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Airborne Microbes and Endotoxinsin the Work Environment of TwoSanitary Landfills in FinlandPäivi Rahkonen a , Matti Ettala a , Mirja Laukkanen b & MirjaSalkinoja-Salonen ba Paavo Ristola Ltd., Consulting Engineers , Terveystie 2,SF-15870, Hollola, Finlandb Department of General Microbiology , University ofHelsinki , Mannerheimintie 172, SF-00380, Helsinki, FinlandPublished online: 08 Jun 2007.

To cite this article: Päivi Rahkonen , Matti Ettala , Mirja Laukkanen & Mirja Salkinoja-Salonen(1990) Airborne Microbes and Endotoxins in the Work Environment of Two Sanitary Landfills inFinland, Aerosol Science and Technology, 13:4, 505-513, DOI: 10.1080/02786829008959465

To link to this article: http://dx.doi.org/10.1080/02786829008959465

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Airborne Microbes and Endotoxins in the Work Environment of Two Sanitary Landfills in Finland

Paivi Rahkonen* and Matti Ettala Paavo Ristola Ltd., Consulting Engineers, Terveystie 2, SF-15870, Hollola, Finland

Mirja Laukkanen and Mirja Salkinoja-Salonen Department of General Microbiology, University of Helsinki, Mannerheimintie 172, SF-00380, Helsinki, Finland

The Andersen sampler was used to study the occur- rence of bacteria and fungi in the working air of two large, intensively used landfills in Finland. Endotoxins were also determined. The concentrations of airborne microbes were high in summer in warm windy weather. Mesophilic bacteria exceeded lo5 colony-forming units (cfu)/m3 and mesophilic fungi l o4 cfu/m3. In 67% of the samples, the concentrations of gram-negative bac- teria exceeded 103/m3, which has been suggested to be the threshold limit value (TLV). The endotoxin levels were all below 0.1 & n 3 . A large part of the colony-

INTRODUCTION

In Finland municipal solid waste is collected from houses and factories and usually taken to landfill disposal sites. In 1984 there were 750 sanitary landfills (Suomela, 1984) with about 1000 workers. A large part of them are very small and there is usually one part-time operator. At bigger landfills there are more workers. The check station atten- dant receives and registers the refuse. The stopper supervises the unloading and the equipment operator spreads and compacts the refuse with the sanitary landfill com- pactor. This disposal method generates dust and aerosols often laden with microorgan- isms, for example, from decomposing food waste (Clark et al., 1983a). Very little is known about the microbial impurities in landfill working air. Our previous study

*To whom correspondence should be addressed.

forming particles were within the respirable size range, about 40% of the bacteria and about 80% of the fungi. The commonly isolated airborne bacteria were Pseudomonas, Enterobacter, and Bacillus spp. The landfill workers should try to work upwind. There are also grounds for using a mask and irrigating the refuse terrace with leachate in dry weather. It is suggested that the traffic on the refuse terrace be decreased by arranging for the private cars to be emptied in the reception area.

(Rahkonen et al., 1987) showed that the dust concentrations at the landfills depended on the quantity and quality of the refuse, traffic, operation of the landfill compactor, and the weather. The highest concentrations were measured at the biggest landfills in summer. In this season the concentrations of airborne bacteria were especially high. Workers hand- ling the refuse may thus inhale large num- bers of airborne bacteria.

According to the report on communal employees in Finland the loading factors for landfill work include the weather, level of hygiene at the working place, dust, smoke, fumes, and chemicals (acid, alkali, oil). Among landfill workers, the frequency of, diseases of the respiratory organs for exam- ple, was higher than average (Llmarinen, 1985).

It was shown earlier that gram-negative bacteria containing endotoxins play an im- portant role in the development of symptoms

Aerosol Science and Technology 13505-513 (1990) O 1990 Elsevier Science Publishing Co., Inc.

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TABLE 1. Characteristics of the Two Landfills

Characteristics at landfills

Parameter 1 2

Landfill size Over 50,000 (inhabitants in area) Machine equipment Sanitary landfill compactor Disposal technology Stratified Coverage of refuse Daily Refuse amount (t/a) 70,000 Special refuse Slaughter refuse, septic

sludges; paint, varnish, colour and surface coating refuse, wastes from impregnation of timber

Wheel loader Stratified 2-3 times a week 23,000 Septic sludges; surface coating, heavy metal, printing industry and tannery wastes, organic and oil refuse

that include fever, chest of the tightness, and diarrhea in workers in environments such as the cotton industries, sewage plants, and compost plants (Clark et al., 1983a; Rylan- der and Morey , 1982; Rylander et al., 1984).

