United States Environmental Protection Agency Atmospheric Sciences Research Laboratory x Research Triangle Park NC 27711 Research and Development EPA/600/S3-87/020 Sept. 1987 &EPA Project Summary The Production of Mutagenic Compounds as a Result of Urban Photochemistry P. B. Shepson, T. E. Kleindienst, and E. 0. Edney A series of atmospheric simulation experiments was conducted to exam- ine the role of urban photochemical processes on the formation and re- moval of potentially hazardous air pollutants. The experiments were con- ducted in a 22.7-m3 Teflon smog chamber, which was coupled to bioas- say exposure chambers. The mutagenic activities of the tested mixtures of organic chemicals and nitrogen oxides were measured before and after irra- diation by direct exposure of Salmo- nella typhimurium (strains TA98 and TA100) to the smog chamber contents. The mutagenic responses of the start- ing materials and the transformed products were quantified by using a modified Ames test. The chemicals examined included ubiquitous urban pollutants (e.g., propylene, toluene, and acetaldehyde), a potentially hazardous chlorinated solvent (ally! chloride), and complex mixtures (wood smoke and auto exhaust) from common urban pollutant sources. In all cases, the irradiated products were more mutagenic than the original chemicals. For the transformed complex mixtures, the bulk of the mutagenicity was found to be associated with the gas-phase products rather than with the aerosol- bound chemicals. Increased mutagen- icity was also observed with increasing photochemical oxidation. The common photochemical pollutant, peroxyacetyl nitrate, was found to contribute signif- icantly to the overall vapor-phase mutagenicity in all of the chemical systems in which it was formed. This Project Summary was devel- oped by EPA's Atmospheric Sciences Research Laboratory. Research Trian- gle Park, NC. to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction During the past several years there has been an increasing awareness that exposure to polluted urban atmospheres may pose an ill-defined but significant human health threat, especially with regard to the incidence of cancer. It has become increasingly apparent that an accurate assessment of the potential human health impact due to exposure to atmospheric pollutants requires an understanding of the influence of atmos- pheric reactions on both hazardous and nonhazardous species. The photooxida- tion products of a wide variety of impor- tant atmospheric aromatic hydrocarbons have been shown to be mutagenic. Researchers have found a variety of oxygenated and nitrogenated species to be mutagenic by using the Ames test, implying that the photooxidation of reactive atmospheric hydrocarbons may have a significant impact on the presence of atmospheric mutagens. Concerns have also been raised with regard to the possible human health threat of exposure to wood stove and fireplace emissions. In addition to the possible presence of mutagens in the emissions themselves, evidence has appeared recently in the literature indicating that reactions of species such as 03 and N2O5 on the surface of atmospheric particulate matter can lead to increases in the mutagenic activity of adsorbed species such as polycyclic ------- aromatic hydrocarbons (PAHs). Consid- erable effort has been expended in studying reactions that occur on the surface of paniculate matter and in studying the impact of particulate-phase photochemistry on the mutagenic activ- ities of adsorbed species. In contrast, there has been a relatively smaller effort aimed at identifying mech- anisms for the production of gas-phase mutagens through atmospheric photo- chemistry. A principal reason for the emphasis on particulate-phase muta- gens is the relative ease of collection and extraction of paniculate matter. To test for the mutagenic activity of gas-phase species by using the standard plate incorporation test, it is necessary to concentrate the gas-phase pollutants into a suitable solvent in some manner. Some limited attempts have been made to measure gas-phase ambient mutagen- icities by using XAD or other adsorbents to concentrate the species present. However, this process poses a number of potential problems, such as loss of volatile species during the extraction and solvent concentration steps. In this report, we present a review of the results of experiments we have conducted by using an alternative tech- nique for measuring the mutagenic activity of gas-phase species generated by photochemical reactions. The product mixtures to be tested were produced in a 22.7-m3 Teflon smog chamber. For this technique, the Ames test plates were dosed by continuously flowing the reac- tion chamber air over the uncovered plates, thereby permitting the soluble species to deposit continuously while the plates are uncovered. Toluene, propylene, and acetaldehyde were chosen for this study because they are all important