Mutagenesis, Vol. 15, No. 5, 391-397,
September 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Mutagenicity of diesel exhaust particles from two fossil and two plant oil fuels
1 Centre of Environmental and Occupational Medicine, Georg-August-University, Waldweg 37, D-37073 Göttingen, 2 Institute of Biosystems Engineering, Federal Agricultural Research Centre, Bundesallee 50, D-38116 Braunschweig and 3 University of Applied Sciences Coburg, Friedrich-Streib-Strasse 2, D-96450 Coburg, Germany
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Abstract |
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Particulate matter of diesel engine exhaust from four different fuels was studied for content of polynuclear aromatic compounds and mutagenic effects. Two so-called biodiesel fuels, rapeseed oil methylesters (RME) and soybean oil methylesters (SME), were compared directly with two fossil diesel fuels with the normal (DF) and a low sulfur content (LS-DF). Diesel exhaust particles were sampled on filters from the diluted and cooled exhaust of a test engine at five different speeds and loads. Filters were weighed for total particulate matter, Soxhlet extracted with dichloromethane and the content of insoluble material determined. The soluble organic fraction was analysed for polynuclear aromatic compounds. Mutagenicity was determined using the Salmonella typhimurium/mammalian microsome assay with strains TA98 and TA100. Compared with DF, the exhaust particles of LS-DF, RME and SME contained less insoluble material, which consisted mainly of the carbon cores of diesel exhaust particles. The concentrations of individual polynuclear aromatic compounds varied widely among the different exhaust extracts, but total concentrations of the compounds were approximately double for DF and SME compared with LS-DF and RME. In TA98 significant increases in mutation rates were obtained for the soluble organic fractions of all fuels for engines running at full speed (load modes A and D), but for DF revertants were 2- to 10-fold more frequent as compared with LS-DF, RME and SME. Revertant frequencies for DF and partly for LS-DF were also elevated in TA100, while RME and SME gave no significant increase in mutations. The results indicate that diesel exhaust particles from RME, SME and LS-DF contain less black carbon and total polynuclear aromatic compounds and are significantly less mutagenic in comparison with DF. A high sulfur content of the fuel and high engine speeds (rated power) and loads are associated with an increase in mutagenicity of diesel exhaust particles.
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Introduction |
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The first diesel engine fuelled with a plant oil (peanut oil) was presented at the world exhibition in Paris in the year 1900 (Diesel, 1913
). Since the oil crisis in 1973, plant oils, mainly from rapeseed and soybean, have been investigated as an alternative, renewable source of liquid fuels. Whereas combustion of the crude oils caused severe damage to normal diesel engines, it was proved that rapeseed oil methylesters (RME) are an adequate substitute for fossil diesel fuel (DF) (Vellguth, 1983; Petersen et al., 1996). Emissions from RME fuelled engines are strikingly different from DF emissions (Krahl et al., 1996a). In the USA research has also focused on soybean oil methylesters (SME) (Schumacher et al., 1996; McDonald and Spears, 1998). Both RME and SME are referred to as biodiesels. The following equation shows the general transesterification of plant oils to the corresponding methyl esters:
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A high sulfur content of fossil DF damages the active surface of oxidation catalysts developed to reduce emissions from diesel engines. Therefore, sulfur in common diesel fuel has been lowered from approximately 2000 to 200–500 p.p.m. in recent years, and there are also fuels available with the sulfur content reduced further down to 1 p.p.m. (Sjögren et al., 1996).
Diesel engine exhausts were classified as carcinogenic to experimental animals and as probably carcinogenic to humans by the International Agency for Research on Cancer (IARC, 1989) and several other scientific and governmental institutions (Health Effects Institute, 1999). The carcinogenic effect of diesel exhaust exposure is mainly ascribed to the inhalation of particles (Scheepers and Bos, 1992b; Health Effects Institute, 1999). The black carbon cores of diesel exhaust particles adsorb many known or suspected mutagens and carcinogens, e.g. polynuclear aromatic compounds, onto their surfaces (Huisingh et al., 1978; Scheepers and Bos, 1992a). Due to their median dynamic diameter (0.01–0.3 µm) most of the particles are readily inhaled and ~10% are deposited in the alveolar region of the lung (Scheepers and Bos, 1992b).
