Environ. Scl. Technol. 1984, 18, 375-382
carbons from Indigenous Species of Vegetation in the Tampa/St. Petersburg, Florida, Area”;U.S.Environmental Protection Agency: 1979; Final Appendix C, EPA 904/977-028. Bufalini, J. J.; Arnts, R. R. “Atmospheric Biogenic Hydrocarbons”;Ann Arbor Science: Ann Arbor, MI, 1981; Vol. 1 and 2, and references therein.
(42) Arnts, R. R.; Petersen, W. B.; Seila, R. L.; Gay, B. W., Jr. Atmos. Enuiron. 1982, 16, 2127. (43) Dimitriades, B. J. Air Pollut. Control Assoc. 1981,31,229. Received for review August 8,1983. Accepted December 5, 1983. This work was supported by National Science Foundation Grant A TM-8209028- 02.
Ames Assay Chromatograms and the Identification of Mutagens in Diesel Particle Extracts Irving 1. Salmeen,” Anna Marie Pero, Rosalyn Zator, Dennis Schuetzie, and Timothy L. Rlley
Scientific Research Laboratory, Ford Motor Company, P.O. Box 2053,Dearborn, Michigan 48 121 Dichloromethane extracts of passenger car diesel exhaust particles were separated into 65 fractions by normal-phase high-performance liquid chromatography (HPLC), and Ames assays (TA98 without activation) were carried out on each fraction. The only prominently mutagenic fractions were ones with elution times approximately coincident with those for 1,3-, 1,6-, and 1,8-dinitropyrenes, l-nitropyrene, and 3- and 8-nitrofluoranthene. These compounds were quantified in one extract sample by gas chromatography-mass spectrometry and gas chromatography coupled with a nitrogen-phosphorus detector. Among five extract samples studied, 30-40% of the total mutagenicity recovered from the columns could be attributed to these six nitroarenes, 15-20% was recovered in the as yet uncharacterized polar fraction, and the remainder was distributed roughly equally over 63 fractions. About 70% of the mutagenicity applied to the HPLC columns was recovered from the columns. The Ames assay mutagenicities of the unfractionated extracts were shown to be additive in the mutagenicities of the components. Diesel engines emit small particles (mass median diameter 99.5%). The 1-nitropyrene contained less than 0.02% dinitropyrene as judged by GCNPD and GC-MS analysis and Ames assay activity.
Results and Discussion Ames Assay Chromatograms. In the following, the general features of the Ames assay chromatograms will be discussed first, and then the fractions that show most of
z
n
SI
0
Flgure 1. (Top) Ames assay chromatogram for sample S1 showing all 30-sfractions starting at 17.5 min. Material eluted before 17.5 min not active. Lower panels designated S1, S2, and S3 are expanded scale displays of the 19-29 min range of Ames chromatograms obtained for samples S1, S2 (another sample collected from the same vehicle used to generate sample Sl),and S3,a sample provided by US. EPA (see text). The horizontal bars show the range of elution times for some nitroarenes when chromatographed as dilute neat solutions. Abbreviations are NF (3-and 8-nitrofluoranthene), 1-NP (1-nitropyrene) 1,3-,1,6-,and 1,8DNP are isomers of dinitropyrene. The letters A-H identify peaks for purposes of discussion in text. Note revertants per fraction scale for S1 and 52 are the same but different from that for S3. Polar fraction not shown (see text). Top panel reprinted with permission from ref 15. Copyright 1983 Battelle Press.
Table I. TA98 Ames Assay Data for Unfractionated Extracts
S1
wg/plate rev/plate’
S2
wg/plate rev/plate”
S3
wg/plate rev/plate”
S4
pg/plate rev/plate”
S5
pg/plate rev/plate”
0 22 35 0 63 68 0 52 33 0 45 51 0 56 40
8 87 112 8 64 77 12 277 288 10 64 74 15 99 86
15 139 137 16 110 98 24 804 589 19 86 78 30 120 112
80 40 356 751 841 360 64 32 383 190 204 433 75 48 2390 1800 1900 1980 50 100 200 126 145 111 75 150 498 269 236 447
150 1665 1686 130 986 909 130 2385 2878 180 284 248 300 814 709
a Number of revertants (TA98) per plate. Duplicate plates per dose.