reactive components of urban air that contribute significantly to the evolution of photochemical smog. None of these species are, themselves, mutagenic in the Ames test. Allyl chlo- ride was chosen to investigate whether chlorinated hazardous air pollutants (HAPs) might yield photooxidation pro- ducts that are more mutagenic than their nonchlorinated analogues. This particu- lar HAP was also chosen because it is the chlorine-substituted analogue of propylene, one of the hydrocarbons we have studied in detail. In urban photochemical smog systems, the air pollutants exist as a mixture of gas-phase species and organic-laden particulate matter. It is therefore impor- tant to determine the ultimate distribu- tion of mutagenic compounds between the gas and paniculate phases for complex mixtures. Thus, we conducted irradiations of wood smoke/NOx and automobile exhaust/NOx mixtures in the smog chamber and measured the muta- genic activities of the reactants and the gas- and particulate-phase products. Throughout these experiments, we have attempted to describe the nature of the photochemical processes that produce the mutagenic products and the specific photooxidation products that contribute significantly to the observed mutagenic activities. Measurements of the mutagenic activity of peroxyacetyl nitrate (PAN) and other peroxyacyl nitrates were made in an attempt to identify photochemically derived muta- gens. PAN is particularly important because it is a product of the photoox- idation of a variety of atmospheric hydrocarbons, and it is present in sig- nificant concentrations in urban air. From the results of the studies of the individual hydrocarbons, we attempted to determine their relative potential contri- bution to atmospheric mutagen production. Procedures For the individual hydrocarbons stu- died, we irradiated mixtures of ~ 1 ppm hydrocarbon in the presence of ~ 0.5 ppm NOx in a 22.7-m3 Teflon smog chamber. The smog chamber can be operated in a flowing mode in which reactants are continuously added at constant concentration, resulting in a steady-state product distribution in the chamber. The product distribution is dependent on the residence time T (where T = chamber volume/total flow rate) of the gases on the chamber. With this method, a constant composition mixture of pollutants can be maintained for long periods of time. A schematic diagram of the reaction chamber and exposure chamber system is shown in Figure 1. For studies of the simple HC/ NOx mixtures, in which the chamber was operated in a dynamic mode, the reac- tants were mixed and diluted in a 140- L stainless steel inlet manifold and then transferred to the chamber through the end plate. For the N2Os/N03 experi- ments, Ns05 was prepared in a small mixing bulb from reaction of N02 with O3 To study irradiated wood smoke/NO, mixtures, we added diluted wood smoke (from an oak-burning wood stove) directly to the reaction chamber by using a metal bellows pump. Automobile exhaust, added directly to the chamber from the tail pipe by using a metal bellows pump, was obtained from a 1980 Toyota Corolla (catalyst equipped). In the wood smoke and automobile exhaust experiments, the initial total hydrocarbon concentra- tions were ~ 20 and 12 ppmC, respec- tively. In both cases the initial NO, concentration was ~ 0.7 ppm. Four 190-L Teflon-coated exposure chambers were used for exposure of the bacteria to the various air streams. These four exposure chambers enabled mea- surements of the mutagenic activity of the following types of air masses: the starting materials (reactants), photooxi- dation products (filtered or unfiltered), clean air, and for the wood smoke experiments, ambient air. For the wood smoke and automobile exhaust experi- ments, particulate matter was collected from the reactant, product, and ambient- air exposure chamber air streams on Teflon-impregnated, glass-fiber filters. The experiments described in this report were conducted by exposing the Ames test bacteria Salmonella typhi- murium to the test gases. Strains TA100 and TA98 were used, both with and without metabolic activation. The expo- sures were conducted by allowing the air to flow through the exposure chambers loaded with, approximately, 50 covered petri dishes containing the bacteria in a nutrient agar. We exposed the bacteria to the components of the chamber air by uncovering the dishes for a specific period of time. This allowed the agar- soluble species to deposit onto the test plates as the air mass flowed through the exposure chamber. Because the agar is mostly water, those species that are water soluble (i.e., polar) are expected to deposit to the test plates. All experiments began with a conven- tional static-mode smog chamber irradi- ation. Once the temporal variation of reactant and product concentrations could be determined and the desired extent of reaction for the dynamic experiments chosen, the dynamic-mode exposures