In animal studies lung tumours were observed reproducibly in rats but not in other rodents after chronic inhalation of diesel engine exhausts at high concentrations (Heinrich et al., 1986; Mauderly et al., 1987). The carcinogenicity of diesel engine exhausts was attributed to a tumour promoting effect of the particles, because inhalation of carbon black and titanium dioxide produced similar results in rats (Heinrich et al., 1995; Nikula et al., 1995). Nevertheless, the formation of DNA adducts has also been observed in rats after inhalation of diesel exhaust particles (Wong et al., 1986; Gallagher et al., 1994), indicating initiation of carcinogenic effects by the soluble organic fraction of diesel exhaust particles. However, the relevance of the studies in rats is questionable for humans, because rats developed tumours only when the lung was `overloaded' with particles. Furthermore different tissues were involved: tumours of the alveolar region were induced in rats, whereas bronchial carcinomas occurred in man (Pott and Roller, 1997; Mauderly, 1998).
Epidemiological studies revealed significant relative risks of 1.2–1.6 for lung cancer after long-term occupational exposure to high concentrations of diesel engine exhausts (reviewed in Mauderly, 1994; Health Effects Institute, 1995, 1999; Bhatia et al., 1998). In two recently published German studies even higher risks were observed for workers in the potash mining industry (RR 2.2, 95% CI 0.8–6.0) and heavy equipment operators (OR 2.31, 95% CI 1.44–3.70) who had been highly exposed to diesel engine exhausts (Säverin et al., 1999; Brüske-Hohlfeld et al., 1999).
Diesel engine exhausts also contribute substantially to environmental air pollution with particles. A recently published prospective cohort study reported a strong association of particulate matter (PM10) with lung cancer for male non-smokers (RR 2.38, 95% CI 1.42–3.97). Independently of PM10 , sulfur dioxide, another combustion product from diesel engines, showed strong associations with lung cancer mortality for males and females (Abbey et al., 1999).
Organic extracts of diesel exhaust particles collected on filters act as mutagens in bacterial and mammalian in vitro assays. Most investigations have been performed using the Salmonella typhimurium/mammalian microsome assay (Huisingh et al., 1978; Claxton, 1983). To study the chemical and biological characteristics of diesel exhaust particles from SME, RME and a low sulfur content diesel fuel (LS-DF), the polynuclear aromatic compound content was determined by HPLC and mutagenicity was tested in the Salmonella typhimurium/mammalian microsome assay (Ames test). The results were compared directly with those of diesel exhaust particles from a diesel fuel with a normal content of sulfur (DF). The influence of different conditions during the combustion process was investigated by altering the speed and load of the test engine.
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Materials and methods |
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Fuels and chemicals
RME was from Connemann (Leer, Germany), LS-DF from Beta Refinery (Wilhelmshaven, Germany) and DF from Shell AG (Hamburg, Germany). SME was kindly provided by L.Schumacher (University of Missouri, St Louis, MO). The fuels were analysed and met the specifications. In Germany biodiesel quality has to meet standard E DIN 51606. The sulfur content of DF was 370 p.p.m. and of LS-DF was 1 p.p.m. The biodiesel fuels had contents of sulfur below the detection limit of 0.1 p.p.m. DF contained 20.6 vol% aromatic compounds and LS-DF 4.9 vol%. No aromatics were detected in RME and SME.
Nutrient media and most chemicals for the mutagenicity test system were obtained from Difco Laboratories (Detroit, MI) and Serva (Heidelberg, Germany). Methyl methanesulfonate (CAS no. 66-27-3), 2-aminofluorene (CAS no. 153-78-6) and ß-naphthoflavone (CAS no. 6051-87-2) were obtained from Aldrich (Milwaukee, MN) and phenobarbital (CAS no. 50-06-6) from Sigma (Deisenhofen, Germany). Dimethyl sulfoxide (DMSO), spectrometric grade (CAS no. 67-68-5) was provided by Merck (Darmstadt, Germany).
Engine test procedures and sampling of particulate
The test engine was an air cooled one cylinder four stroke diesel engine with direct injection (Farymann 18 D, 4.2 kW). Load was provided using an engine dynamometer. To investigate the influence of different speeds and loads on the mutagenicity of diesel exhaust particles, the stationary five mode test (Figure 1) was chosen as the engine test cycle. It simulates the typical load modes of agricultural tractors in Germany (Welschof, 1981). Due to its easy and rapid handling, this stationary engine was suitable for direct comparison of the emissions of the four tested fuels, although it was not equipped with a catalytic converter and did not comply with current EU emission standards in the 13 mode test (ECE R49) when fuelled with DF.
