Mutation Research, 245 (1990) 177-183

177

Elsevier

MUTLET 0416

Evaluation of the mutagenicity of combustion particles from several common biomass fuels in the Ames/Salmonella microsome test Douglas A. Bell* and Richard M. Kamens Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27514 (U.S.A.) (Received 16 January 1990) (Revision received 18 June 1990) (Accepted 19 June 1990)

Keywords: Biomass fuels; Indoor air; Combustion particles; Dichloromethane extracts

Summary We have evaluated the mutagenicity of dichloromethane extracts of combustion particles from several biomass fuels that are commonly used in developing countries in Salmonella strains TA98+__$9 and TA100 + $9. Combustion-particle extracts from dried cow dung and crop residue exhibited mutagenic potencies similar to wood-smoke extracts (0.0-1.0 rev.//zg extract). However, extracts from coconut-shell-smoke particles showed relatively potent direct-acting mutagenicity (1.6 rev.//~g, TA98 - $9). Results from testing this sample in nitroreductase- and acetylase- deficient strains TA98NR and TA98 (1,8-DNP-6) revealed no contribution from nitroarenes.

Biomass fuels are significant energy sources for heating and cooking in the developing countries of the world. Historically, wood has been the primary biomass fuel but with deforestation in the densely populated areas of Asia and Africa, the use of agricultural residues such as dried animal dung and crop by-products has become increasingly common (Barnard, 1985). Hughart (1979) estimated that more than 800 million people in the developing * Present address: Laboratory of Biochemical Risk Analysis, C3-03, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.). Correspondence: Dr. D.A. Bell, C3-03, Laboratory of Biochemical Risk Analysis, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.).

countries routinely use agricultural residues for fuel. These fuels, including dried cow dung, coconut shell and husk, rice and other cereal straws, tobacco stalks, and jute sticks are often burned in open fires or unvented cookstoves inside the home. The resulting human exposures to combustion gases and particles from indoor burning can be 100-fold higher than pollutant levels typically experienced in polluted urban air (Smith et al., 1983; Mumford et al., 1987). Human health effects from exposure to high indoor-smoke levels include acute respiratory disease, chronic bronchitis and other chronic respiratory illnesses, and infant mortality (de Koning et al., 1985; Pandey, 1985). The genotoxic effects from exposure to smoke from agricultural

0165-7992/90/$ 03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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residues are largely unknown. However, recent work has established that smoke from wood, peat, cereal straw and rice straw combustion is mutagenic in Salmonella (Kamens et al., 1984; Bell et al., 1985; Mast et al., 1984; Alfhiem et al., 1985). In addition, wood-smoke extracts have been found to initiate tumors in mouse-skin painting studies (Mumford et al., 1987). We report here on the mutagenicity of combustion particle extracts from several biomass fuels: dried cow dung or coconot husk and shell that were burned in a simulated village hut, East West Center (EWC), Hawaii, and dried cow dung or crop residue burned at the Central Institute of Agricultural Engineering (CIAE) in Bhopal (India). Dichloromethane extracts of the combustion particles were evaluated for mutagenicity using the Salmonella tester strains TA98, TA98NR, TA98(1,8DNP6) and TA100 with and without rat-liver microsomal activation ($9). Materials and methods

Sample generation. Fuels were burned in an Indian-styled cook stove (chula) made from fire bricks and burned at a burn rate of 5-15 kg/h (Smith et al., 1983). The burning and sampling of these fuels took place in a small hut, typical of villages in developing countries, located either at CIAE or at EWC (Smith et al., 1984). Combustion particles were collected for 16-50 min on glassfiber filters using a Hi-Vol sampler at a flow rate of 0.7 m3/min) located - 1.5 m from the stove. The particle concentration in the room at the time of sampling ranged from 10 to 50 m g / m 3. The filters were soxhlet extracted for 18 h with dichloromethane (DCM), and the extracts were concentrated by rotary and dry nitrogen evaporation. Aliquots of the concentrated DCM extracts were evaporated on preweighed pans and weighed to determine the extracted mass. Concentrated extracts were solvent exchanged into dimethyl sulfoxide (DMSO) for the mutagenicity assay. Mutagenicity assay. Samples were tested by the plate-incorporation assay with or without a 10°70 rat-liver homogenate mixture ($9) prepared from