The aim of this study was to investigate the occurrence of harmful microbes in the working air of a landfill. It has been usual to make total microbe counts and identify groups of microbes, but determinations have seldom been made of the microbe genus. Objective of this study was to determine the bacteria to the genus and species level. The culture media used chiefly favoured gram- negative bacteria. The results of many pre- vious studies (Clark et al., 1983a, b; Lund- holm et al., 1986) led to the choice of the Andersen sampler, which allows determina- tion of the size distribution.

MATERIALS AND METHODS

Study Areas and Periods

Two sanitary landfills, which were typical of the waste amount and the technology in Finland, were selected for the study. Their characteristics are presented in Table 1. Samples were taken during the periods July 1 to October 10, 1986 and May 28 to July 23, 1987 in both the forenoon and after- noon, as near as possible to the stopper's working area. The background samples were taken in every day windward of the terrace.

Measuring Methods

Microbes. Airborne bacteria and fungi were collected from the refuse terrace about 1.5 m above the ground using a six-stage Andersen sampler for 1-15 min. The sam- pler was equipped with plastic Petri dishes containing 25 mL of the appropriate media. After the plates had been incubated at cer- tain temperatures for different times (Table 2), the total colonies on each plate were counted and the positive hole correction was applied (Andersen, 1958). The results are presented as colony-forming units per cubic meter of the air (cfu m- 3).

The predominant microorganisms isolated on the CD (117 isolations) and TGY agar plates (22 isolations) were purified and gram staining was performed. Gram-negative bac- teria were identified using both API kits (API 20E, 20EC, 20NE, API Laboratory Products Ltd., Farnborough, Henis) and whole-cell fatty acid analysis by gas liquid chromatography, which consists of a 5 % phenyl methyl silicone capillary column, a automatic sampler, an integrator, and a computer (Hewlett Packard 5898A, Micro- bial Identification System, MIS). Gram-posi- tive bacteria were identified by whole-cell fatty acid analysis.

Endotoxin. Samples were collected from the same places as those for microbes. The specimens were collected on 37-rnm diame-

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Microbes and Endotoxins in Sanitary Landfills

TABLE 2. Cultivation Conditions

Incubation temperature Duration of incubation Microbes Mediuma ("c) (days)

Bacteria TGY-agar a 28,50 2 CD-agar

Fungi M-agar ', 4,22, 37 30, 7, 2-3 ~ ~ ~ - a ~ a r

'Tryptone-glucose-yeast extract-agar (Difco 0479-01-1). * Condradi-Drigalski-agar (Difco 0083-71-1). '20 g of malt extract (Difco 0024-Ol), 15 g of bacto-agar (Difco 0140-Ol), 1000 mL of distilled water. d10 g of peptone (Difco 0118-OM), 20 g of maltose (Difco 0168-17), 12 g of bactoagar (Difco 0140-I), 1000 mL of distilled

water.

ter sterile filters with a pore size of 0.45 pm (Millipore, type HA), using a stationary sampler with a flow rate of 1-2.5 L/min for 1-2.5 h. Filters were washed off with 10 mL of sterile pyrogen-free distilled water containing 0.01 % wt/wt Tween 80. Coatest (Kabi), a chromogenic LAL test assay (Fri- berger, 1985), was used to determine the amount of endotoxin.

Other Measurements During every sampling the air temperature and the relative humidity were recorded with hydrometer (Humicor, type IHRT) and the wind velocity with a thermoanemometer (Alnor, type GGA-65 P). The number of refuse loads and the extent of the operation of the sanitary landfill compactor on the refuse terrace were also recorded. Data on

TGY28 TGYSO CD28 CD50 M,ASM 4,22,37 Culture media and temperature, +'C

FIGURE 1. Concentrations of microbes in the working air of the landfills and in the background air.

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508 Rahkonen et al.

TABLE 3. Number and Frequency of Occurrence of Identified Bacterial Genera by Two Identifying Methods in the Working Air of Two Landfills in Summer 1986 and 1987

Number of strains (no.) and frequency (%) of isolation from samples of

Mesophilic bacteria Thermotolerant bacteria Identifying method at landfill nos. at landfill nos.

Whole-cell fatty 1 2 1 2

acid analysis" API analysisb No. % No. % No. % No. %

-

Pseudomonas Xanthomonas Xanthomonas

- Enterobacter Enterobacter Escherichia Acinetobacter Acinetobacter Hafnia Flavobacterium Flavobacterium Citrobacter Moraxella

- Bacillus Micrococcus Staphylococcus Enterococcus Brevibacterium Corynebacterium Clavibacterium

Pseudomonas Pseudomonas Pseudomonas

-

Enterobacter Enterobacter Klebsiella Enterobacter Enterobacter

Rahnella Flavobacteriurn

- -

Achromobacter

Unidentified gram-negative Unidentified gram-positive

'Similarity index > 0.2. *percentage of identification 2 80%. Frequency of isolation = no. of occasions isolated/no. of air samples X 100.

the precipitation were obtained from Lahti meteorological station, situated about 7 km from landfill 1 and Kotka meteorological station, situated on the coast about 78 km from landfill 2 (Finnish Meteorological In- stitute, 1986, 1987).

