Envlron. Scl. Technol. 1985, 19, 270-273
Ames Assay Chromatograms of Extracts of Diesel Exhaust Particles from Heavy-Duty Trucks on the Road and from Passenger Cars on a Dynamometer Irving T. Salmeen," Robert A. Gorse, Jr., and Wllllam R. Plerson
Research Staff, Ford Motor Company, Dearborn, Mlchlgan 48121 Particles from diluted exhaust of heavy-duty diesel trucks were collected on filters in the Allegheny Mountain Tunnel on the Pennsylvania Turnpike. Dichloromethane extracts of these particles were separated into 65 fractions by normal-phase high-performance liquid chromatography, and Ames assays (TA98 without activation) were carried out on each fraction. The Ames assay chromatograms for the Allegheny Tunnel sample had the same general shape as the chromatograms of dilution tube samples from diesel-powered passenger cars run on a dynamometer. This suggests that the direct-acting mutagens in Allegheny Tunnel samples from heavy-duty diesels are mainly the same as those in laboratory samples from diesel-powered passenger cars and that the relative proportions of these compounds are similar in the two types of samples. If so, then the implication is that differences in the direct-acting Ames assay mutagenicities of unfractionated extracts reflect differences in the absolute concentrations of all of the mutagens and not in the distribution of mutagens. Almost all experiments designed for chemical characterization of diesel engine exhaust particles have involved what we will call laboratory samples, that is, samples collected on filters in a laboratory using a dynamometerdilution tube system ( I ) , For many reasons, summarized elsewhere (2),the validity of laboratory samples as representative of exhaust in the real environment is questionable. With respect to mutagenicity and mutagenic compounds in emissions, several investigators have conducted field experiments (2-4) and chamber experiments (5, 6) in an effort to deal with the validity problem. Pierson and collaborators have attempted to address this question by characterizing particle samples that were collected on filters in the Allegheny Mountain Tunnel on the Pennsylvania Turnpike (2). Traffic composition through this tunnel fluctuates periodically such that samples could be collected during periods dominated by heavy-duty diesel-powered trucks and during periods dominated by spark-ignition vehicles. They found that the heavy-duty diesel particles strongly resembled diluted-exhaust particle samples collected in the laboratory from diesel-powered passenger cars. The criteria for comparison were the following: particle emission rate and size distribution; percent of mass extractable by organic solvents; the liquid chromatograms, molecular weight distributions, and Ames assay mutagenicities of the organic solvent extracts. Of the criteria for comparing samples of diesel exhaust particles, the Ames assay data are especially important to studies of the potential health effects of diesel engine exhaust (1). Unfortunately, the similarity between the Ames assay mutagenicities of the Allegheny Tunnel and laboratory samples does not necessarily imply that the two types of samples contained the same mutagens, for the following reasons. First, Gorse et al. (7) found for 1981 Allegheny Tunnel samples that l-nitropyrene accounted for less than 1% of the TA98 (without activation) Ames assay mutagenicities, compared to more than 10% ob270
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tained in previous studies (8,9)of laboratory samples from various diesel-powered passenger cars. Second, organic solvent extracts of other types of particles, e.g., from wood combustion (10,11) and from ambient airborne particles (10, 12),yielded Ames assay mutagenicities comparable to those obtained for diesel particle extracts, even though their major chemical components were different from those of the diesel particle extracts. In this paper, we describe experiments which suggest that the direct-acting (Le., tissue homogenate is not required for activation) mutagens in the Allegheny Tunnel sample are, indeed, mainly the same compounds as those in laboratory samples from diesel-powered passenger cars and that the relative proportions among these compounds are similar in the two types of samples. In these experiments, dichloromethane extracts of particles from the Allegheny Tunnel and from diesel-powered passenger cars in the laboratory were separated into 65 fractions by high-performance liquid chromatography (HPLC), and Ames assays (TA98 without activation) were carried out on each fraction, thereby generating Ames assay chromatograms as described previously (13). The conclusions presented herein are based on the general shapes of these Ames assay chromatograms and on order-of-magnitude quantitative considerations. The experiments concerned only direct-acting TA98 mutagens in particles collected on filters and extracted into dichloromethane. We were concerned only with the following question: What are the similarities or differences between Ames assay chromatograms of two types of samples-one representative of laboratory conditions and the other more representative of real environmental conditions-both collected and treated as similarily as experimental conditions allowed? The reasons for focusing on direct-acting Ames assay mutagens are discussed in a previous paper (13).
