors and outdoors at the 11 sewage treatment plants, in winter and in summer

 

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Table 2. Mean ± SE of temperature and relative humidity measured indoors and outdoors at the 11 sewage treatment plants, in winter and in summer

 
Difference between outdoor and indoor samples
In both seasons, we found significantly more cultivable bacteria (total and Gram-negative) indoors than outdoors (Table 1) and endotoxin levels were higher indoor than outdoor only in winter (Table 1). The mean number of fungi was not significantly different between indoor and outdoor samples for both seasons (Table 1).

Effect of sampling strategy
The mean concentration of endotoxins collected by personal sampling during special tasks was significantly greater than the mean concentration of endotoxins collected by personal sampling during tasks assumed not to involve peak exposures (mean ± SE = 98.25 ± 32.6, range: 1.4–500 EU m–3 versus 6.0 ± 1.9, range 0.1–21 EU m–3 air; Wilcoxon signed rank test: Z = 2.9, P = 0.003, n = 11). The sampling strategy comparisons for endotoxin evaluation showed that stationary sampling gave higher results (59.3 ± 16.3 EU m–3) than personal sampling during a usual workday (mean of samples collected for 4 h: 6.0 ± 1.9 EU m–3). This difference was significant (Wilcoxon signed rank test: Z = –2.8, P = 0.004, n = 11).

Values in regard to recommendation
In summer, >50% of the sewage treatment plants exceeded the recommended Swiss occupational threshold of 103 CFU m–3 of fungi (Anonymous, 2003Go) (Fig. 1). Indoors, ~30% of the plants exceeded the recommended Swiss occupational threshold for total cultivable bacteria and Gram-negative bacteria (respectively, 104 and 103 CFU m–3) for both seasons (Figs 2 and 3). In summer, only two sewage plants exceeded the indoor value of 100 EU m–3 recommended by some authors (Heederik and Douwes, 1997Go; Douwes et al., 2003Go) for endotoxin (Fig. 4).



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Fig. 1. Mean ± SE of cultivable fungi per cubic metre of air sampled indoors near the grid basin and outdoors near the aeration basin of different sewage treatment plants. White bars represent summer samples and black bars represent winter samples. The horizontal line shows Swiss recommendations.

 


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Fig. 2. Mean ± SE of cultivable total bacteria per cubic metre of air sampled indoors near the grid basin and outdoors near the aeration basin of different sewage treatment plants. White bars represent summer samples and black bars represent winter samples. The horizontal line shows Swiss recommendations.

 


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Fig. 3. Mean ± SE of cultivable Gram-negative bacteria per cubic metre of air sampled indoors near the grid basin and outdoors near the aeration basin of different sewage treatment plants. White bars represent summer samples and black bars represent winter samples. The horizontal line shows Swiss recommendations.

 


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Fig. 4. Concentration of endotoxin per cubic metre of air sampled indoors near the grid basin and outdoors near the aeration basin of different sewage treatment plants. White bars represent summer samples and black bars represent winter samples. The horizontal line shows level recommended by many authors.