Aroclor-induced rats as described by Maron and Ames (1983). Samples were tested twice (unless noted otherwise), and each dose was tested in triplicate with and without $9. Revertant colonies were counted after incubation for 72 h at 37°C. Mutagenic potency was determined by performing a simple linear regression on all dose-response data points unless toxicity was observed. The assays were conducted in the laboratories of the Genetic Toxicology Division, U.S. Environmental Protection Agency, Research Triangle Park, NC. Results and discussion

Mass recovery. The percent extractable mass of the two samples produced at the East-West Center were substantially different. Dichloromethane extracted 97°/0 (370.0 mg extract/379.8 mg particles) o f the mass of the cow-dung-smoke particles and 35.3°7o (50.0 mg extract/141.8 mg particles) of the coconut-shell-smoke particles. In studies at UNC it was observed that soft, slow-burning fuels often produce particles with higher extractable organic content than do hard, fast-burning fuels. A similar relationship was also observed when wood or peat were combusted at low and high burn rates (Knight, 1981; Bell et al., 1984). Combustion particles generated under high burn rate conditions or from fast-burning fuels have undergone more complete combustion and, thus, have a lower organic content. Unfortunately, it was not possible to determine the particle mass loading on the filter samples collected in India because the filters were not preweighed before sampling. Thus, although the extract mass was determined as described in Materials and Methods, the percent extractable mass could not be calculated. Mutagenicity.

The tester strains TA98 and TA100 were chosen for use because these strains have been shown to be sensitive to combustion emissions and ambient air particle samples (Daisey et al., 1980). The mutagenicity dose-response curves for the particle extracts are shown in Figs. 1-4. The slope values (potency) for each sample, expressed as revertants per #g o f extract, and the

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Fig. 3. M u t a g e n i c i t y o f I n d i a n dried c o w - d u n g - c o m b u s t i o n particle extracts. N e g a t i v e a n d positive c o n t r o l d a t a for I n d i a n s a m p l e s ( m e a n o f 3 plates): T A 9 8 , E x p t . 1, D M S O - $9 = 24 + 2; D M S O + $9 = 38 + 1; 2 - n i t r o f l u o r e n e (3/zg) = 222 + 7; 2 - a m i n o a n t h r a c e n ¢ (0.5 t~g + $9) --- 609 + 10; E x p t . 2, D M S O - $9 = 24 + 2; D M S O + $9 = 47 + 4; 2 - n i t r o f l u o r e n e (3 pg) = 379 ± 10; 2 - a m i n o a n t h r a c e n e (0.5 p g + $ 9 ) = 6 9 9 + 15; T A 1 0 0 , E x p t . 1, D M S O - S 9 = 1 1 7 + 11; D M S O + $ 9 = 111 + 7 ; s o d i u m azide (3 #g) = 6 4 4 + 18; 2 - a m i n o a n t h r a c e n e (0.5 pg + $9) = 447 + 24; E x p t . 2, D M S O - $9 = 200 + 7; D M S O + $9 = 169:1:11; s o d i u m azide (3/zg) = 362:1: 19; 2 - a m i n o a n t h r a c e n e (0.5 ~g + $9) = 625 + 17.