the mutagenicity will be considered in detail. UV chromatograms will not be discussed because they have been presented previously (5, 9). A representative chromatogram over all of the 30-s fractions is shown in the upper panel of Figure 1for one of the samples (designated S1) collected in our laboratory. [This chromatogram was the subject of a preliminary account of this work (15).] Five samples were studied: four collected by us and one provided by the U.S. EPA (see Materials and Methods). This EPA sample was the same one we used in previous experiments in our laboratory and designated as “EPA” in ref 7, “sample A” in ref 9, and “sample N1” in ref 21. Chromatograms were obtained on several aliquots of each sample; the data shown were selected arbitrarily from among several possible ones. The mutagenicities of unfractioned samples were substantially different from each other as shown by the dose-response data in Table I. The range in mutagenicity is important for the characterization of diesel particle extracts, but it will not be discussed here. All of the chromatograms were qualitatively similar in Environ. Sci. Technol., Vol. 18, No. 5, 1984 377
Table 11. Distribution of TA98 Mutagenicity among Chromatogram Fractions
mass loaded
revertants recoveredaf'or fractions sample
17-29 rnin
29-49 rnin
49-85 rnin
c
in columns, mg
s1
31700 (64%)b 39571 (42%) 77500 (65%) 9800 (70%) 12300 (67%)
9800 (20%) 14250 (15%) 21000 (17%) 2000 (14%) 2000 (11%)
7900 (16%) 41000 (43%) 21500 (18%) 2200 (16%) 4100 (22%)
49 400 95 000 120 000 14 000 18400
13.5 14.7 16.5 15.7 15.0
s2 s3 s4 s5
a Mutagenicities (spontaneous subtracted) summed over
indicated fractions. bPercentagesare with respect to sum of mutagenicities
for all fractions.
the sense that they showed a cluster of peaks between 17 and 29 rnin and a featureless distribution between 29 and 49 min. There were, however, quantitative differences among chromatograms which are discussed later in this section of the paper. None of the fractions were cytotoxic at the doses tested as far as could be detected in the background lawn. The material collected prior to 17.5 min was not active. Previous experiments (5) showed that this material accounted for around 60-70% of the extract mass and included predominantly aliphatic hydrocarbons and two to five ring unsubstituted and alkyl-substituted PAH. The upper panel of Figure 1 does not show the polar fraction (49-85 min) because this fraction was collected over a 36-min period of the solvent program, whereas the other fractions were collected over 30-s periods. The polar fraction typically accounted for about 20-30% of the extract mass (5). The total mutagenicityof the polar fraction was determined as follows. Ames assays of this fraction were carried out at several doses (see Material and Methods). We observed that the dose-response curves were linear over the doses assayed, therefore, the total number of revertants in the polar fraction was given by the product of the slope (revertants per volume) times the total collected volume of the fraction. The Ames assay mutagenicity for the various samples was distributed among the major portions of the chromatograms as summarized in Table 11. The percentages shown in Table I1 are with respect to total recovered mutagenicity determined by adding the mutagenicities for all of the fractions. We note that about 11-20% of the recovered mutagenicity was distributed over 40 fractions (29-49 min) and each fraction contributed such a meager portion of the total that further investigation of directacting mutagens in these fractions was impractical. Another 16-22% (excluding S2) was recovered in the polar fraction. Very little is known yet about compounds which elute in this fraction except that polar organics may be very difficult to analyze with current techniques because they may not be amenable to gas chromatographyand they tend to be unstable under mass spectrometer injection conditions. We will discuss below the contribution of specific nitroarenes to major peaks in the chromatogram between 17 and 29 min. Before discussing these fractions, however, we comment on the absence of dose-response data for individual chromatographic fractions. In general, Ames assay dose response curves are not linear, and the relative mutagenicities of the various chromatographic fractions could depened on the doses at which the fractions were assayed. Nonlinearities occur because several processes that affect the appearance of mutant colonies take place concurrently and depend on mutagen dose such as cell killing, mutagen transport into cells, and metabolism within the bacteria to produce active mutagens. None of the chromatographic fractions were 378 Environ. Sci. Technol., Vol. 18,No. 5, 1984