 
Concentration and species of Gram-negative bacteria
The mean percentage of the total number of cultivable aerobic bacteria identified as Gram-negative (Gram-negative bacteria/total cultivable bacteria) was 8.4 ± 2.1% in summer and 4.4 ± 1.0% in winter. This difference was not significant (Mann–Whitney test: U = –0.72, P = 0.46). Among the Gram-negative bacteria we have identified at least once in the majority of plants (9/11) the genera Pseudomonas (P. aeruginosa, P. pseudoalcaligenes, P. putida, P. luteola, P. indologenes, P. acidovorans, P. orzyhabitans, P. pickettii, P. cepacia) as the predominant cultivable bacteria (number of colonies >20% of total cultivable Gram-negative bacteria). In seven plants we have detected the genera Klebsiella (K. oxytoca, K. pneumoniae, K. terrigena) and in six plants the genera Enterobacter (E. cloacae, E. aerogenes, E. agglomerans) as a frequent cultivable bacteria compared with the other bacteria present on the nutrient agar (number of colonies >10% of total cultivable Gram-negative bacteria). And finally, Acinetobacter baumanii and junii, Serratia ficaria and liquefaciens, Chromobacterium violaceum, Ochrobactrum anthropi, Aeromonas hydrophila and Salmonella arizonae were found at least in one plant also as a frequent cultivable bacteria compared with the other bacteria present on the nutrient agar. Escherichia coli was found in three plants in very low numbers.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Effects of seasons
Climatic parameters seem to have a significant influence only on the mean airborne concentration of fungi. Indeed, in summer the mean level of fungi is approximately eight times greater than in winter. The mean concentration of total cultivable bacteria, cultivable Gram-negative bacteria and airborne endotoxins was not significantly influenced by climatic parameters. Other studies in occupational environments have demonstrated a seasonal influence in bioaerosol concentrations. For instance, in swine confinement buildings in Canada, cultivable bacteria counts were lower in summer than in winter with no observable difference for fungi (Duchaine et al., 2000Go). In the USA, a lower cultivable bacterial level was observed during winter/spring when compared with summer/fall levels in swine buildings (Kiekhaefer et al., 1995Go). In Denmark, Moller-Nielsen and co-authors (Moller-Nielsen et al., 1997Go) have observed that, for waste collection, there was a significant seasonal influence on fungi and endotoxin concentrations with the lowest concentrations in winter, while the bacterial concentrations showed no clear seasonal variation. The discrepancy of results may be due to the differences among these different countries in terms of the range of outdoor temperature during summer and winter and probably in terms of the ventilation rate of buildings. Moreover, it is known that the composition of organic dust and other volatile components are very different from one occupation (swine confinement house) to another (sewage treatment plant). Thus, the influence of climatic parameters on bioaerosol concentrations is real but rarely reported and the change of bioaerosol loads with respect regard to seasons is unpredictable without analyses since it varies according to the countries and the worksites.

Difference between outdoor and indoor samples
In both seasons, we found significantly more cultivable bacteria (total and Gram-negative) indoors than outdoors and endotoxin levels were higher indoors than outdoors only in winter. An earlier study carried out in a sewage treatment plant in Switzerland during the winter found similar patterns of bacterial counts for indoor and outdoor samples (Schira et al., 1987Go). Our results are between those of two other studies carried out in sewage treatment plants with indoor and outdoor sampling. In Poland (Prazmo et al., 2003Go), a recent investigation in a medium-size treatment plant gave similar results for endotoxin concentrations indoors and outdoors but the mean number of cultivable bacteria observed was ~2–4 times smaller and the number of fungi was 100–200 times smaller than those in this study. In a Finnish wastewater treatment plant (Laitinen et al., 1994Go), endotoxins, Gram-negative bacteria and total bacteria were present, respectively, in concentrations of 3–20, 8–15 and 3–4 times higher than those in this study. The discrepancy of results should be examined in terms of the differences in sampling strategy and analyses and the differences in the type of sewage plant. However, we have to point out that the three studies agree that more bioaerosols are generated indoors near the water inlet (grid basin) than outdoors near the aeration basin.

Effect of sampling strategy
We show that the results of stationary sampling for endotoxin analyses (59.3 EU m–3 on average) are not representative of results obtained with personal sampling either during routine work (6.0 EU m–3 on average) or during special short-time tasks (98 EU m–3 on average). A recent study of sewage treatment plants in Sweden (Thorn et al., 2002aGo) has shown that the amount of airborne endotoxins recorded with personal sampling among workers carrying out repair work were in the range 61–272 EU m–3 and endotoxin levels recorded with stationary samplers were in the same range as those in this study (11–22 EU m–3). In contrast to our results, the amounts of endotoxins present at worksites/tasks were similar when both sampling techniques (stationary and personal) were used in parallel (which is not the case in this study and which can explain the difference). Thus, in certain cases, stationary sampling seems to reflect personal exposure, but this is not universal. In agreement with Thorn et al. (2002a)Go we have shown that the most important exposure to endotoxin in wastewater plants occurs during special work tasks that involve exposures of the employee for a limited time.

