the cytotoxic potential of household waste during composting

Waste Management & Research (1997) 15, 189–196

THE CYTOTOXIC POTENTIAL OF HOUSEHOLD WASTEDURING COMPOSTING

Vibe Roepstorff and Torben Sigsgaard

Department of Occupational and Environmental Health, University of Aarhus,Bldg. 180, 8000 Aarhus C, Denmark

(Received 2 February 1996, accepted in revised form 21 April 1996)

During the last 10 years an increasing number of plants for re-use of refuse havebeen constructed in Europe and the U.S.A. During the same period several cases ofoccupational respiratory diseases among workers have been reported in the recyclingindustry. The aim of this project was to show, in vitro, if there is any change in thecytotoxic potential of garbage dust during the process of converting household wasteto compost. Two cell lines have been exposed to extracts of waste fuel pellets andcompost, taken from three different time periods in the composting process. Sig-nificant differences were found in the cytotoxic potential of extracts of householdwaste (P<0.05). Extracts of 0.48 mg well−1 raw compost, fresh compost and maturedcompost show a cytotoxic effect at 97, 41 and 44%, respectively of unexposed cells.In conclusion, these results show the greatest cytotoxic potential when the microbialactivity seems to be at its height in the composting process. Earlier, studies on theeffect of endotoxin (lipopolysaccharide, LPS) on the cells, and with pure endotoxindid not find any cytotoxic effect in the assay. Further investigations are needed inorder to find which micro-organisms or components from these are responsible forthe cytotoxic potential. 1997 ISWA

Key Words—Cytotoxic effect, in vitro, A 549, VERO, cell culture, compost, organicdust.

1. Introduction

Industrialized countries with growing populations and consumption face rising problemsdisposing the increasing amounts of waste in urban areas. As a consequence, a greatnumber of plants for re-use of refuse have been constructed in Europe and the U.S.A.during the last 10 years. In these plants, household waste may be converted to wastepellets and used as fuel, composted and used on land, or recycled as raw materials.

Recycling is beneficial for the environment. However, the process might cause adversehealth effects among the workers at the recycling facilities. As long ago as during theSecond World War, it was noted that women sorting old cloth and rubber for re-use encountered symptoms in the lungs and the gastrointestinal tract (Arbejds- ogFabrikstilsynet 1942).

In 1986, a new plant for re-use of refuse was started in Skive, Denmark. This plantconverts household waste to fuel pellets. During the first 8 months, eight out of 15employees developed respiratory symptoms (Sigsgaard et al. 1990). In Sweden, aninvestigation showed that four out of 11 compost workers reported diarrhoea but onlytwo out of 41 drinking water plant workers reported these symptoms (Lundholm andRylander 1980). A survey of the recycling industry in Denmark (Sigsgaard et al. 1994a)showed a significantly higher prevalence of symptoms like chest tightness, nausea,vomiting or diarrhoea and itching eyes, nose and throat, among waste-handling workers

0734–242X/97/020189+08 $25.00/0 wm960075 1997 ISWA

V. Roepstorff & T. Sigsgaard190

compared to water supply workers. It was also found that organic dust toxic syndrome(ODTS) was associated with waste handling. Weber et al. (1993) reported a case wherea 52-year-old male developed fever, myalgia and marked dyspnea 12 h after shovellingcomposted wood chips and leaves. Similar symptoms have been seen in other workingenvironments such as in the cotton industry, the wood industry and among farmers(Donham et al. 1989; Chan-Yeung et al. 1992; Sigsgaard et al. 1992b; Dahlqvist andUlfvarson 1994).

Common to all these working environments is the exposure of the workers to organicdust. Many studies have tried to find the causative agent for respiratory symptomsoccurring among waste handling workers and other workers exposed to organic dusts.In vivo and in vitro investigations have been made (Rylander et al. 1985; Johnson et al.1986; Milanowski & Dutkiewicz, 1992; Gordon & Harkema, 1995) with extracts oforganic dust, single micro-organisms or components with microbial origin found indust from different working environments. However, no firm conclusions have beendrawn from the studies so far.

