EPA/600/3-87/020
                                     June  1987
 THE PRODUCTION OF MUTAGENIC COMPOUNDS
   AS A RESULT OF URBAN PHOTOCHEMISTRY
                    by
  P.B. Shepson, T.E. Kleindienst, and E.O. Edney

 Northrop Services, Inc. - Environmental Sciences
               P.O. Box 12313
       Research Triangle Park, NC 27709
         Contract Number 68-02-4443
              Technical Monitor
               Larry T. Cupitt
     Gas Kinetics and Photochemistry Branch
   Atmospheric Sciences Research Laboratory
     U.S. Environmental Protection Agency
       Research Triangle Park, NC 27711
ATMOSPHERIC SCIENCES RESEARCH LABORATORY
   OFFICE OF RESEARCH AND DEVELOPMENT
  U.S. ENVIRONMENTAL PROTECTION AGENCY
     RESEARCH TRIANGLE PARK, NC 27711

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                                       ABSTRACT
      A series of hydrocarbon/NOx irradiations were conducted in a 22.7-m3 Teflon smog chamber
in which the mutagenic activities (measured using the Ames test bacteria Salmonella typhimurium
Strains TA100 and TA98) of the mixtures were measured before and after the irradiation.  Simple
hydrocarbons including propylene, acetaldehyde, toluene, and allyl chloride, as well as two complex
hydrocarbon mixtures, wood smoke and automobile exhaust, were examined in detail.  For these
systems the irradiated mixtures were much more mutagenic than the reactant hydrocarbons.  For the
complex mixtures the distribution of mutagenic activity between the gas and particulate phase was
measured, and, for all cases, a majority of the mutagenic activity of the product mixture was found in
the gas phase. For each system studied, the observed mutagenic activities were interpreted in terms
of the product species that may have contributed to the observed response.  In most cases (except
allyl chloride), peroxyacetyl nitrate was believed to contribute significantly.
                                            in

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                                  TABLE OF CONTENTS
Abstract	      iii
List of Figures	      vi
List of Tables 	1	      vii
List of Abbreviations  	     viii
Acknowledgements  	      ix

   1   Introduction	       1
   2   Conclusions and Recommendations  	       6
   3   Experimental Procedures	       7
   4   Results	      16
      4.1   Irradiated Toluene/NOx Mixtures    	      16
      4.2  Irradiated Propylene/NOx Mixtures  	      25
      4.3  Irradiated CH3CHO/NOX Mixtures	      36
      4.4  Irradiated Allyl Chloride/NOx Mixtures   	      40
      4.5  Com pi ex Mixture Irradiations   	      47
      4.6  Peroxyacyl Nitrates   	      58
   5   Discussion	      62

References	      73

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                                   LIST OF FIGURES



 FIGURE                                                                          PAGE

  3-1  Schematic diagram of the reaction chamber apparatus   	       8

4.1-1  Static-mode toluene/NOx/H^O/air irradiation   	      16
4.1-2  Reaction profile: toluene/NOx irradiation, 10/26, T = 6.7 h  	      20
4.1-3  PAN concentrations:  toluene/NOx irradiation, 10/26, i = 6.7 h  	      21
4.1-4  Formaldehyde concentrations: toluene/NOx irradiation, 11/30,1; = 3.Oh  	      21
4.1-5  Methylglyoxal profiles: toluene/NOx irradiation, 11/30,i = 3.Oh   	      22
4.1-6  TA100 dose-response curve for toluene photooxidation products: 8/86,i = 6.7h  ._-      24
4.2-1  Time profiles for the major products in the CsHe/NOx irradiation (static mode)	      26
4.2-2  Time profiles for the aldehydes and nitric and formic acids in the CsHe/NOx
      irradiation  	      27
4.2-3  Time profiles for nitrates in the CsHe/NOx irradiation  	      27
4.2-4  Dose-response curve for T = 2.7-h irradiated CaHg/NOx mixture, TA100	      31
4.2-5  Dose-response curve for T = 7.5-h irradiated CaHe/NOx mixture, TA100	      31
4.2-6  Dose-response curve for products of propylene ozonolysis, TA100	      35
4.3-1  CH3CHO/NO/NO2 static-mode irradiation	      37
4.3-2  Dose-response curves for irradiated CHsCHO/NOx exposures    	      39
4.4-1  Experiment A: reaction chamber component concentrations and mixture
      mutagenic activity, CaHjCI/NOx irradiation  	      43
4.4-2  Experiments: reaction chamber component concentrations and mixture
      mutagenic activity, CaHsCI^He/NOx irradiation  	      43
4.4-3  Experiment C: reaction chamber component concentrations and mixture
      mutagenic activity, C2Hg/NOx irradiation	      44
4.4-4  Experiment D: reaction chamber component concentrations and mixture
      mutagenic activity, C3H5CI/C2H6/NOx irradiation  	      44
4.5-1  Wood smoke static irradiation with 500 ppb of additional NOX-  Selected product
      and reactant profiles   	      49
4.5-2  EAA volume distribution of diluted wood smoke before and following
      irradiation in Experiment F  	      50
4.5-3  Dose-response curve for the mutagenic activity (TA100) of the gas-phase
      components of wood smoke (+ 500 ppb NOx) before and following irradiation   ...      51
4.5-4  Dose-response curve for the mutagenic activity (TA98) of the paniculate extracts
      of wood smoke ( +500 ppb of NOx) before and following irradiation   	      52
4.5-5  EAA volume distribution, wood smoke/^Os   	      55
4.5-6  Automobile exhaust/NOx reactant and product concentrations   	      57
4.6-1  Dose-response curve for exposure of TA100 to pure PAN (220 ppb)  	      60