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TABLE 1 C O M P A R A T I V E M U T A G E N I C P O T E N C Y OF BIOMASS FUELS Fuel

Revertants per p.g extract ± S.E. a TA98

TA 100

-$9

+$9

-$9

+$9

0.04 ± 0.02

0.63 ± 0.04

0.02 ± 0.04

0.15 ± 0.05

C o c o n u t shell

1.56 ± 0.14

1.58 ± 0.12

2.61 ± 0.17

0.83 ± 0.10

I ndian Dried cow dung

0.28 ± 0.04

1.00 + 0.24

0.63 -+ 0.06

0.91 ± 0.28

I ndian Crop residue

0.04 ± 0.01

0.28 ± 0.04

0.12 _+ 0.04

0.42 +_ 0.08 b

Pine ¢

0.48 ± 0.08

1.20 ± 0.14

NA f

NA

Red oak a

0.09 ± 0.03

0.81 ± 0.12

0.21 ± 0.05

0.31 ± 0.05

Peaff

0.25

0.38

NA

NA

E- W CenT:er Dried cow dung E-W Center

a Standard error of the slope estimate. b Regression based on single assay, 3 replicate plates at 6 doses. ¢ Data d Data e Data f Data

from Bell and Kamens, 1986; slope values are + 95% confidence intervals. from Rives, 1983. from Bell et al., 1985. not available.

standard error of the slope estimates are given in Table 1. The East-West Center dung-smoke extract produced a dose-dependent mutagenic response ( - 10-fold higher than the spontaneous level at the highest dose) in strain TA98 in the presence of $9 activation (Fig. 1). In TA100 (+ $9) there also was a dose-dependent mutagenic response, which suggested base-pair substitution activity; however, the maximum revertant counts never reached a doubling of the spontaneous revertant level. The pattern of activity [low direct-acting response ( - $ 9 ) for both strains; frameshift response (TA98) > base-pair response (TA100)] for the East-West Center cow-dung-smoke sample in these two strains and the mutagenicity slope values were similar to wood-smoke-particle extracts previously tested at UNC (Table 1). S9-dependent activity in wood smoke was highly relate_d to polynuclear

aromatic hydrocarbon (PAH) concentration, and the total mutagenic contribution of PAH to wood smoke was typically 12-25070 of the S9-dependent mutagenicity in TA98 (Bell and Kamens, 1986; Kamens et al., 1985; Bel, 1988). Chemical analysis of the cow-dung-particle extracts using HPLC and fluorescence detection suggested PAH levels similar to those of wood smoke ( - 2 0 0 ng benzo[a]pyrene/mg smoke particle). Because no fractionation studies were performed, the mutagenic contribution of PAH to cow-dung-smoke mutagenicity could not be quantitated. The mutagenicity of coconut-shell-smoke particle extracts was qualitatively and quantitatively different from the cow dung smoke as shown in Table 1 and Fig. 2. There was a potent direct-acting mutagenic response in both tester strains. It was of interest that coconut shell smoke showed a reduced level of mutagenic activity in TA100 + $9, whereas

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the presence of $9 had no apparent effect in strain TA98. This indicated that there may be at least two classes of mutagens in this sample: direct-acting, base-pair substitution mutagens (TA100) that were inactivated in the presence of $9; and direct-acting frameshift mutagens (TA98) that were unaffected by $9. However, in a complex mixture, S9-mediated mutagenicity is the sum of the unaffected direct-acting mutagens and any metabolically-activated mutagens present. Direct-acting mutagenicity in combustion particle extracts has often been attributed to the presence of nitroarenes (Lofroth et al., 1981; Scheutzle, 1983; Kamens et al., 1985). We used the Salmonella strains TA98NR and TA98 (1,8-DNP-6) (Rosenkranz et al., 1981), which lack either functional nitroreductase (NR) or acetyl transferase (1,8-DNP-6) enzymes, to indicate if there was a large contribution by nitroarenes to the directacting mutagenicity of coconut smoke. Fig. 2c shows the dose-response curves for this extract in TA98-$9 and the two enzyme-deficient strains. The similarity of all 3 curves suggested little, if any, contribution by nitroarenes to the mutagenicity of this sample. The cow-dung-smoke particles collected at the CIAE in Bhopal (India), were mutagenic with and without $9 in both TA98 and TA100 (Fig. 3). Potencies (slope values) were roughly 4-fold higher in the presence of $9 for both strains (Table 1). The potencies for the India dung smoke were also considerably higher and qualitatively different from the East-West Center dung-smoke sample in that the India dung smoke was considerably mutagenic in TA100. These results were not surprising considering the variables involved in the combustion process. In studies at UNC, it was observed that mutagenic potency can vary over more than an order o f magnitude for a given biomass fuel depending on combustion conditions such as burn rate and fuel moisture content (Bell et al., 1985; Bell and Kamens, 1986). One might ponder the numerous additional dietary variables which could make a particular cow-dung sample more mutagenic than another. Fig. 4 shows the dose-response curves from the