were begun. By varying the reaction chamber residence time in the dynamic-mode experiments, we were able to conduct bioassay measurements for steady-state mixtures having product distributions corresponding to different regions of a conventional static-mode irradiation. Because the concentration profiles for many photooxidation pro- ------- Mass 3L ^ Flow Mixing] Controller Bulb Reaction Chamber 22.7m3 Samples and Exhaust Lights Teflon Figure 1. Schematic diagram of the reaction chamber apparatus. ducts are highly dependent on the extent of reaction (e.g., presence or absence of NO), the change in the mutagenic activity of the mixture as the reaction proceeds can be interpreted in terms of changes in the product distribution. In these experiments, plates were exposed for varying periods of time (typically 2 to 20 h), depending on the sensitivity of the bioassay test. After the exposures, the plates were incubated at 37°C for 48 h, and the number of revertants/plate were counted. The particulate-phase filter samples obtained in the wood smoke and automobile exhaust experiments were soxhlet extracted with methylene chloride, and the extracts were tested for mutagenic activity by using the standard plate incorporation test. For the wood smoke and automobile exhaust experiments, the smog chamber was operated in a static mode, in which the gas-phase exposures were conducted after the chamber lights were turned off. To improve our understanding of the nature of the photochemical processes occurring in the smog chamber that lead to the production of mutagenic products, we quantitatively measured a wide variety of reactant and product species during the chamber irradiations. These measurements were made by using a wide variety of techniques, including continuous gas monitors, gas, liquid, and ion chromatography, and gas chromatog- raphy/mass spectrometry. From infor- mation about changes in the mutagenic activity of the product mixture with respect to changes in product concen- tration (such as PAN), it is possible to discover the possible causes of observed increases in mutagenic activity. Results and Discussion A typical static-mode smog chamber HC/NOx irradiation for toluene is pres- ented in Figure 2. For all hydrocarbons studied, the removal of the hydrocarbon occurred through OH radical reaction, as long as NO was present. After the NO was removed, O3 began to accumulate. For propylene and ally! chloride, signif- icant reaction with Oa occurred at long extent of reaction. The static mode plots for propylene, acetaldehyde, and ally! chloride are similar to that in Figure 2. We conducted Ames test exposures at short extent of reaction and at long extent of reaction, near the ozone maximum. In all cases, a relatively small increase in the number of revertants/plate was observed for the short extent of reaction, whereas at long extent of reaction, a much larger response was observed. In the case of toluene, roughly 500 induced revertants/plate (i.e., in excess above the clean air or reactant controls) were observed with Strain TA100. Similar results were observed for propylene and acetaldehyde. In all cases, the mutagenic activities determined with Strain TA100 were considerably larger than with TA98. For many of the exposures conducted, groups of plates were covered during the exposure period, effectively stopping the dosage of those plates at that point. This method allowed construction of a dose- response curve for each exposure. A typical dose-response curve (with TA100) is shown in Figure 3 for the products of the photooxidation of propyl- ene at the long extent of reaction. The curve ultimately bends over because of ------- 1.0 -\ 0.9- Dilution -8000 O Toluene ONO D /VO.-A/O A 03 • PAN x CNC 23456 Time, h Figure 2. Static-mode toluene/NO*/HiQ/air irradiation. 700- 0 5 W 15 20 Exposure Time, h Figure 3. Dose-response curve for T= 7.5-h irradiated Ctft/NO, mixtures, TA 100. toxicity effects. The observation (using the Ames test) that these relatively low molecular weight, nonmutagenic, organic pollutants are converted to mutagenic products in the course of their photooxidation should be regarded as significant given their important rotes in urban photochemistry. Thus, it is impor- tant to attempt to determine the cause of the observed mutagenic activities. To determine which chemical species may have caused the mutagenic activity resulting from exposure of the Ames assay plates to these product mixtures, it is necessary to know the dose of each product in the plates and the mutagenic activity of each product. It is possible to estimate the dose of each chemical by measuring its gas-phase concentration in the exposure chambers before and after the plates are uncovered. If we know the dosage and the product mutagenic activities in revertants/yumol, then the contribution of each product to the total observed mutagenic activity can be determined. This type of calculation