Environ. Sci. Technol. 1992, 26, 622-624
COMMUNI CAT IONS Formation of Mutagenic Nitrodibenzopyranones and Their Occurrence in Ambient Air Detlev Helmlg, Janet Arey, *,+ Wllllam P. Harger, Roger Atklnson,+ and JosQ L6pez-Cancio Statewide Air Pollution Research Center, University of California, Riverside, California 9252 1
Introduction Nitrated polycyclic aromatic hydrocarbons (nitro-PAH) are strong direct-acting bacterial mutagens which have been estimated to contribute up to 10% of the direct mutagenic activity of ambient air particulate extracts ( I ) . The majority of ambient nitro-PAH are believed to be formed in the atmosphere (2-5) from the gas-phase radical-initiated reactions of the parent PAH, mainly with the hydroxyl radical (2,643). The two- to four-ring PAH most abundant in ambient air have been studied, and the nitro-PAH yields from their OH radical-initiated reactions are 55% (6,8,9). It has been postulated, therefore, that the other PAH reaction products formed might also contribute significantly to ambient air mutagenicity (10). We present here the identification of mutagenic nitrodibenzopyranones from the OH radical-initiated reaction of phenanthrene in the presence of NO, and show that these, and probably other nitropolycyclic lactones, are present in ambient air particles. Experimental Section Reactions of gas-phase phenanthrene with the OH radical in the presence of NO, were carried out in a 6400-L all-Teflon chamber using the photolysis of methyl nitrite as the source of OH radicals at concentrations 100 times those found in ambient atmospheres (8, 11). Initial reactant concentrations (in molecules cm”) were as follows: CH,ONO, -5 X lo1,; NO, -2.4 X lo1,; and phenanthrene, (2-4) X 1Ol2. Irradiations were carried out for 10 min at the maximum light intensity (corresponding to an NOz photolysis rate of -7.5 X low3s-l), resulting in significant NO to NOz conversion and hence the expected formation of 0, and NO, radicals. After the reactions, products were collected by sampling 2OOO-L volumes onto polyurethane foam with subsequent extraction, normal-phase (compound polarity increasing with elution time) high-performance liquid chromatography (HPLC) fractionation and analyses by gas chromatography/mass spectrometry (GC/MS) with parallel mutagenicity assays (10). Mutagenicity bioassays of the HPLC fractions were performed using Salmonella typhimurium strain TA98, without exogenous (S9) activation. The bioassay sensitivity was enhanced by using a microsuspension preincubation modification (12, 13) of the histidine reversion test of Ames and co-workers (14), as described previously (10). Ambient particulate matter was collected on the Riverside campus of the University of California during the summer of 1988 using an ultrahigh-volume sampler as detailed elsewhere (4). The dichloromethane extract of the particles was prefractionated by open-column chromatography on silica, using successively pentane, di-
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t Also Department of Soil and Environmental Sciences, University of California, Riverside, CA 92521.
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chloromethane, and methanol as eluents. Over 80% of the total mutagenic activity of the crude extract was recovered in the dichloromethane open-column fraction, which was then subjected to HPLC fractionation (10). Results Of the nine 9-min fractions collected from the HPLC silica column separation of the phenanthrene reaction products, fraction 6 was found to be the most mutagenic (10). GC/MS analysis of fraction 6 (see Figure 1)showed the presence of two compounds tentatively identified on the basis of their mass spectra as nitrophenanthrene lactones (nitrodibenzopyranones). After further characterization (15) these compounds were identified as 2-nitro6H-dibenzo[b,d]pyran-6-one (I) and 4-nitro-6H-dibenzo[b,d]pyran-6-one (11).