Materials and Methods Four kinds of data were obtained on samples prepared by dichloromethane-Soxhlet extraction of particles collected on filters. One was the Ames assay mutagenicity of the dichloromethane extracts. The second was the distribution of extract mass among 8 fractions obtained by normal-phase HPLC; the third was the distribution of Ames assay mutagenicity among 65 fractions obtained by following the same normal-phase chromatographic scheme but collecting more frequent samples; the fourth was UVabsorbance chromatograms recorded during all of the fractionations. The particle sample from the Allegheny Mountain Tunnel was collected between 2100 eastern daylight time (EDT), July 28, 1981, and 0800 the next morning, in a manner already described (2, 7). The traffic composition was 39.6% spark-ignition vehicles (predominantly passenger cars) and 60.4% diesel vehicles (predominantly heavy trucks, average weight -27 metric tons, with direct-injection 2- and 4-stroke engines, both turbocharged and naturally aspirated). Traffic speed was approximately constant at -80 km/h. The average exhaust dilution ratio
0013-936X/85/0919-0270$01.50/0
0 1985 American Chemical Society
was several hundred and more. Particles were collected on standard (8 in. X 10 in.) HiVol glass fiber filters concurrently beside the right and left lanes several meters inside the exit portal of the eastbound tunnel and also in the intake air going into the tunnel. Average residence time of emissions in the tunnel, from emission to collection, was about 2.5 min. The samples were stored at -78 "C in the dark until time for extraction. The samples were Soxhlet extracted in dichloromethane under nitrogen for 17 h in the dark. Following extraction, the right and left lane tunnel samples were combined, yielding 55.9 mg of extract. (The intake sample was comparatively small, 6.4 mg, and was not analyzed further.) Particle samples from diesel passenger cars were collected on Teflon-coated glass fiber filters (20 in. X 20 in. Pallflex TX40-HI20) by using a chassis dynamometer and dilution tube as described elsewhere (14). Average exhaust dilution ratio was about 7 and average residence time of emissions in the dilution tube about 2 s. The vehicles were run repetitively through the 1974 Federal Test Procedure-Hot cycle by using Phillips Control Diesel no. 2 fuel. The filter samples were extracted with dichloromethane as above. The passenger car data chosen for display in Figure 1were for a 1981Peugeot equipped with a 2.3-L 4-cylinder prechamber naturally aspirated diesel engine and manual transmission. The data are from the same sample as that deaignated S4 in a previous (13) paper. In earlier work from our laboratory on samples from passenger cars (9,13-15),extracts were characterized by analytical-scale normal-phase high-performance liquid chromatography with a W- (254-nm) absorbance detector and the elution solvent program diagrammed in Figure 1A. The UV chromatograms showed eight distinctive periods, as designated by the numbers in the upper part of Figure 1A. When at least 15 mg of extract was fractionated on semipreparative-scale columns by using this solvent program, each of the eight fractions contained enough material for accurate weighing. Thus, the mass distribution among these eight fractions affords an empirical characterization of the extracts. Accordingly, we determined this mass distribution for the Allegheny Tunnel and for passenger car samples. The eight fractions had the following properties (13,15).Fraction 1is comprised of aliphatics and accounts for roughly half of the total extract mass. Fraction 2 is comprised of mostly two- to five-ring unsubstituted and alkyl-substituted PAH and accounts for as much as 10% of the total mass. Fractions 1and 2 are not directly active in the Ames assay. Fractions 3-7 each contain only a few percent of the total mass, but together they contain around 80% of the direct-acting Ames assay activity. The identity of the compounds in these fractions is still under investigation. Fraction 8 contains the remaining portions of the mass and