tested at doses for which cytotoxicity was manifested in the photomicrographsof the background lawn. The doses of some of the most active fractions (e.g., A, B, C, G, and H, Figure 1)conceivably could have been in a range where the dose-response curves were approaching a maximum or a plateau. A plateau or near maximum could reflect a low percentage of cell killing not detectable as a change in the background lawn, or it could reflect saturation of metabolic processes. If the doses of the most active fractions had correspondedto this nonlinear range of their respective dose-response curves, then the mutagenicities of these fractions may have been somewhat underestimated relative to other fractions. The underestimate, if it obtained, however, would not qualitatively change the Ames assay chromatograms. Conceivably the doses of some of the weakly mutagenic fractions could have corresponded to points on their dose-response curves for which the shape was an ascending curve with a positive second derivative. Since the doses consisted of the entire fractions, the observed mutagenicities were the maximum they could possibly have relative to the other fractions. We next consider in detail the most active portion of the Ames assay chromatograms, i.e., the 17-29-min range, as shown in the small panels in the lower part of Figure 1. The letters (A-H) on the figure are labels used later in the paper. The bars are not labels for the chromatogram fraction but rather indicate elution times of specific compounds discussed in the following paragraph. The data shown in the small panels (Figure 1) illustrate the similarities and differences between chromatograms obtained for different samples from the same vehicle (Sl and S2) and from a different vehicle (S3). The gross shapes of the three chromatograms are obviously similar, but the peaks occur at slightly different elution times, and the detailed relative amplitudes differ. The large absolute amplitude differences between the chromatograms for S3 and those for S1 and S2 reflect quantitative differences in sample composition (see below). Differences among S1, S2, and S3 in elution times of peaks and small differences in relatiue amplitudes more likely reflect well-known irreproducibilities inherent to the chromatography. For example, even the elution times of neat compounds observed over many runs on the same column varied over a range of about h30 s. These ranges are illustrated for several nitroarenes by the horizontal bars on the small panels of Figure 1. The length of the bars correspond to the extremes in elution times observed for the various compounds (see caption). Relative mutagenicities of fractions may differ between two chromatograms because the line widths of the chromatographic elution bands were comparable to the 30-s sample periods. For example, the full width at halfmaximum observed for the elution bands of 1-nitropyrene (1pg loaded onto the column) and of the isomers of di-
Table 111. Nitroarene Concentrations in Extract Sample SI" PPm 1-nitropyrene 1,3-dinitropyrene 1,6-dinitropyrene
PPm
75 f lob 1,8-dinitropyrene 0.3 f 0.2b 3-nitrofluoranthene 0.4 f 0.2b 8-nitrofluoranthene
*
0.5 0.3' 0.7 f 0.2d 3 f 2d 2 f Id
" Data from ref 20 and 21. The same values were obtained from GC-NPD and GC-MS data. GC-MS data. GC-NPD data.
nitropyrenes (1pg of each) were 10 and 20 s, respectively. Therefore, compounds were distributed among neighboring fractions. Because of the fluctuations in elution times, the distribution differed among chromatographic runs. (For one fractionation of S2, we found by GC-NPD that 1nitropyrene was about equally distributed between two adjacent fractions.) Thus, small differences in details among chromatograms are less important than gross similarities. Quantification of Nitroarenes and Assignments of Chromatogram Peaks. We now consider the data that suggest assignments for some of the major peaks in the Ames assay chromatograms. The proximity of the elution times for the six nitroarenes noted on the lower panels of Figure 1 and the elution times for various peaks in the chromatograms suggested that the mutagenicities of these fractions may be due to predominantly these six compounds. Therefore, the six nitroarenes were quantified for one of the samples (Sl). The analysis of sample S1 was described in a preliminary account of these experiments (15) and in detail in ref 21. These results are needed together with the Ames assay mutagenicities of the nitroarenes in order to interpret the chromatograms. Table I11 lists the results from our previous chemical analysis work. Table IV lists our Ames assay data for these six nitroarenes. Data for these compounds have been published by others (6,18),but because of the uncertainties inherent in interlaboratory experiments, the Ames assays of these compoundswere repeated in order to have data which were obtained under conditions comparable to those under which the Ames assay chromatograms were obtained. First consider fractions A-C (sample S1, Figure 1). The elution times of 1-nitropyrene and 3- and 8-nitrofluoranthene were so close to each other that the three compounds