was conducted for all the known products of the photooxidation of toluene and propylene we were able to measure. Although some products (e.g., HCHO, H202, glyoxal, methylglyoxal) have been found to be mutagenic, none were mutagenic enough to account for more than 5 to 10% of the total observed response. The observation that a very significant mutagenic activity existed for the pro- ducts of the photooxidation of acetalde- hyde at short extent of reaction, when NO was present, provided a very impor- tant clue to the identity of a major mutagenic product. Under these condi- tions, the only organic products present are PAN, HCHO, and, to a much lesser extent, CH3ONOz. However, we had already determined that HCHO and CH3ON02 were not mutagenic enough to contribute significantly to the observed response with the Ames test. These observations led us to conduct an in- depth study of the mutagenic activity of PAN. We have subsequently observed that pure PAN, at concentrations ranging from ~ 100to 500 ppb, yields a measured reversion rate (in our test system, using TA 100) of ~ 14 revertants/h, independ- ent of PAN concentration. The PAN concentrations measured in the toluene, propylene, and acetaldehyde exposures at long residence time were 198, 181, and 171 ppb, respectively, and the measured reversion rates were 27, 24, and 20 revertants/h, respectively. There- ------- fore, it seems likely that PAN accounts for a large fraction of the total observed mutagenic activity of the products in these experiments. We found that the mutagenic activity of the photooxidation products of ally! chloride was highly dependent on the extent of Cl-atom reaction with ally! chloride. Under conditions in which Cl- atom reactions were significant, the mutagenic activity of the products was extremely high. When ally! chloride/NO, irradiations were conducted in the presence of a Cl-atom scavenger to simulate better its actual tropospheric chemistry, the mutagenic activity of the products was substantially less. Even so, the mutagenic activity of this product mixture is roughly 30 times greater than that produced from the photooxidation of propylene. The mutagenicity in this latter case was accounted for by the major photooxidation product, chloroacetalde- hyde. Therefore, this particular chlori- nated HAP yields much more mutagenic photooxidation products than does its nonchlorinated analogue. We conducted a series of wood smoke/ NOx and automobile exhaust/NO, irra- diations to determine the extent to which mutagenic products can be produced in more complex HC/NOX mixtures. We also sought to determine the phase distribu- tion of the mutagenic products that were produced. The mutagenic activities of the gas- and particulate-phase species were measured before irradiation of the mixtures and after the ozone maximum was reached in the irradiations. For both wood smoke and automobile exhaust, the mutagenic activities of the gas-phase species increased dramatically as a result of the irradiation. The measured reversion rates after the irradiation, using TA100, were 174 and 70 rever- tants/h for wood smoke and automobile exhaust, respectively. PAN therefore can account for 10-20% of the total observed (gas phase) mutagenic activities of these two product mixtures. For both wood smoke and automobile exhaust, irradia- tion of the mixtures resulted in a dramatic increase in the total particulate-phase mass. The increase in the paniculate phase mass was the result of nonvolatile photooxidation products adsorbing onto existing paniculate matter. To compare the mutagenic activities of the gas- and particulate-phase species in these complex mixtures, the muta- genic activities for both phases must be expressed in a common set of units. The most convenient set of units is rever- tants/m3. The gas-phase mutagenic activities can be converted to these units if the collection efficiencies of the test plates for the mutagenic gases that pass through the exposure chambers are known. These collection efficiencies were determined in experiments in which the exposures were conducted by using two exposure chambers in series. Typical collection efficiencies were found to be ~ 60 to 70% for both strains. Once mutagenic activities for both the gas- and particulate-phase species are obtained in revertants/m3, they can also be con- verted to revertants/jug using the density (fjg/m3) of the gas- or particulate-phase species in the reaction chamber. The results of these calculations for irra- diated wood smoke/NOx mixtures are presented in Table 1 and are shown graphically in Figure 4. Similar results were obtained for the automobile exhaust/NO, mixtures. The data suggest that, on a revertants/m3 basis, the overwhelming majority of the mutagenic activity of these product mixtures resides in the gas phase. It is therefore clear that in any assessment of the production of mutagenic species from irradiated com- plex HC/NO, mixtures, it