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During the runs the exhaust gas was led through a dilution tunnel (length 5.50 m, diameter 0.5 m) and the particulate matter of each run (sampling time 20 min) was collected onto one filter. Samples were isokinetically taken from the 1:10 diluted and cooled (<50°C) exhaust on glassfibre filters coated with PTFE (Teflon) (T60A20; Pallflex Products, Putnam, CT). Each mode of the test cycle (A, B, C, D and E) was tested three times with each fuel, resulting in a total of 60 filters. The filters were conditioned (20°C, relative humidity 50%) and weighed before and after sampling to determine the total particulate matter. Filters were stored at 5°C and the three filters for each load mode using the same fuel were pooled and extracted with 150 ml dichloromethane (Merck, Darmstadt, Germany) in a Soxhlet apparatus (Brand, Wertheim, Germany) for 12 h in the dark to extract the soluble organic fractions. For further evaluation and comparison with results of other studies, 50 mg of a standard reference material of diesel particles (SRM 1650) developed and distributed by the US National Institute of Standardisation were extracted in parallel with the other samples and analysed using the same procedures. The extracts were reduced by rotary evaporation (Heidolph, Kehlheim, Germany) and dried under a stream of nitrogen. The residues were redissolved in DMSO for analysis of polynuclear aromatic compounds and for the mutagenicity test.
HPLC analysis of polynuclear aromatic compounds
The four extracts of load mode A were analysed for the 16 polynuclear aromatic compounds identified by the US Environmental Protection Agency as priority pollutants (EPA 610, polynuclear aromatic compounds mix; standard supplied by Supelco, Bellefonte, PA). The analysis of extracts of load mode A was chosen since it has the highest impact (31%) on the weighted average of the five mode test. HPLC analysis was carried out according to Supelco Application Note no. 99 (Supelco, Literature no. 396099) on a Kontron HPLC system with UV-DAD detection (Kontron, Neufahrn, Germany). The compounds in the EPA test mix dissolved in DMSO were calibrated in the range 5–4000 ng/100 µl injection volume.
Initial peak identification in the sample extracts was achieved by comparison of retention times with the authentic standards. Subsequently, defined amounts of the authentic standards were added and checked for co-elution with the predefined peaks. Positively identified peaks were quantified.
Mutagenicity assay
The Salmonella typhimurium/mammalian microsome test (Ames et al., 1973) detects mutagenic properties of a wide spectrum of chemicals by reverse mutation of a series of Salmonella typhimurium tester strains. The Ames test is the most frequently used test system world wide to investigate mutagenicity of complex mixtures like combustion products. This study employed the revised standard test protocol (Maron and Ames, 1983) with tester strains TA98 and TA100. A detailed description of some modifications has been published previously (Bünger et al., 1998a).
Briefly, each dry extract was redissolved in 4 ml of DMSO immediately before use and the following dilutions (with DMSO) were tested: 1.0, 0.5, 0.25, 0.125 and 0.06. To 2 ml of molten top agar were added 100 µl of the test concentrations and 100 µl of a fresh overnight culture containing 1–2x108 microorganisms. These were mixed gently and poured onto minimal agar plates. The mutagens methyl methanesulfonate (1 µl/plate) and 2-aminofluorene (10 µg/plate) were used as positive controls. Each extract was tested in duplicate. The assays were repeated within 2 weeks.
Tests were performed with and without metabolic activation by the microsomal mixed function oxidase system (S9 fraction). For test series with exogenous metabolic activation, 500 µl of 4% S9 was additionally pipetted into the mixture. Higher concentrations of S9 are known to reduce the number of revertants (Courtois et al., 1992; Bünger et al., 1998a). Preparation of the liver S9 fraction from young male Sprague–Dawley rats was carried out as described by Maron and Ames (1983). For induction of liver enzymes, phenobarbital and ß-naphthoflavone (5,6-benzoflavone) were used instead of Arochlor 1254 (Matsushima et al., 1976). The activity of cytochrome P450 enzymes was determined using a test battery of four kinetic assays converting different substrates to resorufin (Schulz-Schalge et al., 1991; Edwards et al., 1994; Schulz et al., 1996). Protein content was determined using the BCA protein kit (Pierce, Rockford, IL) with 20 mg/ml microsomes. The results demonstrate that both CYP1A1/1A2 and CYP2B1/2 were successfully induced in the S9 mix used for the experiments (Table I).