were assumed to have been distributed among fractions A-C. The distribution was unknown, so the fractions were analyzed together. These fractions together yielded about 10000 revertants (sample Sl). From the data listed in Tables 11-IV and considering that only half of each fraction was applied to the Ames assay plates, we calculated that 1-nitropyrene, $nitrofluoranthene, and 8-nitrofluoranthene would contribute about 2100 f 150,555 f 372, and 876 f 438 revertants, respectively, to the chromatogram. Together these three nitroarenes account for about onethird of the combined mutagenicity of fractions A-C. We have been unable to identify other compounds which could account for the remaining two-thirds of the mutagenicity of fractions A-C. Consider next, fractions D-F. We assume these fractions contain all of the 1,3- and 1,6-dinitropyreneeven though in the chromatogram for S1, these three peaks occurred about 1min before the elution times observed for neat 1,3and 1,6-dinitropyrenes. We attribute this shift in elution times to well-known "matrix effects". Shifts in elution times increase for compounds as their concentration in the mixtures decreases. The shifts were not as apparent for sample S3 because the concentrations of the nitropyrenes in sample S3 (9,21) were about 5-10 times higher than those in sample S1. We calculated, as above, that 1,3dinitropyrene would yield about 3600 f 2700 revertants and 1,6-dinitropyrene about 1400 f 840 revertants. These two compounds together would contribute about 5000 f 3540 revertants. The observed sum of the activities for fractions D-F was 4700. Thus, the amounts of these two dinitropyrene isomers were sufficient to account for all of the mutagenicity in these fractions. Finally, consider fractions G and H and assume that together they contain all of the l,&dinitropyrene. We calculated, as above, that 1,8-dinitropyrene would yield about 14000 f 7800 revertants. The observed mutagenicity for G and H together was 9500 revertants. Thus, the amount of 1,8-dinitropyrene was sufficient to account for all of the combined mutagenicity of fractions G and H. The large absolute differences between the mutagenicities of fractions A-H for samples S3 and those for S1 and S2 correspond to the higher concentrations of nitropyrenes in S3 (9,21). For S3, the 1-nitropyrene concentration was 2300 ppm, and the concentration of the three dinitropyrenes together was about 5 ppm. Ames Assay Chromatograms with TA98NR and TA98DNP. The foregoing assignments of chromatogram
Table IV. TA98 Ames Assays for Pure Nitroarenes
0 37 45 0 50 30 0 50 30 0 37 45 0 37 45 0 37 45
0.04 71 99 4 82 99 2.5 103 92 0.19 99 108 0.14 66 72 0.13 256 214
0.08 0.18 0.40 0.90 172 384 736 1826 452 125 789 1972 3-nitrofluoranthene ng/plate 20 9 44 100 rev/plate 134 212 602 1192 252 119 621 1290 8-nitrofluoranthene ng/plate 5.0 10 25 60 rev/plate 170 189 2219 896 180 351 2376 857 1,3-dinitropyrene ng/plate 0.39' 0.18' 1.56' 3.12' 6.24' rev/plate 102 388 1396 -3110" 6000" 151 363 1318 3328" -6200" 1,6-dinitropyrene ng/plate 0.28' 0.56' 1.12c 2.24' 4.6' rev/plate 144 102 342 624 1798 134 89 377 1631 660 1,8-dinitropyrene ng/plate 0.25" 0.50' 1.0' 2.OC 4.0' rev/plate 771 336 4400" 1970 7600" 345 735 1900 -3700" 7000" Estimated by counting colonies within 4-cm2area and normalizing to plate area. bDuplicate plates per dose. 'Cytotoxic as reflected in decreased background lawn density. 1-nitropyrene
wz/plate rev/plateb
-
-
-
--
(I
Environ. Sci. Technol., Vol. 18, No. 5, 1984
379
Table V. TA98 Ames Assay-Reconstitution Experiment revertants/ platen for chromatographic fractions
dose/plate, fiL
3
4
5
6
7
8
sum of fractions 3-8
10 20 30 40
190 393 617 641*
97 218 351 386'
17 50 86 159
10 9 24 33
78 140 150 196
225 228d 283 274
617 1038 1511 1689
reconstituted mixture, rev/date (&)plate)
unfractionated extracte (dose/plate)
517 (80 pL) 1003 (160 WL) 1546 (240 p L ) 1711 (320 gL)
918 (80 pL) 1667 (160 fiL) 2400 (240 fiL) NT
"Fractions 1and 2 were not active, but they were included in the reconstituted mixture. Entries are average of two plates. Spontaneous revertants [(33 + 46)/2 = 401 have been subtracted from every entry in the table. The difference between the two plates was less than 3% of average except where noted. b T ~ plates o were 854 and 429. c T w plates ~ were 461 and 312. d T ~ plates o were 130 and 326. eAmount of material sufficient only for single plate per dose.