direct-acting Ames assay activity. The compounds in the fraction are very polar, as judged by the polarity of the solvent required to elute them, but they have not yet been identified. In our earlier work (13) with passenger car laboratory samples, and here with the Allegheny Tunnel samples, we determined the distribution of direct-acting Ames assay mutagenicity among the compounds that elute between 17.5 and 49 min of the chromatogram, i.e., the period of the chromatogram that accounts for roughly 80% of the total direct-acting mutagenicity. This was accomplished by collecting fractions every 30 s and carrying out Ames assays on each fraction (63 fractions all together). Two more fractions were collected, but not over 30-s periods. One of these was the material that eluted between 49 and 85 min: this material could not be fractionated into 30-s
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Flgure 1. (A) Complete UV chromatogram (254-nm absorbance) for diesel passenger car sample. Solvent program Is shown by lines superimposed on the UV chromatogram. Abbreviations: dlchloromethane (DCM) and acetonitrile (ACN). Period marked between 17.5 mln and 49 mm 1s that over which fractions were collected every 30 s In order to obtain Ames assay chromatograms shown in lower panels. The eight periods deslgnated by numbers are those over which fractions were collected for mass dlstributlon (seeTable I). These periods were chosen to correspond to main UV-absorbing peaks. (B) Expanded-scale display of UV chromatogram for Allegheny Tunnel and diesel passenger car samples. The arrows with symbols designate the elution times of reference standards run following the chromatography of the samples. These are shown only to indicate the approximate (* 1 mln) elution times of the compounds under the specific solvent program and do not necessarily indicate that the compounds are found in specific fractions. The compounds are the following: NP, 1-nitropyrene; DNP, 1,8-dinltropyrene; 9F, 9-fluorenone; 9-OHF, 9hydroxyfluorene. These chromatograms were recorded while the 30-s fractions were being collected for the Ames assay chromatograms. For both samples, the mass loaded onto the column was about 15.7 mg. The recorder gain and display scales are the same for both plots. (C) Ames assay chromatogram for diesel passenger car sample and (Inset) dose-response data for unfractlonated passenger car (PC) and Allegheny Tunnel (AT) samples; TA98 without tissue homogenate. Spontaneous revertants have been subtracted from the observed number of revertants per fraction. The slope of the line for the passenger car data is 1.3 revlpg; that for the Allegheny Tunnel sample Is 0.5 rev/wg. (D) Ames assay chromatogram for Allegheny Tunnel sample. Letters A, B, C, and 0 are used to facilitate reference in the text to specific fractions. These letter deslgnatlons are the same as those used in ref 13.
periods because the compounds that eluted in the 49-85min period did not separate very well from each other on normal-phase columns. Therefore, this 36-min period was collected as one fraction and the Ames assay carried out on it. We also collected the material that eluted prior to 17.5 min as one fraction and carried out the Ames assay of it in order to be sure that this usually inactive fraction was inactive for every sample. All together 65 fractions were collected. The Ames assay was carried out concurrently on each fraction at a single dose per fraction as described previously (13).The Ames assay chromatograms are histograms obtained by plotting activity per fraction as a function of fraction number for the 63 fractions collected over the 30-9 periods. The reasons for carrying out Ames assays at a single dose per fraction have been disEnviron. Scl. Technol., Vol. 19, No. 3, 1985 271
cussed previously (13). The lack of dose-response data can cause uncertainties when Ames assay data are quantified (131,but the main conclusions herein depend only on the shapes of the Ames assay chromatograms and on order-of-magnitude calculations. The quantitative uncertainties do not affect these main conclusions.