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The number of revertant colonies on the plates was recorded after 48 h incubation in the dark at 37°C. The background bacterial lawn was checked regularly by microscopy, as high doses of the extracts proved to be toxic to the tester strains, resulting in a thinning out of the background. Counting was performed using an electronic colony counting system (Cardinal, Perceptive Instruments, Haverhill, UK) and 10% of the plates were checked routinely by hand counting. According to the criteria given by Maron and Ames (1983), results were considered positive if the number of revertants on the plates containing the test concentrations was more than double the number of spontaneous revertants and a reproducible dose–response relationship was observed. The number of revertants was calculated from the slope of the initial linear portion of the dose–response curve (Krewski et al., 1992
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Results |
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The collected masses of total particulate matter differed widely depending on the fuels. Under all engine load modes (A–E) the collected masses from RME and SME exhaust were substantially higher than those from DF. LS-DF had the lowest emissions of total particulate matter. Higher percentages of the sampled masses were extractable from the filters for the biodiesel fuels than from those for petroleum diesel fuels (Figure 2
). The resulting soluble organic fractions were analysed for polynuclear aromatic compounds and tested for mutagenic effects.
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The contents of polynuclear aromatic compounds of the soluble organic fractions from RME, SME and DF under load mode A were determined using the EPA test mix as a standard and compared with a reference material of diesel particulate matter (SRM 1650) from the National Institute of Standardisation of the USA. DF and SME showed significantly higher total concentrations of polynuclear aromatic compounds than RME and LS-DF. Compared with reference levels of SRM 1650 (evaluated in the IPCS collaborative study by Savard et al., 1992), our results for DF and LS-DF were slightly higher, while RME and SME had lower contents of polynuclear aromatic compounds per gram of particles (Table II
). As the fuels led to highly different emissions of total particulate matter, results were recalculated on the basis of particle emissions per hour of engine running time for a more exact comparison between the tested extracts (Figure 3).
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For comparison with SRM 1650, evaluated in the IPCS collaborative study on complex mixtures (Claxton et al., 1992
), revertants per milligram particles were calculated from linear scatter plots for all loads with and without metabolic activation (Tables III and IV). In the absence of S9, our results with SRM 1650 were higher in TA98 as compared with the reference data but were within the 80% margins of the certification document. In the presence of S9 the results were below the standard deviation for TA98 and for TA100. This difference was possibly due to modification of the induction method for the rat liver enzymes (Matsushima et al., 1976).
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Due to the different emissions of total particulate matter from the tested fuels, for direct comparison the calculation of revertants per hour of engine running time was chosen, which is less dependent on the amounts of total particulate matter. A direct comparison of revertants per milligram particulate matter would have resulted in an overestimation of the benefit of fuels with high emissions of particles (RME and SME) and an underestimation of the benefits of LS-DF, emitting less particulate matter. In the tester strain TA98 without S9 activation a significant increase in mutation rate was obtained for all fuels under load modes A and D. However, for DF the revertant frequency was 2- to 10-fold higher as compared with LS-DF, RME and SME. Under load modes B, C and E only the soluble organic fraction of DF increased the number of revertants significantly. Compared with results without S9, assays in the presence of S9 led to an additional increase in revertants for the biodiesel fuels under load mode A and to a slight increase for DF under load modes D and E (Figure 4
).
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In TA100 without S9 a significant increase in revertants was induced by DF under all load modes, while LS-DF was effective only under load modes A, B and D. The biodiesel fuels did not increase the number of revertants significantly. Testing with metabolic activation lowered the number of revertants induced by the soluble organic fraction of DF under all load modes (Figure 5
).
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A dose-dependent increase but no doubling of mutations was observed in most assays, which therefore were considered not to show a significant response of revertants. In addition, under load modes B, C, D and, to a lesser extent, E, the SME extracts were toxic to the tester strains at high concentrations. The soluble organic fraction of RME showed a slight toxicity under load modes D and E.
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Discussion |
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As recently stated in literature reviews, the combustion of biodiesel fuels leads to a substantial reduction in solid material (insoluble carbonaceous fraction of total particulate matter) and polynuclear aromatic compounds in diesel exhaust particles compared with DF (Krahl et al., 1998
; Mauderly, 1998). These observations were supported by our results, although the analysis of polynuclear aromatic compounds was restricted to extracts under load mode A and the 16 compounds of the EPA test mix. However, the total particulate matter is increased in some stationary engine test cycles, as in the present study (Krahl et al., 1998). This increase in the soluble organic fraction is most probably due to unburned plant oil methylester, because the tested engines were optimized for combustion of DF. A more efficient combustion of biodiesel fuels can be achieved by altering the engine management systems (Syassen, 1998). The more than 10-fold higher mutation rates of the particulate matter of the reference material (SRM 1650) cannot be explained. A possible reason is the different sampling conditions. The SRM 1650 material was collected from the heat exchangers of a dilution tube facility, following 200 engine h of particle accumulation of heavy duty diesel engines, whereas the particles of the tested fuels were sampled on filters from diluted exhaust. However, the results for mutagenicity of SRM 1650 were produced only for validation of our extraction method and the mutagenicity assay and not for direct comparison with other results.