peaks to specific compounds can be checked for consistency by using strains TA98NR and TA98DNP because the sensitivities of these strains to mutations by some of the nitroarenes are substantially different from those of TA98. Estimates of the relative mutagenicities among the strains (based on the following: ref 5,6, and 18; G. Lofroth, unpublished results; our data, unpublished results) are the following
r' ,yJ
1-NP 3NF 8NF 1,3DNP 1,GDNP 1,8DNP TA98 1 TA98NR 0.08 TA98DNP 0.5-1
1
1
0.2
0.2 0.03-0.2 0.5 0.02
0.2
1
1 0.2
1
0.3-1
0.2
0.02
The following experiments used a different sample (236) from that used for quantification of the nitroarenes. The general similarities among the Ames assay chromatograms suggest, however, that the conclusions based on sample S6 should apply qualitatively to S1. Figure 2 shows the 19-29-min portion of the Ames assay chromatograms using TA98, TA98NR, and TA98DNP. For these experiments, only the 17-29-min fractions were assayed. The chromatographic column was new, and in order to minimize effects of long-term column changes, the chromatography to generate the three samples for the three strains was carried out one run immediately after the other. The same mass was loaded onto the column each time. The obvious qualitative differences between the TA98 chromatogram for S6 and those for S1, S2, and S3 (cf. Figures 1 and 2) are assumed to reflect mostly fluctuations inherent to the chromatography, as discussed above. We will assume that the peak marked DEF in Figure 2 is due to the sum of 1,3- and 1,6-dinitropyrenes and that these isomers did not separate during the chromatography. When the mixture of standards was run following sample fractionation, these isomers eluted within 20 s of each other. First, consider fraction GH. The data in Figure 2 strongly support the conclusion reached from the analysis of sample S1 (see above) that all of the TA98 mutagenicity of fraction GH is due to l&dinitropyrene. This compound is a t least 50 times less mutagenic with TA98 DNP than it is with TA98. If all of the 700 TA98 revertants of fraction GH (Figure 2) were due to l,8-dinitropyrene, then the mutagenicity of these fractions should decrease by at least 50-fold and be barely detectable with strain TA98DNP. This was observed. The available literature data for l,&dinitropyrene with strain TA98 NR are somewhat disparate. Mermelstein et al. (18)report a ratio of 0.3 and Pederson and Siak (6) about 1 for the ratio of TA98NRITA98 mutagenicity. We observed 600 revertants for fraction GH with TA98NR which is consistent with expectations based on data of Pederson and Siak (6). Those data of Figure 2 for fractions A-C cannot be unequivocally interpreted because, by analogy with sample 380
Environ. Sci. Technol., Vol. 18, No. 5, 1984
TIME ( M I N I
TIME (MINI
21 23 25 TIME ( M I N I
27
Flgure 2. Ames assay chromatograms using strains TA98, TA98NR, and TA98DNP and aliquots of sample (S6) obtained from the same vehicle as used to obtain S1 and S2 (see Figure 1). The bars are the same as in Figure 1. The letter designatlons for A-H are as in Figure 1. We assume that the peak labeled DEF corresponds to the three peaks as labeled in Figure 1 (see text). The revertants per plate scale is the same for the three chromatograms.