In an earlier investigation, a new assay was presented to detect cytotoxic effect oncell lines after incubation with extracts of Bacillus sp. as well as crude cotton dust(Sigsgaard et al. 1993, 1994b). This method is able to show increases in the cytotoxicpotential of dust extracts with rising concentrations and with increased exposure time.The aim of this project is to show, in vitro, if there is any change in the cytotoxicpotential during the conversion of household waste to compost.

2. Material and methods

2.1 Plant description

2.1.1 Plant IThe raw materials in this plant are household and industrial waste. After the waste hasbeen sorted, the organic component is converted into pellets for use as fuel. When thisplant started in 1986 there was an epidemic of respiratory problems among the employees(Sigsgaard et al. 1990). Extracts have been made from comminuted waste fuel pelletsfrom this plant.

2.1.2 Plant IIThis plant receives presorted organic household waste. Substantial composting takesplace in a rotating drum. After 24 h in a rotating drum of the DANO type at a finaltemperature of 40°C, the rest of the composting process takes place in five aeratedroofed piles. The compost stays in each pile for 10 days, before it is turned and movedto the next pile. The last pile is outdoors and the process takes 2–3 months. Extractshave been made from compost aged 48 h, 35 days and 77 days after initial composting.The temperatures of the compost at the time of sampling were approximately 40°C,70°C and 50°C, respectively.

2.2 Extraction protocol

Converted waste (1:10 w/v) or compost suspended in 0.125 M ammonium hydrogencarbonate at pH 8.3 was gently rotated (20 rpm) for 18 h at 4°C. After filtering througha fibre glass filter (Gelman Sciences type A/E Glass) the extract was freeze dried beforebeing dissolved in a small amount of pyrogen-free water (conc. 1.25 g dust ml−1). This

Cytotoxic potential of household waste 191

20.8

120

0

0.04

mg well–1

%

1.3

100

80

60

40

20

0.080.1

60.3

30.6

5 2.6 5.2 10.4

(b)

20.8

120

0

0.04

mg well–1

%

1.3

100

80

60

40

20

0.080.1

60.3

30.6

5 2.6 5.2 10.4

(d)

20.8

120

0

0.04

mg well–1

%

1.3

100

80

60

40

20

0.080.1

60.3

30.6

5 2.6 5.2 10.4

(a)

20.8

120

0

0.04

mg well–1

%

1.3

100

80

60

40

20

0.080.1

60.3

30.6

5 2.6 5.2 10.4

(c)

Fig. 1. Cytotoxic effect of extracts of compost 35 days after homogenization on (a) VERO cells after 2 hand (b) 24 h of incubation, and on (c) A 549 cells after 2 h and (d) 24 h of incubation.

final solution was filtered through a 0.2 lm filter (Schleicher & Schuell FP 030/3) andstored at −80°C until use.

2.3 Cell cultures

Cultures of monkey kidney cells (VERO from American Type Culture Collection ATCCno: CCL 81 pass: 125–130) were grown in Earle’s medium 199 (95%) with fetal bovineserum (5%, endotoxin content <0.02 ng ml−1; Gibco-BRL) and penicillin/streptomycin(100 IU/100 lg ml−1; JRH-Biosciences) at 37°C, 5% CO2. For the assay 5×103 cellswell−1 were seeded.

Cultures of human lung carcinoma cells (A 549 from American Type CultureCollection ATCC no: CCL 185 pass: 98–103) were grown in HAM’s F 12K medium(90%) with fetal bovine serum (10%, endotoxin content <0.02 ng/ml; Gibco-BRL) andpenicillin/streptomycin (100 IU/100 lg ml−1; JRH-Biosciences) at 37°C, 5% CO2. Forthe assay 4×103 cells well−1 were seeded.