Statistical Methods All the statistical analyses were performed with the statistical program package SPSS (Norusis, 1986). Differences in the microbe concentrations at the landfills were tested with the t-test. After that Duncan's multiple comparison test was used to determine the

correlations between the weather and other conditions to microbe concentrations. Con- clusions were drawn at the 0.050 risk level.

RESULTS

Microbes

The concentrations of bacteria and fungi in the working air of the two sanitary landfills are presented in Figure 1. The concentra- tions were 5-20 times higher than the back- ground concentrations and did not differ no- ticeably between the forenoon and afternoon or between the two landfills. The concentra-

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Microbes and Endotoxins in Sanitary Landfills 509

TABLE 4. Number and Frequency of Bacterial Species Identified by Two Different Identifying Methods in the Working Air of Landfills in Summer 1986 and 1987

Number of strains (no.) and frequency (%) of isolation from samples

Whole-cell fatty analysisa

Species No. % No. %

Pseudomonas vesicularis 4 15.4 Pseudomonas Jluorescens 2 7.7 Pseudomonas paucimobilis 1 3.8 Pseudomonas putida 1 3.8 Pseudomonas maltophilia 2 7.7 Pseudomonas agarici 1 3.8 Pseudomonas syringae 1 3 .8 Enterobacter agglomerans 2 7.7 8 30.8 Enterobacter cloacae 2 7.7 1 3.8 Acinetobacter calcoaceticus 2 7.7 Acinetobacter IwoJii 1 3.8 Flavobacterium indologenes 1 3.8 Moraxella phenylpyruvica 1 3.8 Achromobacter sr. VD 1 3.8 Hafnia alvei 1 3.8 Bacillus licheniformis 3 11.5 Bacillus thuringiensis 1 3.8 Bacillus sphaericus 1 3 .8 BaciNus pumilus 2 7.7 Micrococcus luteus 2 7.7 Micrococcus kristinae 1 3.8 Enterococcus faecalis 1 3 .8 Corynebacterium flaccum faciens 1 3.8

"Similarity index > 0.6. b~ercentage of identification > 95.0%. Frequency of isolation = no. of occasions isolated/no. of air samples X 100

tions of mesophilic bacteria varied from 3.5 x lo2 to lo5 cfu/m3. In 67% of the sam- ples, the concentrations of gram-negative bacteria exceeded 103/m3, which has been suggested to be the threshold limit value (TLV) (Rylander et al. , 1984).

The concentrations of fungi varied from lo2 to 3.4 X lo4 cfu/m3. These concentra- tions were 2-30 times higher than the back- ground concentrations. They did not differ noticeably between the forenoon and after- noon, between the landfills, or between the culture media and temperature.

The bacterial genera identified in the working air of the landfills are listed in Table 3 and the species in Table 4. Of the isolated bacteria, 8 1 were gram-negative and 58 gram-positive.

Size Distribution Size distributions of each plate can be seen in Figure 2. The proportions of bacteria in the respirable size range were about 40% and fungi almost 80% (Task Group on Lung Dynamics, 1966). The mesophilic bacteria (< 5 pm) had average concentrations of lo4 cfu/m3 and the mesophilic fungi 7 x lo3 cfu/m3.

Endotoxin

The concentrations of endotoxin in the working air of the two sanitary landfills varied from 0.4 to 29 ng/m3 ( n = 20), aver- age 5.3 ng/m3. These levels are all below 0.1 pg/m3, which can be considered safe (Rylander et al., 1984). The concentrations

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Rahkonen et al.

1 2 5 Aerodynamic diameter, dA (urn)

Bacterla (n-99) Fungi (n- 144) - --.-..

FIGURE 2. Size distributions at the working air of landfills.

did not differ noticeably between the forenoon and afternoon or between the land- fills.

Other Observations

Air temperature varied from 10°C to 3 1 "C, relative humidity from 30% to 94%, wind velocity from 0.5 to 3 m/s and deposited refuse loads from 4 to 14. The sanitary landfill compactor operated 19 times from 26 during the sampling. The distance be- tween the compactor and the sampling point was 5 to 30 m.

The microbial concentrations correlated with air temperature, relative humidity, and rainfall. On the other hand, they showed poor and generally negative correlations with

the transport of refuse loads and the opera- tion of the sanitary landfill compactor on the refuse terrace during the measurements.

