THE CYTOTOXIC POTENTIAL OF HOUSEHOLD WASTE DURING COMPOSTING

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

THE CYTOTOXIC POTENTIAL OF HOUSEHOLD WASTE DURING 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 have been constructed in Europe and the U.S.A. During the same period several cases of occupational respiratory diseases among workers have been reported in the recycling industry. The aim of this project was to show, in vitro, if there is any change in the cytotoxic potential of garbage dust during the process of converting household waste to compost. Two cell lines have been exposed to extracts of waste fuel pellets and compost, taken from three different time periods in the composting process. Significant differences were found in the cytotoxic potential of extracts of household waste (P<0.05). Extracts of 0.48 mg well−1 raw compost, fresh compost and matured compost show a cytotoxic effect at 97, 41 and 44%, respectively of unexposed cells. In conclusion, these results show the greatest cytotoxic potential when the microbial activity seems to be at its height in the composting process. Earlier, studies on the effect of endotoxin (lipopolysaccharide, LPS) on the cells, and with pure endotoxin did not find any cytotoxic effect in the assay. Further investigations are needed in order to find which micro-organisms or components from these are responsible for the cytotoxic potential.  1997 ISWA Key Words—Cytotoxic effect, in vitro, A 549, VERO, cell culture, compost, organic dust.

1. Introduction Industrialized countries with growing populations and consumption face rising problems disposing the increasing amounts of waste in urban areas. As a consequence, a great number 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 waste pellets 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 adverse health effects among the workers at the recycling facilities. As long ago as during the Second World War, it was noted that women sorting old cloth and rubber for reuse encountered symptoms in the lungs and the gastrointestinal tract (Arbejds- og Fabrikstilsynet 1942). In 1986, a new plant for re-use of refuse was started in Skive, Denmark. This plant converts household waste to fuel pellets. During the first 8 months, eight out of 15 employees developed respiratory symptoms (Sigsgaard et al. 1990). In Sweden, an investigation showed that four out of 11 compost workers reported diarrhoea but only two out of 41 drinking water plant workers reported these symptoms (Lundholm and Rylander 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

190

V. Roepstorff & T. Sigsgaard

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 where a 52-year-old male developed fever, myalgia and marked dyspnea 12 h after shovelling composted wood chips and leaves. Similar symptoms have been seen in other working environments 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 and Ulfvarson 1994). Common to all these working environments is the exposure of the workers to organic dust. Many studies have tried to find the causative agent for respiratory symptoms occurring 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 of organic dust, single micro-organisms or components with microbial origin found in dust from different working environments. However, no firm conclusions have been drawn from the studies so far. In an earlier investigation, a new assay was presented to detect cytotoxic effect on cell 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 cytotoxic potential 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 cytotoxic potential during the conversion of household waste to compost. 2. Material and methods 2.1 Plant description 2.1.1 Plant I The raw materials in this plant are household and industrial waste. After the waste has been sorted, the organic component is converted into pellets for use as fuel. When this plant 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 pellets from this plant. 2.1.2 Plant II This plant receives presorted organic household waste. Substantial composting takes place in a rotating drum. After 24 h in a rotating drum of the DANO type at a final temperature of 40°C, the rest of the composting process takes place in five aerated roofed piles. The compost stays in each pile for 10 days, before it is turned and moved to the next pile. The last pile is outdoors and the process takes 2–3 months. Extracts have 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 hydrogen carbonate at pH 8.3 was gently rotated (20 rpm) for 18 h at 4°C. After filtering through a fibre glass filter (Gelman Sciences type A/E Glass) the extract was freeze dried before being dissolved in a small amount of pyrogen-free water (conc. 1.25 g dust ml−1). This

Cytotoxic potential of household waste

80

80 %

100

60

(c)

60 40

20

20

0

0 0. 0 0. 4 0 0. 8 1 0. 6 3 0. 3 65 1. 3 2. 6 5. 2 10 .4 20 .8

40

mg well–1

mg well–1 120

(b)

100

100

80

80 %

60

60

65 1. 3 2. 6 5. 2 10 .4 20 .8

0.

0.

0.

0.

33

0 16

20

0 08

40

20 04

40

0.