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The mutagenicity profile from the ambient particulate extract is shown in Figure 2. GC/MS analysis of HPLC fraction 6, which as for the chamber reaction products possessed the highest mutagenic activity, showed the presence of the identical nitro-6H-dibenzo[ b,d]pyran-6-one isomers as observed from the chamber OH radical-initiated reactions of phenanthrene (Figure 1). Mutagenicity assays of authentic samples of 2- and 4nitro-6H-dibenzo[ b,d]pyran-6-one gave activities of 240 000 and 2000 revertants pg-l, respectively, in the microsuspension assay (the activity of the positive control mutagen, 2-nitrofluorene, was 30 000 revertants pg-l). Combining these activity values with the measured concentrations of the 2- and 4-nitro-GH-dibenzo[b,d]pyran-6-ones showed that the activity of HPLC fraction 6 of the ambient Riverside particulate extract could be accounted for by the 2-nitro-GH-dibenzo[ b,d]pyran-g-one present, whereas the contribution from the 4-nitro-GH-dibenzo[b,d]pyran-6-one was low. Similarly, in the fractionated chamber OH radical-initiated reaction sample, the 2-nitro-GH-dibenzo[ b,dlpyran-6-one accounted for the majority of the mutagenic activity present in the HPLC fraction 6. Mutagenic HPLC fractions of the Riverside particulate extract, fractionated with a modified procedure using sequentially a normal- and a reversed-phase column separation (16), were also analyzed by GC/MS. In addition to the 2- and 4-nitro-GH-dibenzo[b,d]pyran-6-ones, five compounds which appear from their molecular ions and fragmentation patterns to be methyl derivatives of the nitrodibenzopyranones (MW 255) were observed, together with two further compounds with mass spectra suggestive
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Flgure 1. GUMS selected ion traces of the molecular ion (mlz = 241) and a characterlstlc fragment ion ( m l z = 139) of nitro-6H-dibenzo[b,d]pyran-6-ones (NDBP) (75)observed in HPLC fraction 6 of an ambient particulate extract collected in Riverside, CA (upper) and a sample collected from a chamber reactlon of gas-phase phenanthrene with the OH radical in the presence of NO, (lower). Injectlons were made dlrectly onto a 60 m X 0.245 mm DB-1701 column, film thickness 0.25 pm (Jaw Scientific, Inc.), which was held at 50 OC and then temperature and pressure programmed. Compound identification for the NDBP isomers was achieved by using mass spectral and retention index data (also determined on a DB-5 column) obtained from purchased and synthesized standard compounds, which had been fully characterized by 'H NMR and high-resolution mass spectral data (75).
mutagenicity in the Salmonella test could be accounted for by the measured nitrofluoranthenes and nitropyrenes ( I , 20). However, as seen from Figure 2 and in agreement with previous reports (18,21,22),most of the mutagenicity of ambient particulate matter resides in fractions more polar than fraction 4. As noted above, 2-nitro-6H-dibenzo[b,d]pyran-6-one accounts for the activity in fraction 6, the most mutagenic fraction, and indeed contributes an estimated -20% of the total activity of the crude Riverside ambient particulate extract. Among the PAH present in ambient air, those of lower molecular weight are more abundant (3, 7, 20), and the two- to four-ring PAH are expected to be in the gas phase in the atmosphere (3, 23, 24). Whereas ambient measurements indicate that the atmospheric reaction products of the two-ring PAH, such as the nitronaphthalenes and methylnitronaphthalenes, remain predominantly in the gas phase (3,7,9,20,25), certain of the gas-phase atmospheric reaction products of the three- and four-ring PAH condense onto particles in the atmosphere (4, 17-20). The three- and four-ring PAH are expected to react in the atmosphere with the OH radical, leading to lifetimes on the order of a few hours (7). While phenanthrene is more abundant in ambient air than anthracene, fluoranthene, and pyrene (3, 7, 20), the nitrofluoranthenes and nitropyrenes are generally the most prevalent nitro-PAH in ambient particles and nitrophenanthrenes, presumably due to their lower formation yields (6),are not observed (3,20). However, this work shows that phenanthrene reaction products are indeed present in ambient air particles and account for a significant percentage of the mutagenicity of the more polar extract fractions. Results of on-going analyses, including those of an urban dust standard reference material (the National Institute of Standards and Technology SRM 1649) and ambient particulate samples from several locations in the United States, by us and other investigators (26),suggest that the presence of 2- and 4nitro-6H-dibenzo[b,d]pyran-6-one in urban atmospheres is a general phenomenon. Quantitative results from several urban samples (including gas/particle distribution data) will be presented in detail elsewhere (27). The high bacterial mutagenic activity of this newly identified class of PAH atmospheric transformation products, the nitropolycyclic lactones, indicates that additional assays to assess the potential human health effects of these compounds are certainly warranted. Acknowledgments
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Fraction Number Flgure 2. Mutagenic actlvlty in the microsuspension assay to Salmonella typhlmurhm strain TA98 (-S9) of Individual HPLC fractions of a Rlverside amblent particulate extract fractlonated on a semipreparative Regis Spherisorb S5W silica (5-pm) column as described previously ( 70), after prefractionation by opencolumn chromatography. Note that compound polarity increases wlth the fractlon number.