is very impor- tant to consider gas-phase mutagens, as well as those that are particle bound. The extent to which mutagens can be produced from NOs reactions with reac- tive atmospheric hydrocarbons was also assessed. We studied the reactions of propylene and wood smoke with NO3/ N205. For the C3He/N205 experiment, the mutagenic activity increased substan- tially. It was unclear, however, which products caused the observed mutagenic activity. In the wood smoke/NzOs exper- iment, the mutagenic activity increased substantially for both the gas- and particulate-phase species after the reaction, as measured with both TA100 and TA98. It is very interesting to note that, even for reactions with NgOs, both strains indicated at least as much mutagenic activity (in revertants/m3) in the gas phase as in the paniculate phase. These measurements indicate that night- time chemistry involving N03 and N205 can also lead to the production of both gas- and particulate-phase mutagenic products in the atmosphere. In addition to our studies of these irradiated mixtures, we also have con- ducted measurements of the mutagenic activities of a series of peroxyacyl nitrates, such as PAN. In this study, it was found that peroxypropionyl nitrate (PPN), peroxybutyryl nitrate (PBN), and peroxybenzoyl nitrate (PBzN) were all mutagenic as determined with Strain TA100. In addition, PBzN was found to be considerably more mutagenic (on a revertants-h"1-ppb"1 basis) than PAN. Both PPN and PBzN have been measured in ambient air. Conclusions and Recommendations The experiments described in this report indicate that mutagenic com- pounds (as determined by using the Ames test) are produced as a result of atmospheric photochemistry. For many HC/NO, systems studied, including propylene and toluene, the photooxida- tion products were much more muta- genic than the reactant hydrocarbons. Not all HC/NO, irradiations produced mutagenic products, however. The pho- tooxidation products of ally! chloride, a chlorinated HAP, were shown to be extremely mutagenic. Some HAPs may be important in terms of atmospheric mutagenesis, even though their ambient concentrations are relatively low. For complex reactive mixtures such as wood smoke, irradiation can produce substan- Table 1. Comparison of the Gas- and Paniculate-Phase Mutagenic Activities for Wood Smoke Before and After Irradiation, Strains TA100 and TA98 TAJ 00 TA98 Gas Paniculate Gas Paniculate Rev/m3 Rev/ug Rev/m3 Rev/ug Rev/m3 Rev/ug Rev/m3 Rev/ug Reactants Products <230 1 7.300 <0.4 1.6 100 180 0.30 0.27 <100 3,230 <0.17 0.30 80 730 0.22 0.94 ------- tial increases in the mutagenic activities of both gas- and particulate-phase species. It was found that the majority of the mutagenic activity of the product mixture of wood smoke is associated with gas-phase species. Much of the increase in mutagenic activity for the particulate phase in our experiments is probably the result of adsorption of gas-phase (low volatility) mutagenic products onto existing particulate matter, rather than a reflection of reactions occurring on the surface of the particulate matter. For most of the product mixtures investigated, a significant portion of the mutagenic activity observed may have been caused by PAN, which has been shown to be mutagenic. The absolute value of the mutagenic activity of this species is currently uncertain, however, and much more work is necessary to determine the mutagenic potential of PAN at ambient concentration levels. Because PAN is ubiquitously present in polluted urban atmospheres, it would also be desirable to determine the biological impact of PAN on other bio- logical systems by using additional bioassay techniques. At this point, laboratory experiments have shown that pollutants commonly found in urban air can be transformed into mutagenic products as a result of typical urban photochemical processes. These results suggest the need to extend this effort in both laboratory and field measurement areas. Ambient measure- ments of mutagenic activities in urban air masses would be helpful in determin- ing the possible correlation of mutagenic activity of the air mass with the presence of photochemical oxidants such as PAN, and may aid in the overall assessment of the contribution of urban photochem- istry to the presence of atmospheric mutagens. 775- 150- 125- 100- I 50H 25- 0~\ TA 100 | | Gas Phase f"':"\ Particulate Phase TA98 Before Irradiation After Irradiation Before Irradiation After Irradiation Figure 4. Comparison of the gas- and particulate-phase mutagenicity of dilute wood smoke in air. P. B. Shepson, T. E. Kleindienst, and E. O. Edney are with Northrup Services, Inc.-Environmental Sciences. Research Triangle Park. NC 27709. L. T. Cupitt is the EPA Project Officer (see below). The complete report entitled "The Production of Mutagenic Compounds as a Result of Urban Photochemistry." 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