Results and Discussion Over a period of several years, we have obtained UV chromatograms, mass distributions, and Ames assay chromatograms for many laboratory samples from several diesel passenger cars. Here, we want to compare these data with data obtained for Allegheny Tunnel samples. For illustration, we have arbitrarily selected data for one of these laboratary samples. We stress that data for any of the other laboratory samples could have served as well. Figure 1A shows the complete UV chromatogram for a sample from the diesel passenger car. Figure 1B shows an expanded-scale display for the 17-49-min range of the UV chromatogram for this passenger car laboratory sample and for the Allegheny Tunnel sample; this is the range over which the Ames assay chromatograms were obtained. The UV chromatograms were recorded while collecting the 65 fractions used to obtain the Ames assay chromatograms discussed below. The U V chromatogramsfor the laboratory samples from the diesel passenger cars were similar to those for the Allegheny Tunnel samples, with prominent peaks near 3, 13, and 25 min, a nondescript series of relatively weak peaks between 25 and 49 min, and an intense very broad peak starting at 50 min. Only the passenger car samples, however, showed a prominent peak near 18 min; the data now available are insufficient to interpret this difference. There are differences in minor features, all well within the uncertainties attributable to retention time variations inherent in the chromatographic scheme. For example, retention times of neat reference compounds under the same chromatographic scheme varied by at least f l min (13). Furthermore, the retention times and resolution of components in a mixture can depend in unpredictable ways on their concentrations relative to other components in the mixture (so-called matrix effects). The Ames assay chromatograms (Figures 1C,D) are the focus of this study. The figures do not show data for the polar fraction (4!+85 min) because, for reasons noted above under Materials and Methods, it was collected as one fraction rather than in 30-9 subfractions. This polar fraction contained about 17% of the recovered mutagenicity (ie., the 65-fraction sum) in the case of the passenger car sample and about 35% in the case of the Allegheny Tunnel sample. We are not sure that there is a difference between the 17% and the 35% in the polar fractions because of the difficulties, mentioned above (Materials and Methods), in separating polar compounds by normal-phase chromatography. The general shapes of the Ames assay chromatograms for the laboratory (diesel passenger car) and Allegheny Tunnel samples are similar to each other, but the individual fractions in the former (Figure IC) are 3-4 times more active than the corresponding fractions in the latter (Figure 1D). This difference is reflected in the dose-response data for unfractionated extracts (inset to Figure 1C) which show that the unfractionated laboratory sample is about 3 times more mutagenic (revertants (rev) per microgram of extract) than the Allegheny Tunnel sample. The general similarities between the two chromatograms (Figure lC,D) hold also when Figure 1D is compared with chromatograms reported previously (13)for other laboratory samples from diesel passenger cars. These general 272
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similarities among the Ames assay chromatograms strongly suggest that the extracts of the Allegheny Tunnel and laboratory samples contained mainly the same kinds of direct-acting mutagens and in approximately the same proportions to each other. We note furthermore that the Ames assay mutagenicities of unfractionated extracts span a range from 0.5 (Allegheny Tunnel samples) to about 25 rev/pg (sample S3, ref 13)) yet the Ames assay chromatograms all have the same shape; the mutagenicity per chromatographic fraction is roughly proportional to the mutagenicity of the whole extract. It appears that the abundances of the mutagenic compounds in various samples, relative to the rest of the sample mass, vary together. Such behavior leads us to speculate that somehow all of the direct-acting mutagens are chemically related to one another as if, for example, they were all nitroarenes. The only peaks in the Ames assay chromatograms that can be wholly or partly assigned, with any confidence, to specific compounds are the peaks designated by letters A, B, C, and G in Figure 1D. In previous experiments with passenger car laboratory samples, we found that 1-nitropyrene was distributed among fractions A-C. In the one sample (Sl, ref 13) for which both Ames assay chromatography and 1-nitropyrene quantification were carried out, the 1-nitropyrene accounted for about 20% of the mutagenicity of fractions A-C together (Figure 1of ref 13). As we now explain, the measured 1-nitropyrene concentration in the Allegheny Tunnel sample accounts for a comparable percentage of peaks A-C in the Ames assay chromatogram of the