of nitrophenanthropyranones (MW 265). Discussion The presence of 2-nitrofluoranthene and 2-nitropyrene, atmospheric transformation products of fluoranthene and pyrene, respectively (2, 6, 8 ) , in ambient air samples worldwide has previously been reported by this laboratory (2,4,17), and this finding was subsequently confirmed by other investigators (18, 19). The nitro-PAH elute in fraction 4 for the HPLC conditions noted on Figure 2 and thus the mutagenicity profile as shown is consistent with previous reports that up to 10% of ambient particulate
We thank Ms. Patricia A. McElroy for valuable technical assistance. Literature Cited (1) Arey, J.; Zielinska, B.; Harger, W. P.; Atkinson, R.; Winer, A. M. Mutat. Res. 1988,207, 45-51. (2) Arey, J.; Zielinska, B.; Atkinson, R.; Winer, A. M.; Ramdahl, T.; Pitts, J. N., Jr. Atmos. Enuiron. 1986, 20, 2339-2345. (3) Arey, J.; Zielinska, B.; Atkinson, R.; Winer, A. M. Atmos. Enuiron. 1987, 21, 1437-1444. (4) Zielinska, B.; Arey, J.; Atkinson, R.; Winer, A. M. Atmos. Enuiron. 1989, 23, 223-229. (5) Arey, J.; Atkinson, R.; Aschmann, S. M.; Schuetzle, D. Polycyclic Arom. Compd. 1990, 1, 33-50. (6) Arey, J.; Zielinska, B.; Atkinson, R.; Aschmann, S. M. Int. J. Chem. Kinet. 1989, 21, 775-799. (7) Arey, J.; Atkinson, R.; Zielinska, B.; McElroy, P. A. Enuiron. Sei. Technol. 1989, 23, 321-327. (8) Atkinson, R.; Arey, J.; Zielinska, B.; Aschmann, S. M. Int. J. Chem. Kinet. 1990,22, 999-1014. (9) Helmig, D.; Arey, J.;Atkinson, R.; Harger, W. P.; McElroy, P. A. Atmos. Enuiron., in press. Environ. Sci. Technol., Vol. 26, No. 3, 1992
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(10) Arey, J.; Harger, W. P.; Helmig, D.; Atkinson, R. Mutat. Res., in press. (11) Atkinson, R.; Arey, J.; Zielinska, B.; Aschmann, S. M. Environ. Sci. Technol. 1987, 21, 1014-1022. (12) Kado, N. Y.; Langley, D.; Eisenstadt, E. Mutat. Res. 1983, 121, 25-32. (13) Kado, N. Y.; Guirguis, G. N.; Flessel, C. P.; Chan, R. C.; Chang, K.-I.; Wesolowski, J. J. Environ. Mutagen. 1986, 8, 53-66. (14) Ames, B. N.; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347-364. (15) Helmig, D.; Arey, J. Int. J. Enuiron. Anal. Chem., in press. (16) Atkinson, R.; Arey, J.; Harger, W. P.; Helmig, D.; McElroy, P. A. Final Report to the California Air Resources Board Contract No. A732-154, Sacramento, CA, 1991. (17) Ramdahl, T.; Zielinska, B.; Arey, J.; Atkinson, R.; Winer, A. M.; Pitts, J. N., Jr. Nature 1986, 321, 425-427. (18) Nishioka, M. G.; Howard, C. C.; Contos, D. A.; Ball, L. M.; Lewtas, J. Enuiron. Sei. Technol. 1988, 22, 908-915. (19) Ciccioli, P.; Cecinato, A.; Brancaleoni, E.; Draisci, R.; Liberti, A. Aerosol. Sei. Technol. 1989, 10, 296-310. (20) Atkinson, R.; Arey, J.; Winer, A. M.; Zielinska, B.; Dinoff, T. M.; Harger, W. P.; McElroy, P. A. Final Report to the California Air Resources Board Contract No. A5-185-32, Sacramento, CA, 1988.
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(21) Schuetzle, D.; Lewtas, J. Anal. Chem. 1986, 58, 1061A1075A. (22) Lewtas, J.; Chuang, J.; Nishioka, M.; Petersen, B. Znt. J. Environ. Anal. Chem. 1990, 39, 245-256. (23) Bidleman, T. F. Environ. Sci. Technol. 1988,22,361-367. (24) Coutant, R.W.; Brown, L.; Chuang, J. C.; Riggin, R. M.; Lewis, R. G. Atmos. Environ. 1988, 22, 403-409. (25) Zielinska, B.; Arey, J.; Atkinson, R.; McElroy, P. A. Enuiron. Sci. Technol. 1989, 23, 723-729. (26) Nishioka, M. G. Battelle Columbus Division, Columbus, OH; Lewtas, J., Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC, private communication, 1991. (27) Helmig, D.; L6pez-Canci0, J.; Harger, W. P.; Arey, J.; Atkinson, R. Environ. Sei. Technol., to be submitted for publication.
Received f o r review September 12, 1991. Revised manuscript received November 18, 1991. Accepted November 22, 1991. J.L.-C. thanks the DGZCYT (MEC)of Spain f o r financial assistance. This work was funded through the California Air Resources Board (Contract A732-154) and the U.S. Enuironmental Protection Agency, Officeof Research and Development, through Assistance Agreement R814857-01.