S1, only part of the TA98 mutagenicity of these fractions was attributable to specific nitroarenes and we do not known what to expect for the unidentified compounds when they are tested with the other strains. We would expect, however, a very small contribution from l-nitropyrene and 3- and 8-nitrofluoranthene to the TA98NR mutagenicity of fractions A-C. The TA98NR mutagenicity observed for fractions A-C was 2750, almost all of which therefore must be due to the unidentified TA98 mutagens. Finally, consider fraction DEF. The data with sample S1 showed that all of the TA98 mutagenicity of fraction DEF could be due to the sum of the mutagenicities of 1,3and 1,6-dinitropyrenes. The observed TA98 mutagenicity of fraction DEF for sample S6 (Figure 2) was 1000 revertants. The mutagenicity of fraction DEF expected for strain TA98NR should be less than half of that observed for TA98. The observed TA98NR mutagenicity was 600 revertants. When the experimental uncertainties are considered the observed and calculated mutagenicities are in fair agreement. On the other hand, the calculated mutagenicity for TA98DNP is far from the observed. By calculation, 1,3- and 1,6-dinitropyrene should yield less than 200 revertants with TA98DNP, whereas the observed mutagenicity for TA98DNP was 600. In view of the general consistency for other compounds, fractions, and bacteria strains, this discrepancy for the TA98DNP data is surprising and remains for future investigation. In summary, Figure 2 confirms that all of the TA98 mutagenicity of fraction GH is due to l,&dinitropyrene, suggests that the majority of the mutagenicity of fractions A-C is due to the combination of yet unidentified compounds which are approximately equally active in strains TA98, TA98NR, and TA98DNP, and is not inconsistent with the conclusion from analysis of sample S1 that the mutagenicity of fraction DEF is due to the combination
Table VI. Summary of TA98 Mutagenicity Data
sample
s1 s2 s38 s4 s5
% recovered mutagenicitp due to identified compounds 1-NP, 3NF, 1,3DNP,c 8NFb 1,GDNP 1,8DNPd
6 3 6 12 14
9 6 11 10
19 11 17 17 10
broadly distributed, probably not assignable: % fractions 17-29 rnin
fractions 29-49 rnin
polarf fraction, %
30 22 30 32 41
20 15 17 14 11
16 43 18 16 27
3 a The average mutagenicity recovered from columns was about 70%. These three compounds were found together in fractions A-C of Ames assay chromatogram (see Figure 1 and text). 1/3 of mutagenicity of A-C was due to these three compounds for sample S1. This ratio was assumed to hold for all samples. Abbreviations: 1-nitropyrene(1-NP);3- and 8-nitrofluoranthene (3,8NF). Fraction DEF: 1,3- and 1,6-dinitropyrene(1,3- and 1,GDNP). Fraction GH: l&dinitropyrene (1,8DNP). e Fractions 29-49 rnin from Table 11. Fractions 17-29 min determined from the sum of mutagenicity for all fractions between 17 and 29 min minus the contribution due to six nitroarenes. fFrom Table 11. gConcentrations of all six identified nitroarenes were about 10 times higher in S3 than in S1 (see text, ref 9). The mutagenicity of unfractionated extract of sample S3 was about 10 times higher than that of S1 (see Table I). It is likely that Ames assays of fractions A-H were carried out at doses well into the plateau regions of the respective nitroarene dose-response curves, and relative contributions of these fractions were substantially underestimated. of 1,3- and 1,6-dinitropyrenes. Additivity of Mutagenicity of Extracts and Recovery from Columns. We next consider the additivity experiments (see Methods and Materials). The results from one of the experiments (using Sample Sl) are summarized in Table V. Similar data were obtained on aliquota of sample 52 and another sample for which data are not shown here. The activities for the reconstituted mixture calculated at various doses from the sum of the activities determined for the fraction agree remarkably well with those measured for the reconstituted mixture. Thus, at least for these samples, the Ames assay mutagenicities were additive. Kaden et al. (23)have also demonstrated additivity for the bacterial mutagenicity, measured by a tissue homogenate dependent forward mutation assay, of a complex mixture, i.e., one derived from kerosene soot. The data to be considered last are those for estimating how much of the Ames assay activity in the original samples was recovered from the columns. Concurrently with obtaining data for the reconstituted samples, we carried out Ames assays on the unfractionated material a t the same doses as those used for the reconstituted mixture (see Table V). Figure 3 shows a plot of the number of revertants observed for unfractionated vs. reconstituted extract for various experiments. The mass recoveries for the experiments are listed in the caption. The Ames assay activity recovered ranged among different experiments from about 50% to about 100%. We did not investigate possible reasons for the low recoveries because they seemed to occur sporadically. Even the same sample on one occasion yielded 50% and on another 90% recovery. Mass recoveries ranged from about 80% to more than 90%, but in most cases the fraction of Ames assay activity lost was greater than the fraction of mass lost. In reviewing elution times of standards run before and after the chromatography and histories of the columns, we could not find any unusual occurrences in those experiments that resulted in low recoveries. It is unlikely that the losses were in the 17-29-min range because mono- and dinitropyrenes were 100% recovered as shown by recovery experiments in which perdeuterionitropyrenes were added to the samples prior to chromatography and the perdeuterio compounds quantified by mass spectrometry after chromatography. It is possible that polar mutagens were not recovered from the normal-phase columns, but this possibility could not be tested with the available data. We do not know why the recoveries were low in some cases.