Allegheny Tunnel sample. Gorse et al. (7)found that Allegheny Tunnel samples, collected during the same 2-week period and under the same traffic conditions as the Allegheny Tunnel sample examined here, contained 2.3 1.0 ppm of 1-nitropyrene in the CHzClzextracts. The present sample was presumably the same. The mass of sample used to generate the Ames assay chromatogram was 15.7 mg. Thus, the amount of 1-nitropyrene in the sample represented in Figure 1D was 36 f 16 ng. The mutagenicity of 1-nitropyrene in our laboratory was about 1.8 rev/ng (13).Therefore, 1-nitropyrene would have contributed about 65 f 30 revertants to the mutagenicity of the Allegheny Tunnel sample. Fractions A, B, and C (Figure 1D) yielded 90,240, and 120 revertants, respectively. Thus, the 1-nitropyrene in the Allegheny Tunnel sample would account for 14 f 7% of the combined mutagenicity of fractions A-C. Fraction G (Figure 1D) in the Ames assay chromatogram for the Allegheny Tunnel sample corresponds in elution time to a peak that we previously attributed to 1,8-dinitropyrene in the Ames assay chromatograms of passenger car samples. Gorse et al. (7)did not measure dinitropyrene concentrations, but if we attribute peak G of Figure 1D to l,&dinitropyrene, then the Ames assay chromatogram yields an order-of-magnitude estimate of the 1,Sdinitropyrene concentration in the Allegheny Tunnel sample. The mutagenicity of l$-dinitropyrene is between 800 and 1600 rev/ng (13).Fraction G (400 revertants) would then correspond to between 0.25 and 0.5 ng of 1,Sdinitropyrene. This amount of 1,Sdinitropyrene amounts to between 0.02 and 0.04 ppm of extract, or between l/looand l / M that of 1-nitropyrene. In laboratory samples from passenger car diesels for which the dinitropyrene concentrations were high enough to be quantified, the dinitropyrene concentrations were in the order of l/lao that of 1-nitropyrene (13, 16). Thus, the range 0.02-0.04 ppm of 1,8-dinitropyrene estimated for the Allegheny Tunnel sample from the Ames assay chromatogram would be consistent with concentra-
*
Table I. Distribution of Mass among Chromatographic Fractions HPLC fraction 1 2 3 4 5 6 7 8
70 of total massa Allegheny Tunnel passenger car
(53.7 i 0.3)b 10.7 3.3 4.0 1.3 2.1 3.5 21.5
66.8 5.3 2.9 4.4 2.3 1.2 3.5 13.7
a Percentage with respect to total mass recovered from columns. For Allegheny Tunnel sample: 16.82 injected onto column and 15.69 mg recovered. For passenger car sample: 15.95 mg injected and 15.73 recovered. *The uncertainty in every entry is about *0.3 as a result of i 3 0 fig uncertainty in tare mass.
tions expected based on the 1-nitropyrene concentration. In addition to the mutagenicity data described in the foregoing paragraphs, we also determined the distribution of mass among the eight chromatographic fractions as defined under Materials and Methods. The mass distribution (Table I) for the Allegheny Tunnel sample was similar to that of a typical laboratory sample from a diesel passenger car (different sample from the same vehicle as in Figure 1). The differences in absolute mutagen concentrations (0.5 rev/pg for the Allegheny Tunnel sample and 2 rev/pg (data not shown) for this laboratory sample) were not manifested in the mass distribution. Thus, the predominant mutagens in the Allegheny Tunnel sample must have comprised a very small portion of the extract mass. In view of the similarities among Ames assay chromatograms over extremes of experimental conditions, we speculate that experimental variables such as vehicle type, engine type, driving cycle, and laboratory vs. field collections do not have a very large influence on the relative composition of the direct-acting mutagens in the extracts, and therefore, for the purposes of investigating these mutagens, it does not matter whether heavy-duty or light-duty, field or laboratory samples are used. It remains to be seen, however, whether future work with laboratory heavy-duty diesel samples supports this speculdtion. In this study we have not concerned ourselves with artifact mutagenicity, for example, the possibility that arenes already collected on the filter or in the collected particles might react with nitrating agents (e.g., "OB, NO2) to produce spurious mutagenic nitroarenes. One might imagine that the difference in dilution ratio between Allegheny and the dilution tube could affect the extent of filter artifact formation by altering the portion of the parent species (e.g., pyrene in the case of nitropyrene) that stays in the gas phase and does not get collected on the filter. The issue of artifact nitroarenes is still not resolved (17, 18);strictly speaking, our results deal with mutagenicity and mutagenic species without distinction between real and (if any) artifact formation processes. It is probably
useful to add that if chemical reaction is occurring between the point of emission and the point of collection, then the differences in dilution ratios between Allegheny and dilution tubes could be critical.