DISCUSSION

Table 5 lists the levels of airborne microbes and endotoxins in different environments. The levels of bacteria and fungi in the land- fills are equivalent to those in other environ- ments. The bacteria cultered in CD media (favors gram-negative bacteria, Lundholm and Rylander, 1983) and endotoxin gener- ally had lower levels in the landfill samples. In a previous study (Rahkonen et al., 1987) lower microbial concentrations were found, possibly because the landfills were smaller and the sampling periods more humid. The effects of the transport of refuse loads and the operation of the landfill compactor may not always have been evident in the statisti- cal calculations, because of the distance from the sampling point and the small air veloc- ity.

Landfill 1 serves over 50,000 inhabitants and landfill 2 10,000-50,000 inhabitants. The size of the landfills studied is not repre- sentative, because only 12% of the 750 sani- tary landfills in Finland serve more than 10,000 inhibitants. However, the study ar- eas represent the landfills at which the dis- posal technique is based on the official guidelines and the amount of waste at these landfills is 73% of the total amount of waste in Finland (Ettala, 1988a; Finnish State Computer Center, 1985). Because of simi- larities in waste disposal technology, refuse quality and hydrological conditions the re- sults are comparable to the sanitary landfills in Scandinavia (Ettala, 198813).

The bacteria isolated on selective CD medium from the working air of landfills are mostly gram-negative. The medium selected mesophilic gram-negative bacteria better than the thermotolerant bacteria. The bacte- rial colonies cultured at 50°C were purified and later subcultured 28°C to determine if this group comprised facultatively thermo-

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TABLE 5. Concentrations of Airborne Mesophilic Bacteria (Anderson Sampler) and Endotoxins in Different Environments

Bacteria cultured in

Bacteria CD media Endotoxin Fungi Work place (cfu/m3) (cfu/m3) ( d m 3 ) (cfu/m3) Reference

Landfill Landfill

Waste sorting plant a

b

Composting plant Wastewater treatment plant

Poultry confinement building Swine confinement building Cotton mill

Farm

7 x lo2-3 x lo4 Present study 2 x 10'-6 x lo3 Rahkonen

et al., 1987

Malmros and Petersen, 1988 Petersson and Vikstrom, 1984;Clark et al., 1983a

lo3- > lo5 Nevalainen; Lundholm and Rylander 1983; Makela et al., 1982

2 x lo2-2 x lo3 Clark et al., 1983b

lo2-4 X lo2 Clark et al., 1983b

lo3-2 x lo4 Cinkotai et al., 1977; Rylander and Morey, 1982

8 x lo5-3 x lo6 Lundholm et al., 1986; Kotimaa et al., 1984

m E 8 X ti' Y)

ti'

'Before encapsulation of the conveyor belts and establishment of a central vacuum cleaning device. *~fIer rebuilding the plant.

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512 Rahkonen et al.

tolerant bacteria and no, or only selected, obligately thermotolerant bacteria.

A large part of the colony-forming parti- cles were within the respirable size range. Particles of this size accounted for about 40% of the bacteria and about 80% of the fungi. The bacteria which are not in the respirable size range are cleared out of the respiratory tract and enter the gastrointesti- nal tract. The size distribution of bacteria was the same as in waste treatment plants (Rylander et al., 1984).

Many different species were present in the airborne bacteria. The most commonly isolated bacteria were Pseudomonas, En- terobacter , and Bacillus spp. They and other bacteria identified are common in soil, water, plants, and food (Krieg et al., 1984; Sneath et al., 1986). Most of them are op- portunistic pathogens, which cause infec- tions if human resistance is reduced. The same kind of bacteria have been identified in waste treatment plants (Rylander et al., 1984).

Most of the bacteria encountered during this study could be called environmental bacteria. Two methods were used to identify bacterial genera in this study. The accuracy for the level of bacteria general by API analysis is 1 80% and by whole cell fatty acid analysis > 20%. For the level of bacte- rial species the corresponding accuracies are > 95% and > 60%.

Though there were no potent pathogens in the working air of landfills, the concentra- tions of bacteria were high. Thus there are grounds for decreasing the dust concentra- tions in landfills. The stopper should try to work upwind and use a mask in dry weather. The refuse terrace should also be irrigated with leachate in dry weather. Traffic on the refuse terrace can be decreased by arranging for the private cars to be emptied in the reception area.

We wish to thank the communes and their landfill workers for their cooperation. This study was financed by The Finnish Work Environment Fund. Of the authors, Mirja

Laukkanen, M.Sc., and Prof. Mirja Salkinoja-Salonen were responsible the methods for sampling and analysing microbes. Matti Ettala, D. Tech., was responsible for the technology of waste management. PGvi Rahkonen, M.Sc., was responsible for the sampling and analysing of mi- crobes and wrote the report.

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