Values in regard to recommendation
During special short-term tasks (spray removal from basins, tank walls, grids or rakes), which do not reflect the mean personal daily exposure of workers, we measured endotoxins levels up to 500 EU m–3 with personal samplers, which is 10 times greater than the most strict health-based exposure limit of 50 EU m–3, proposed in The Netherlands by the Dutch Expert Committee on Occupational Standards (Heederik and Douwes, 1997Go), but it does not exceed the threshold of 1000 EU m–3 recommended by the Swiss National Insurance Fund for Occupational Diseases (Anonymous, 2003Go). However, it has been demonstrated that an annual decline in forced expiratory volume during the first second (FEV1) was significantly associated with endotoxin exposure in pig farmers (Vogelzang et al., 1998Go). For instance, an endotoxin occupational exposure of 250 EU m–3 is already associated with an annual decline of 40 ml of FEV1 (Vogelzang et al., 1998Go). In our case, workers are not exposed on a daily basis to such endotoxin concentrations, but repeated short exposures to this range of concentrations could influence workers' health and so protective respirators should be worn during special tasks.

Concerning micro-organisms, 50% of the plants tested in summer for fungi and 30% of the plants tested indoors for both seasons exceed the proposed occupational exposure threshold values in Switzerland (Anonymous, 2003Go). However, we have to take into account that these assessments of bioaerosols were carried out with stationary samplers and for very short periods of time. Short-term samples are frequently more vulnerable to bias due to temporal variations in concentration than long-term samples. Consequently, they do not necessarily reflect the mean personal daily exposure of workers.

Species community
The percentage of viable bacteria observed to be Gram-negative ranged from 8.4 ± 2.1% in summer and 4.4 ± 1.0% in winter for viable counts. The small concentration of airborne cultivable Gram-negative bacteria is primarily due to the short survival periods in the airborne state (Müller et al., 1981Go). At the same time, not all Gram-negative bacterial species are capable of growth on the MacConkey medium used in this study. A broad spectrum of different species within the Pseudomonadaceae (ubiquitous bacteria) and the Enterobacteriaceae families were predominant in nearly all plants investigated (9/11). This is in agreement with other authors (Lunholm and Rylander, 1983Go; Laitinen et al., 1994Go; Prazmo et al., 2003Go). Within the family of Enterobacteriaceae, the genera Klebsiella and Enterobacter dominated. These bacteria are found in the soil and in water (Gauthier and Archibald, 2001Go). Bacteria from human faeces, such as E. coli (potentially pathogenic), were generally present in very low numbers. Potentially human pathogenic bacteria (Aeromonas hydrophila and Salmonella arizona) were isolated in only two plants and in very low numbers. However, the presence of such airborne pathogenic bacteria, even in very low concentrations, is worrying since immunosuppressed workers can be exposed.


    CONCLUSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
We have observed that all bioaerosols were frequently above the recommended values of occupational exposure. Moreover, workers carrying out special tasks such as cleaning tanks were exposed to very high levels of endotoxins compared with those carrying out routine work. An influence of season was detected only for airborne fungi load, which were in higher concentrations in summer than in winter. We also found that there was more bacteria in the enclosed area, near the particle grids for incoming water, than in the unenclosed area near the aeration basins. A broad spectrum of different species of airborne bacteria within the Pseudomonadaceae and the Enterobacteriaceae families were predominant in nearly all plants investigated.


    ACKNOWLEDGEMENTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Thanks to P. Hotz and two anonymous reviewers for insightful comments which greatly improved this manuscript. Karen Parker is thanked for help with English. This work was supported by the Swiss National Science Foundation, grant 3200BO-104246 to A.O.

Received October 21, 2004; in final form December 9, 2004


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 INTRODUCTION
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 DISCUSSION
 CONCLUSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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