extract of the crop residue smoke in TA98 ___$9 and TA100 + $9. The mutagenic response in TA98 + $9 was relatively low (0.28 rev.//~g, see Table 1) compared to the other samples tested. However, this was a reproducible positive result. As shown in Fig. 4b, there was a dose-dependent mutagenic response in T A 1 0 0 + S 9 and at the highest dose a 2-fold increase over background activity was observed. Until detailed chemical analysis and bioassaydirected fractionation studies are performed, no definite conclusions may be made about the possible contribution of P A H , aromatic amines, nitroarenes, or other more polar compounds to the mutagenicity of these combustion samples. The positive mutagenicity results of these samples indicate a need for further characterization of the genotoxicity of combustion products from these, and other agricultural residue fuels. Furthermore, the magnitude and extent of human exposures to these emissions suggest that, in addition to increased risk of respiratory disease, there may be increased genotoxic or carcinogenic human health risks associated with domestic burning of agricultural residue fuels.

Acknowledgements The authors would like to thank Kirk R. Smith, Resources Systems Institute, East-West Center, Honolulu, Hawaii for providing the samples for this study. We acknowledge the support and encouragement of Drs. Larry Claxton and Joellen Lewtas, Genetic Toxicology Division, U.S. E . P . A . , in the completion of this work. DAB was supported by cooperative agreement CR 812514 from the US-EPA. We thank M. Shrestha and P. Menon, U. of Ha. for technical support during the sampling part o f this study.

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of exhaust from diesel and gasoline powered motor vehicles, Environ. Int., 5, 255-261. Maron, D., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Mast, T.J., D.P. Hsieh and J.N. Seiber (1984) Mutagenicity and chemical characterization of organic constituents in rice straw smoke particulate matter, Environ. Sci. Technol., 18, 338-348. Mumford, J., X.Z. He, R.S. Chapman, S.R. Cao, D.B. Harris, X.M. Li, Y.L. Xian, W.Z. Jiang, C.W. Xu, J.C. Chuang, W.E. Wilson and M. Cooke (1987) Lung cancer and indoor air pollution in Xuan Wei, China, Science, 231,217-220. Pandey, M.R. (1984) Domestic smoke pollution and chronic bronchitis in a rural community of the hill region of Nepal, Thorax, 39, 337-339. Ramdahl, T., and M. Moiler (1983) Chemical and biological characterization of emissions from a cereal burning furnace, Chemosphere, 12, 23-34. Rives, G., M.S. Thesis, Dept. of Envir. Sci. and Eng., Univ. of N. Carolina, Chapel Hill, NC. Rosenkranz, H.S., E.C. McCoy, R. Mermelstein and W.T. Speck (1981) A cautionary note on the use of nitroreductasedeficient strains of Salmonella typhimurium for the detection of nitroarenes as mutagens in complex mixtures including diesel exhausts, Mutation Res., 91, 103-105. Scheutzle, D. (1983) Sampling of vehicular emissions for chemical analysis and biological testing, Environ. Health Perspect., 47, 65-80. Smith, K., A. Aggarwal and R. Dave (1983) Atmospheric Environ., 17, 2343-2362. Smith, K.A., M. Apte, P. Menon and M. Shrestha (1984) Carbon monoxide from cooking stoves: Results from a simulated village kitchen, Third International Conference on Indoor Air Quality and Climate, Aug. 20-24, Stockholm, Sweden Communicated by J.W. Allen