2.4 Cytotoxic assay

CellTiter 96TM Non-Radioactive Cell Proliferation/Cytotoxicity assay (Promega) wasused. Cells were grown in microtitre plates for cell cultures (Greiner) for two days. Onday 3 when the cells were in the log-phase of growth the medium was exchanged withextracts in concentrations 0.04–20.8 mg well−1 suspended in medium and incubated for2 or 24 h. At this time the cells were nearly confluent and the medium was exchanged


TABLE 1Cytotoxic effect on both cell lines after 2 and 24 h of incubation with alkaline

soluble extracts, expressed as percent of unexposed cells. Median and range

A 549 VERO

mg well−1 2 h 24 h 2 h 24 h

0.48 99† 90 96†§ 79Fuel 92–106 81–99 87–105 70–88pellets 15.6 101†‡ 54 82 23

95–107 47–61 75–89 21–25

0.48 103 97† 79 81∗Compost 99–107 92–102 70–88 77–8548 h 15.6 103‡ 45 73‡ 21

101–105 39–51 57–89 19–23

0.48 77 41 49 39Compost 74–80 40–42 46–52 36–4235 days 15.6 32 1 4 0

30–34 0–2 1–7 0–0

0.48 81 44 56 43Compost 77–85 43–45 52–60 40–4677 days 15.6 31 1 7 0

33–29 1–1 4–10 0–0

∗ 2 h and 24 h are not significantly different (P<0.05).† Extract is not significantly different from control (P<0.05).‡ No difference between extract 0.48 and 15.6 mg well−1 (P<0.05).§ VERO cells not significantly more sensitive than A 549 cells (P<0.05).

once again with medium containing dye (15% tetrazolium-salt). After another 4 h ofincubation solubilizer was added into the dye medium and the suspension was leftovernight at room temperature in a dark humid container. The results were read witha 570-nm messenger and 630-nm reference filter in an ELISA microplate reader (BIO-RAD 450), and plotted as a percentage of non exposed cells (Fig. 1).

2.5 Statistics and results

For significance testing Mann–Whitney and Wilcoxon’s Signed rank test (SPSS statisticalPackage for the Social Sciences/IBM) was used. The measurement of 0.48 mg dustwell−1 was the mean of 16 wells expressed as a percentage of untreated control cells(eight readings at concentration 0.32 mg dust well−1 and eight readings at concentration0.64 mg dust well−1). The measurement of 15.6 mg dust well−1 was the mean of 16 wellsexpressed as a percentage of untreated control cells (eight readings at concentration10.4 mg well−1 and eight readings at concentration 20.8 mg well−1).

3. Results

Two cell lines were tested against extracts of four different stages in the convertingprocess of household waste. The two cell lines reacted similarly to the exposure.However, the VERO cells are more sensitive than the A 549 cells (Table 1).


120

0

mg well–1

%

96

72

48

24

0.48 15.6 0 0.48 15.6 0 0.48 15.6 0 0.48 15.6

Fig. 2. Cytotoxic potential on A 549 cells of extracts of four different types of household waste. The cytotoxiceffects are expressed as a percentage of unexposed cells. Dotted lines after 2 h of incubation and straightlines after 24 h of incubation. (Α), waste pellets; (Χ), compost (48 hours); (Φ), compost (35 days); (Ε),

compost (77 days).

Moderate cytotoxic potential was found from extracts of waste pellets and compostat 40°C, 48 h after initial mixing. After 2 h of incubation there was no cytotoxic effect(Fig. 2), but a significant (P<0.05) cytotoxic effect was seen after 24 h of incubation.

A strong cytotoxic effect was seen after 2 h of incubation with extracts from compost35 days and 77 days after initial mixing. The cytotoxic potential was markedly increasedafter 24 h of incubation with these two extracts of compost. The effect of compost 35and 77 days after mixing at concentration 0.48 mg well−1 was significantly (P<0.05)higher than the effect of waste pellets at 15.8 mg well−1. There are no differences of thecytotoxic effect of extracts of compost at 70°C (35 days) and 50°C (77 days).

4. Discussion

In this study significant differences were found in cytotoxic potential of extracts ofhousehold waste taken at different time periods during the converting process, or fromdifferent end points in the recycling process.