The substantially lower mutagenicity of the soluble organic fraction from diesel exhaust particles of LS-DF as compared with DF was probably due to the much lower content of sulfur and aromatic compounds. Very detailed studies on the fuel characteristics and exhaust emissions of 10 different fossil diesel fuels with sulfur contents ranging from 1 to 2000 p.p.m. showed a strong positive correlation of the sulfur content of the fuels with the mutagenic response of the extracts (Sjögren et al., 1995, 1996). Despite the very different fuel characteristics of SME and RME compared with DF and LS-DF, the lower mutagenicity of diesel exhaust particles from the biodiesel fuels is considered to be partially a result of their extremely low sulfur content as well (Bagley et al., 1998). A possible mechanism of the contribution of sulfur to mutagenic effects of extracts of diesel exhaust particles is the production of polynuclear aromatic compounds containing sulfur in the process of combustion. Yet there are no studies available on mutagenic effects of such compounds from diesel exhaust particles. The sulfur-containing polynuclear aromatic compounds chryseno [4,5-b,c,d]thiophene and its sulfone metabolite chryseno[4,5-b,c,d]thiophene-4,4-dioxide were less mutagenic to TA98 and TA100 in the presence of S9 than the structural analogue benzo[a]pyrene (Sinsheimer et al., 1992).
In addition, biodiesel fuels contain almost no aromatic compounds, whereas fossil diesel fuels include 3–40 vol% aromatics, including polynuclear aromatic compound contents up to 340 mg/l (Sjögren et al., 1995; McDonald and Spears, 1998). Mutagenicity of extracts of diesel exhaust particles increases with content of aromatic compounds of the fuels (Crebelli et al., 1995). Sjögren et al. (1996) observed a significant correlation of mutagenic effects with the content of polynuclear aromatic compounds of the fuels, particularly of picene, phenanthrene, 2-methylanthracene, 3-methylphenanthrene and fluoranthene. This is probably a second explanation for the observed substantial reduction in mutagenicity of RME and SME versus DF in the present study.
The results indicate a strong dependency of the number of revertants on the number of revolutions of the engine (speed). Under load modes A and D (full speed) substantially higher mutagenic effects of the extracts of diesel exhaust particles were observed than under load modes B and C (moderate speed) or E (idling). Similar results were obtained by our study group in a previous study on a passenger car with a turbocharged diesel engine tested on a chassis dynamometer. A reduced mutagenicity for DF and RME was observed in the American FTP-75 test cycle (maximum speed 91.2 km/h) as compared with the European MVEG-A test cycle (maximum speed 120 km/h) in strain TA98 and TA100 (Bünger et al., 1998a).
The results for DF in strain TA98 in the presence of S9 indicate production of more direct acting mutagens under high loads, while under light duties and running idle more indirect acting mutagens are produced. Direct acting mutagenicity is attributed to nitroaromatic compounds in diesel exhaust particles (Pederson and Siak, 1981; Ohe, 1984; Tokiwa and Onishi, 1986). In recent years additional nitrated polynuclear aromatic compounds were proven to have direct acting mutagenic effects and were identified or suspected to occur in diesel exhaust particles (Sera et al., 1994; Ball et al., 1995; Upton and Upton, 1999).
The extracts of the plant oil methylesters showed no significant mutagenic activity under most load modes at the tested concentrations. Tests with even higher concentrations were not possible due to toxicity. A higher toxicity of the soluble organic fraction of biodiesel fuels as compared with DF was reported earlier by our study group (Bünger et al., 1998a,b). It is possibly caused by a higher content of carbonyls in diesel exhaust particles of the biodiesel fuels (Krahl et al., 1996b).
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Notes |
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4 To whom correspondence should be addressed. Tel: +49 551 394950; Fax: +49 551 396184; Email: jbuenge{at}gwdg.de
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References |
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Received on January 14, 2000; accepted on January 18, 2000.
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