I
Oo0
T
t
600 1000 1400 REVERTANTS RECOMBINED FRACTIONS
200
Flgure 3. Recovery of TA98 Ames assay activity from columns. Vertical axis is revertants observed for unfractionated extract, and horizontal axis is revertants observed for reconstituted extract when Ames assay carried out concurrently at the same dose per plate for both unfractionated and reconstituted extract. Symbols correspond to different experiments. Solid circles from experiment summarized in Table V; size of symbol encompasses experimental uncertainties except when extended by vertical or horizontal lines. Uncertainty is difference in duplicate plates. Solid line is slope of one line upon which data would fall if recovery were 100 % . Mass recoveries were (open circles) 87 %, (triangles)96%, (squares)84%, (closed circles) 85 % , and (crosses)98 % . Data represented by crosses and by squares were for the same sample run at different times.
Conclusions The main question addressed by the experiments described herein was the following: How much of the direct-acting Ames assay mutagenicity of the extracts can, as a practical matter, be assigned to identified compounds? The answer is summarized in Table VI. Only about eight fractions stood out prominently in the Ames assay chromatograms (A-H, Figure 1). After taking into consideration that compounds were distributed among adjacent fractions, the combined mutagenicity of two of the fractions (G and H) could be attributed to 1,8-dinitropyrene and the mutagenicity of three other fractions (D, E, F) and could be attributed to 1,3- and 1,6-dinitropyrene. About one-third of the combined mutagenicity of fractions A-C was due to the combination of 1-nitropyrene, 3-nitroEnvlron. Sci. Technol., Vol. 18, No. 5, 1984 381
fluoranthene, and 8-nitrofluoranthene, and the other two-thirds was not assignable. The six nitroarenes together accounted for 30-40% of the recovered mutagenicity. Once these few prominent fractions were taken into account, almost half of total recovered mutagenicity was found nearly uniformly distributed over the other 30-9 fractions. When the amount of work and time required to analyze complex mixtures is considered, it appears that detailed analysis of these weakly mutagenic fractions is not worth the effort unless experiments can be designed to uncover general characteristics of the compounds which will explain their direct-acting Ames assay activities. (For example, are they all nitroarenes?) The typically 20% or so of mutagenicity recovered in the polar fraction remains for future investigation. In addition to the main question, the experiments showed that the direct-acting mutagenicities of the complex mixtures here were additive in terms of the mutagenicities of the components. The importance of this conclusion is that questions related to biological effects of the extracts can be directed to specific identified compounds rather than to the intact complex mixtures. Acknowledgments
We are grateful to Robert A. Gorse, Jr., Fred Ferris, and William Okamoto for diesel particulate extracts. GC-NPD data were recorded by Richard Marano and Michelle Paputa-Peck. Loretta Skewes assisted with parts of the HPLC fractionation. Thomas Prater and Christine Hampton helped with the GC-MS experiments. Panos Zacmanidis assisted with various phases of the Ames assay experiments. William Pierson provided many helpful suggestions throughout the work and, by critically reading and discussing drafts, helped write this paper. L i t e r a t u r e Cited Chan, T. L.; Lee, P. S.;Hering, W. E. JAT, J. Appl. Toxicol. .. 1981, I , 77-82. Kotin, P.; Falk, H. L.; Thomas, M. AMA Arch. Ind. Health 1955,11, 113-123. Pepelko, W. E.; Donner, R. M.; Clarke, N. A., Eds. “Health Effects of Diesel Engine Emissions: Proceedings of an International SvmDosium. Dec 3-5. 1979: U.S. Environmental Agency: “Ciicinnati, OH, 1980; EPA-600/9-80-057.
382 Environ. Sci. Technol., Vol. 18, No. 5, 1984
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Received for review August 17,1983. Revised manuscript received December 8, 1983. Accepted December 28, 1983.