(d)

0. 0 0. 4 0 0. 8 1 0. 6 3 0. 3 65 1. 3 2. 6 5. 2 10 .4 20 .8

120

%

120

(a)

100

0. 04 0. 08 0. 16 0. 33 0. 65 1. 3 2. 6 5. 2 10 .4 20 .8

%

120

191

mg well–1

mg well–1

Fig. 1. Cytotoxic effect of extracts of compost 35 days after homogenization on (a) VERO cells after 2 h and (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) and stored at −80°C until use. 2.3 Cell cultures Cultures of monkey kidney cells (VERO from American Type Culture Collection ATCC no: CCL 81 pass: 125–130) were grown in Earle’s medium 199 (95%) with fetal bovine serum (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 cells well−1 were seeded. Cultures of human lung carcinoma cells (A 549 from American Type Culture Collection 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) and penicillin/streptomycin (100 IU/100 lg ml−1; JRH-Biosciences) at 37°C, 5% CO2. For the assay 4×103 cells well−1 were seeded. 2.4 Cytotoxic assay CellTiter 96TM Non-Radioactive Cell Proliferation/Cytotoxicity assay (Promega) was used. Cells were grown in microtitre plates for cell cultures (Greiner) for two days. On day 3 when the cells were in the log-phase of growth the medium was exchanged with extracts in concentrations 0.04–20.8 mg well−1 suspended in medium and incubated for 2 or 24 h. At this time the cells were nearly confluent and the medium was exchanged

192

V. Roepstorff & T. Sigsgaard

TABLE 1 Cytotoxic 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

Fuel pellets

mg well−1

2h

24 h

2h

24 h

0.48

99† 92–106 101†‡ 95–107

90 81–99 54 47–61

96†§ 87–105 82 75–89

79 70–88 23 21–25

103 99–107 103‡ 101–105

97† 92–102 45 39–51

79 70–88 73‡ 57–89

81∗ 77–85 21 19–23

77 74–80 32 30–34

41 40–42 1 0–2

49 46–52 4 1–7

39 36–42 0 0–0

81 77–85 31 33–29

44 43–45 1 1–1

56 52–60 7 4–10

43 40–46 0 0–0

15.6 0.48

Compost 48 h

15.6 0.48

Compost 35 days

15.6 0.48

Compost 77 days

VERO

15.6

∗ 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 of incubation solubilizer was added into the dye medium and the suspension was left overnight at room temperature in a dark humid container. The results were read with a 570-nm messenger and 630-nm reference filter in an ELISA microplate reader (BIORAD 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 statistical Package for the Social Sciences/IBM) was used. The measurement of 0.48 mg dust well−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 concentration 0.64 mg dust well−1). The measurement of 15.6 mg dust well−1 was the mean of 16 wells expressed as a percentage of untreated control cells (eight readings at concentration 10.4 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 converting process 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).

Cytotoxic potential of household waste

193

120

96

%

72

48

24

0

0.48

15.6

0

0.48

15.6

0

0.48

15.6

0

0.48

15.6

mg well–1 Fig. 2. Cytotoxic potential on A 549 cells of extracts of four different types of household waste. The cytotoxic effects are expressed as a percentage of unexposed cells. Dotted lines after 2 h of incubation and straight lines 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 compost at 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 compost 35 days and 77 days after initial mixing. The cytotoxic potential was markedly increased after 24 h of incubation with these two extracts of compost. The effect of compost 35 and 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 the cytotoxic 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 of household waste taken at different time periods during the converting process, or from different end points in the recycling process. The composting process is a microbial process. Compost may be made from many organic materials, for instance grass, wood chips, organic household waste, waste sludge or animal manure. When organic material is composted, the temperature rises and the ubiquitous micro-organisms multiply rapidly when their optimal vital needs are met. Many studies have been performed in order to find the optimal temperature and the type of micro-organisms involved in the degradation of the organic material (Clark et al. 1983; Nakasaki et al. 1985a; Nakasaki and Akiyama 1988). Nakasaki et al. (1985b) found that the number of thermophilic bacteria and thermophilic actinomycetes in composted sewage sludge rose with the temperature until the pile reached 70°C. Another investigation was made on domestic waste (Crook et al. 1988) composted for 1 month at a temperature of 55°C. In this experiment the researchers found that the number of fungi and thermophilic bacteria rose markedly, 400-fold and 4700-fold respectively,