The composting process is a microbial process. Compost may be made from manyorganic materials, for instance grass, wood chips, organic household waste, waste sludgeor animal manure. When organic material is composted, the temperature rises and theubiquitous micro-organisms multiply rapidly when their optimal vital needs are met.Many studies have been performed in order to find the optimal temperature and thetype of micro-organisms involved in the degradation of the organic material (Clark etal. 1983; Nakasaki et al. 1985a; Nakasaki and Akiyama 1988). Nakasaki et al. (1985b)found that the number of thermophilic bacteria and thermophilic actinomycetes incomposted sewage sludge rose with the temperature until the pile reached 70°C. Anotherinvestigation was made on domestic waste (Crook et al. 1988) composted for 1 monthat a temperature of 55°C. In this experiment the researchers found that the number offungi and thermophilic bacteria rose markedly, 400-fold and 4700-fold respectively,


during the first 4 weeks. Other groups have investigated composted wood chips orwood chips mixed with sewage sludge (Millner et al. 1977; Jappinen et al. 1987;Olenchock et al. 1991). All found a noticeable number of fungi, with Aspergillusfumigatus as the dominant species.

In order to control temperatures and to avoid the terrible odour that occurs whenanaerobic bacteria take over, it is necessary to aerate the pile. The plant from wherethe samples were obtained aerates the piles in order to keep the O2 content between 5and 20%. The humidity is kept at 50% and the piles are turned every 10 days. Duringthe first 10 days, the temperature reaches 40–50°C, and during the next 30 days, thepile temperature rises to approximately 70°C. The temperature falls slowly to 30°C atthe end of a further 2-month period.

The compost may be classified in four types: raw, fresh, matured and special,according to the degree of biochemical degradation (Brunt et al. 1985). We have testedcompost corresponding to the first three of these types: raw compost at 40°C, 48 hafter initial mixing; fresh compost 70°C, 35 weeks after mixing, in the early stages ofbiochemical degradation; matured compost at 50°C, 77 days after mixing, which isfully composted with a texture similar to that of good growing-soil and an ‘‘earthy’’smell produced by actinomycetes which grow during the final stage.

Reports about health problems among workers in some recycling plants (Malmroset al. 1992; Sigsgaard et al. 1992a), show that the illnesses occur when the turnover ofthe waste in the plant is low, leading to a long dwell time of the waste, and as aconsequence, increased microbial growth. Another investigation (Sigsgaard et al. 1994a)found that symptoms of ODTS among waste-handling workers was associated withmoving compost or working with highly contaminated waste. An investigation ofbiochemical growth parameters during the composting of sewage sludge (Chino et al.1983) found a great microbial activity after 35 days. They also found high cellulaseactivity during day 23 and 79. Nakasaki et al. (1987) showed that in the beginning of thecomposting process the decomposing of cellulosic material is done by the thermophilicbacteria and actinomycetes. Later on, the thermophilic fungi take over the process.Our results agree with these investigations, the fresh compost 35 days old is the mostaggressive.

The smallest effect is seen after exposure to the extract of the waste pellets. However,the cytotoxic effect is still considerable. At the highest concentration of extract after24 h of incubation, the metabolic rate of the cells is about 50% of unexposed cells.Second in the order is raw 40°C compost 48 h after initial mixing. This waste is organic,it is 1 week old but almost unconverted and the micro-organisms have not reachedtheir optimal growing conditions due to the short dwell time in the compost pile. Wastepellets are composed of compressed, dried, mixed organic and inorganic waste. Micro-organisms can live in and on them. However, the growing conditions are not optimal.These results show that the waste already has a cytotoxic potential when it arrives atthe plant. The strongest cytotoxic effect is seen after exposure to extracts of compostat 70°C (35 days) and 50°C (77 days).

These results show that the greatest cytotoxic potential is seen when the microbialactivity is at its height. The authors have previously studied the effect of endotoxin(lipopolysaccharide, LPS) on the cells, and with pure endotoxin we did not find anycytotoxic effect in our assay (Sigsgaard et al. 1994b). Further investigations are neededin order to find which micro-organisms or components from these are responsible forthe cytotoxic potential. This may give a clue to which components of the dust shall be


focused on in studies of adverse effects. The research team is currently working on thisproblem.

Acknowledgements

This study was made possible by grants from the Danish Work Environment Fund.The authors wish to thank Director H. Gregersen, Vejle Composting Plant and J. C.Jensen, 4S plant Skive for their kind co-operation.

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the cytotoxic potential of household waste during composting

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