Acknowledgments We acknowledge Wanda W. Brachaczek and Ann C. Szkarlat for helping to conduct the field experiment at the Allegheny Mountain Tunnel, Fred C. Ferris for carrying out the extractions of the tunnel and dilution tube samples, Larry D. Wiggins and William K. Okamoto for conducting the dilution tube runs, Rosyln Zator and Loretta Skewes for carrying out the HPLC fractionations, and Anna Marie Per0 for carrying out the Ames assays. We appreciate the cooperation of the Pennsylvania Turnpike Commission in the 1981 experiment at Allegheny and especially the assistance and hospitality of the late Robert J. Hauger and his crew at the Allegheny Tunnel.
Literature Cited (1) Lewtas, J., Ed. "Toxicological Effects of Emissions from Diesel Engines"; Elsevier: New York, 1982. (2) Pierson, W. R.; Gorse,R. A., Jr.; Szkarlat,A. C.; Brachanek, W. W.; Japar, S. M.; Lee, F. S.-C.; Zweidinger, R. B.; Claxton, L. D. Environ. Sci. Technol. 1983, 17, 31-44. (3) Ohnishi, Y.; Kachi, K.; Sato, K.; Tahara, I.; Takeyoshi, H.; Tokiwa, H. Mutat. Res. 1980, 77, 229-240. (4) Handa, T.; Kato, Y.; Yamamura, T.; Ishii, T.; Matushita, H. J . Environ. Sci. Health Part A 1980, 15, 573-599. (5) Albrechcinski, T. M.; Michalovic, J. G.; Wattle, B. J.; (6)
(7) (8) (9)
(10) (11) (12) (13) (14)
Wilkinson, E. P.; Kittelson, D. B. First International Aerosol Conference, Minneapolis, MN, Sept 17-21,1984, Paper 184. Albrechcinski, T. M.; Michalovic,J. G.; Gibson, T. L. Eighth International Symposium on Polynuclear Aromatic Hydrocarbons, Battelle Columbus Laboratories, Columbus, OH, Oct 26-28, 1983. Gorse, R. A,, Jr.; Riley, T.; Ferris, F.; Pero, A. M.; Skewes, L. M. Environ. Sci. Technol. 1983, 17, 198-202. Pederson, T. C.; Siak, J.4. J. Appl. Toxicol. 1981,1,54+3. Salmeen, I.; Durisin, A. M.; Prater, T. J.; Riley, T.; Schuetzle, D. Mutat Res. 1982, 104, 17-23. Gibson, T. L. Atmos. Environ. 1982, 16, 2037-2040. Kamens, R. M.; Rives, G. D.; Perry, J. M.; Bell, D. A,; Paylor, R. F., Jr.; Goodman, R. G.; Claxton, L. D. Environ. Sci. Technol. 1984, 18, 523-530. Daisey, J. M.; Kneip, T. J.; Hawryluk, I.; Mukai, F. Enuiron. Sci. Technol. 1980, 14, 1487-1490. Salmeen, I. T.; Pero, A. M.; Zator, R.; Schuetzle, D.; Riley, T. L. Environ. Sci. Technol. 1984, 18, 375-382. Gorse, R. A., Jr.; Salmeen, I.; Clark, C. R. Atmos. Environ.
1982,16, 1523-1528. (15) Schuetzle, D.; Lee, F. S. C.; Prater, T. J.; Tejada, S. B. Znt. J . Environ. Anal. Chem. 1981, 9, 93-143. (16) Schuetzle, D.; Riley, T. J.; Harvey, T. M.; Hunt, D. F. Anal. Chem. 1982,54, 265-271. (17) Bradow, R. L.; Zweidinger, R. B.; Black, F. M.; Dietzmann, H. M., SAE Tech. Pap. Ser. 1982, No. 820182. (18) Herr, J. D.; Dukovich, M.; Lestz, S. S.; Yergey, J. A.; Risby, T. H.; Tejada, S. B. SAE Tech. Pap. Ser. 1982, No. 820467.
Received for review April 16,1984. Revised manuscript received October 25, 1984. Accepted November 13, 1984.
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