194

V. Roepstorff & T. Sigsgaard

during the first 4 weeks. Other groups have investigated composted wood chips or wood chips mixed with sewage sludge (Millner et al. 1977; Ja¨ppinen et al. 1987; Olenchock et al. 1991). All found a noticeable number of fungi, with Aspergillus fumigatus as the dominant species. In order to control temperatures and to avoid the terrible odour that occurs when anaerobic bacteria take over, it is necessary to aerate the pile. The plant from where the samples were obtained aerates the piles in order to keep the O2 content between 5 and 20%. The humidity is kept at 50% and the piles are turned every 10 days. During the first 10 days, the temperature reaches 40–50°C, and during the next 30 days, the pile temperature rises to approximately 70°C. The temperature falls slowly to 30°C at the 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 tested compost corresponding to the first three of these types: raw compost at 40°C, 48 h after initial mixing; fresh compost 70°C, 35 weeks after mixing, in the early stages of biochemical degradation; matured compost at 50°C, 77 days after mixing, which is fully 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 (Malmros et al. 1992; Sigsgaard et al. 1992a), show that the illnesses occur when the turnover of the waste in the plant is low, leading to a long dwell time of the waste, and as a consequence, increased microbial growth. Another investigation (Sigsgaard et al. 1994a) found that symptoms of ODTS among waste-handling workers was associated with moving compost or working with highly contaminated waste. An investigation of biochemical growth parameters during the composting of sewage sludge (Chino et al. 1983) found a great microbial activity after 35 days. They also found high cellulase activity during day 23 and 79. Nakasaki et al. (1987) showed that in the beginning of the composting process the decomposing of cellulosic material is done by the thermophilic bacteria 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 most aggressive. 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 after 24 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 reached their optimal growing conditions due to the short dwell time in the compost pile. Waste pellets are composed of compressed, dried, mixed organic and inorganic waste. Microorganisms 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 at the plant. The strongest cytotoxic effect is seen after exposure to extracts of compost at 70°C (35 days) and 50°C (77 days). These results show that the greatest cytotoxic potential is seen when the microbial activity 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 any cytotoxic effect in our assay (Sigsgaard et al. 1994b). Further investigations are needed in order to find which micro-organisms or components from these are responsible for the cytotoxic potential. This may give a clue to which components of the dust shall be

Cytotoxic potential of household waste

195

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

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.

References Arbejds- og Fabrikstilsynet (1942). Beretning for aarene 1939–41, Copenhagen, Denmark: Arbejdsog Fabrikstilsynet. pp. 77–78. Brunt, L. P., Dean, R. B. & Patrick, P. K. (1985). Composting (characteristics of composting). In Solid Waste Management Selected Topics (M. J. Suess, ed.), Copenhagen, Denmark: World Health Organization. Chan-Yeung, M., Dimich-Ward, H., Enarson, D. A. & Kennedy, S. M. (1992). Five cross-sectional studies of grain elevator workers. American Journal of Epidemiology 136, 1269–1279. Chino, M., Kanazawa, S., Mori, T., Araragi, M. & Kanke, B. (1983). Biochemical studies on composting of municipal sewage sludge mixed with rice hull. Soil Science and Plant Nutrition 29, 159–173. Clark, C. S., Rylander, R. & Larson, L. (1983). Levels of gram-negative bacteria, Aspergillus fumigatus, dust and endotoxin at compost plants. Applied Environmental Microbiology 45, 1501–1505. Crook, B., Bardos, R. P. & Lacey, J. (1988). Domestic waste composting plants as sources of airborne micro-organisms. U.K. Department of Environment, grant no. 1/MS/126/209/85. Dahlqvist, M. & Ulfvarson, U. (1994). Acute effects on forced expiratory volume in one second and longitudinal change in pulmonary function among wood trimmers. American Journal of Industrial Medicine 25, 551–558. Donham, K. J., Haglind, P., Peterson, Y., Rylander, R. & Belin, L. (1989). Environmental and health studies of farm workers in Swedish swine confinement buildings. British Journal of Industrial Medicine 46, 31–37. Gordon, T. & Harkema, J. R. (1995). Cotton dust produces an increase in epithelial mucosubstances in rat airways. American Journal of Respiratory Critical Care Medicine 151, 1981–1988. Ja¨ppinen, P., Haahtela, T. & Liira, J. (1987). Chip pile workers and mould exposure. Allergy 42, 545–548. Johnson, C. M., Hanson, M. N. & Rohrbach, M. S. (1986). Endothelial cell cytotoxicity of cotton bracts tannin and aqueous cotton bracts extract: Tannin is the predominant cytotoxin present in aqueous cotton bracts extract. Environmental Health Perspectives 66, 97–104. Lundholm, M. & Rylander, R. (1980). Occupational symptoms among compost workers. Journal of Occupational Medicine 22, 256–257. Malmros, P., Sigsgaard, T. & Bach, B. (1992). Occupational health problems due to garbage sorting. Waste Management & Research 10, 227–234. Milanowski, J. & Dutkiewicz, J. (1992). Effect of organic dust-derived microbial agents on the function of alveolar macrophages in vitro. Polish Journal of Immunology 17, 353–366. Millner, P. D., Marsh, P. B., Snowden, R. B. & Parr, J. F. (1977). Occurrence of Aspergillus fumigatus during composting of sewage sludge. Applied Environmental Microbiology 34, 765–772. Nakasaki, K., Sasaki, M., Shoda, M. & Kubota, H. (1985a). Change in microbial numbers during thermophilic composting of sewage sludge with reference to CO2 evolution rate. Applied Environmental Microbiology 49, 37–41. Nakasaki, K., Shoda, M. & Kubota, H. (1985b). Effect of temperature on composting of sewage sludge. Applied Environmental Microbiology 50, 1526–1530. Nakasaki, K., Kato, J., Akiyama, T. & Kubota, H. (1987). Transformations of water soluble

196

V. Roepstorff & T. Sigsgaard

organic materials during thermophilic composting of sewage sludge. Journal of Fermentation Technology 65, 675–681. Nakasaki, K. & Akiyama, T. (1988). Effects of seeding on thermophilic composting of household organic waste. Journal of Fermentation Technology 66, 37–42. Olenchock, S. A., Sorenson, W. G., Kullman, G. J. & Jones, W. G. (1991). Biohazards in composted wood chips. In Biodeterioration and biodegradation 8 (H. W. Rossmore, ed.), London: Elsevier Applied Science, 481–483. Rylander, R., Haglind, P. & Lundholm, M. (1985). Endotoxin in cotton dust and respiratory function decrement among cotton workers in an experimental cardroom. American Review of Respiratory Disease 131, 209–213. Sigsgaard, T., Bach, B., Taudorf, E., Malmros, P. & Gravesen, S. (1990). Accumulation of respiratory disease among employees in a recently established plant for sorting refuse. Ugeskr Læg 152/35, 2485–2488. Sigsgaard, T., Hansen, I. & Sherson, D. (1992a). Asthma, hyperreactivity, and ODTS among recycling workers. European Respiratory Journal 5 (Suppl), 407s–408s. Sigsgaard, T., Pedersen, O. F., Juul, S. & Gravesen, S. (1992b). Respiratory disorders and atopy in cotton, wool, and other textile mill workers in Denmark. American Journal of Industrial Medicine 22, 163–184. Sigsgaard, T., Roepstorff, V., Tuxford, A. F. & Hoult, B. (1993). Cytotoxicity of Bacillus species to vero cells. Proc. Seventeenth Cotton Dust Research Conference (L. N. Domelsmith, R. R. Jacobs & P. J. Wakelyn, eds.), National Cotton Council, Memphis, TN, U.S.A. 258–260. Sigsgaard, T., Malmros, P., Nersting, L. & Petersen, C. (1994a). Respiratory disorders and atopy in Danish refuse workers. American Journal of Respiratory Critical Care Medicine 149, 1407–1412. Sigsgaard, T., Roepstorrf, V. & Tuxford, A. F. (1994b). Cytotoxicity of cotton dust and dust components to A549 and VERO cells. Proc. Eighteenth Cotton Dust Research Conference (R. R. Jacobs, P. J. Wakelyn & L. N. Domelsmith, eds.), 322–325. Weber, S., Kullman, G., Petsonk, E. et al. (1993). Organic dust exposures from compost handling: case presentation and respiratory exposure assessment. American Journal of Industrial Medicine 24: 365–374.