  5-1  Comparison of gas-and particulate-phase mutagenicity of dilute wood smoke in
      air  	      69
                                          VI

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                                    LIST OF TABLES
 TABLE                                                                           PAGE

4.1-1  Bioassay Results, Toluene/NOx Experiments, Average Revertants/Plate(S.D.)   	     18
4.1-2  Average (±20%) Product Concentrations, Toluene/NOx Irradiations   	     19
4.1-3  Water Plate Concentrations, Nanomoles per Plate (10/26, T = 6.7 h, Toluene/NOx
      Irradiation) 	     22
4.1-4  Reactant and Product Concentrations, ppb (Toluene/NOx Irradiation, 8/86)   	     24
4.2-1  Average Reactant and Product Concentrations (ppb) for CsHg/NOx Irradiation
      (Dynamic Mode)  	     28
4.2-2  CaHe/NOx Irradiations: Concentrations of Species Detected from Exposure
      Chamber Decrease (Exp. II) and/or Appearance in the Surrogate Plates    	     29
4.2-3  Measured Mutagenic Activity for Exposure of TA100 to the Experimental Gas
      Streams in Revertants/Plate (± 1o) for the CaHe/NOx Irradiations  	     30
4.2-4  Average Reactant and Product Steady-State Concentrations for the CsHe/^Os
      Exposure	     32
4.2-5  Observed Mutagenic Activities in Revertants per Plate for the CaHg/^Os
      Exposure	     34
4.2-6  Concentrations of Products (CaHe/^Os Exposure) Detected in Micromoles per
      Plate as Calculated from Exp. II    	     34
4.3-1  Average Reactant and Product Concentrations (ppb) for Irradiated
      CH3CHO/NOX Exposures  	     38
4.4-1  Reactant and Product Concentrations (ppb) and Bioassay Results  	     45
4.5-1  Individual Gas-phase Inorganic and Hydrocarbon Concentrations, ppb, for
      Wood Smoke/NOx Irradiations    	     48
4.5-2  Paniculate Extract Data for Wood Smoke/NOx Irradiations, Nanogram of
      PAH/Milligram of Particulate Mass  	     49
4.5-3  Measured Mutagenic Activities of the Gas and Particulate Phases of Irradiated
      Wood Smoke   	     51
4.5-4  Wood Smoke/N2O5 (Experiment G) Reactant and Product Concentrations (ppb) .
      and Product Mutagenic Activities	     54
4.5-5  Irradiated Automobile Exhaust/NOx (Experiment H) Reactant and Product
      Concentrations (ppb) and Mutagenic Activities	     56

  5-1  Mutagenic Activities Measured by TA100	     63
  5-2  Comparison of the Gas- and Particulate-Phase Mutagenic Activities for Wood
      Smoke Before and After Irradiation, Strains TA100 and TA98   	     68
  5-3  Calculated Mutagenic Activities of the Photooxidation Products of Atmospheric
      Hydrocarbons  	     71
                                          VII

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                                   LIST OF ABBREVIATIONS
PAH




PAN




HAP




PPN




PBN




PBzN




CPAN




PGDN




2-HPN




2-NPA




NPPN




EAA




CNC
polycylic aromatic hydrocarbon




peroxyacetyl nitrate




hazardous air pollutant




peroxypropionyl nitrate




peroxybutyryl nitrate




peroxybenzoyl nitrate



chloroperoxyacetyl nitrate




propylene glycol dinitrate




2-hydroxypropyl nitrate




2-nitratopropyl alcohol




nitroxyperoxylpropyl nitrate




electrical aerosol analyzer




condensation nuclei counter
                                            VIII

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                                  ACKNOWLEDGEMENTS
      We thank LT. Cupitt and LD. Claxton of the U.S. Environmental Protection Agency for their
helpful discussions and technical support of this project, and LD. Claxton for his direction of the
bioassay work. We thank G.R. Namie, J.H.  Pittman, CM. Nero, D.N. Hodges, and E.E. Hudgens of
Northrop Services, Inc. - Environmental Sciences for their assistance in conducting the experiments,
and E. Perry and G. Harris of Environmental Health Research and Testing, Inc., for their assistance
with the bioassay work.  Dr.  Bruce Ames (University  of California, Berkeley, CA)  provided the
Salmonella typhimurium tester Strains TA100 and TA98.
                                           ix

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                                        SECTION 1
                                     INTRODUCTION
      Over the last several years, there has been an increased level of concern that exposure to
polluted urban atmospheres may pose a "significant, although not yet precisely defined" threat to
human health (1).  Risk assessments (2,3) have tended to focus on the threat of developing cancer
from exposure to vapor-phase air toxics that are emitted directly from urban sources.  Efforts at
chemical and biological characterization of ambient samples, however, have primarily examined the
aerosol-bound species (4-6).  This analytical emphasis on paniculate  pollutants was probably due to
the greater ease of sampling,  chemically analyzing, and conducting bioassays of the easily
concentrated aerosol-bound organic compounds. Limited data on the biological impact of vapor-
phase pollutants (7) in actual ambient air masses do  indicate a significantly  increased level of
mutagenic activity in  heavily polluted and industrialized areas.   Clearly, any  comprehensive
assessment of the potential health impact resulting from exposure to air toxics must consider both
the gaseous and particulate-bound chemicals.

      In addition, it has become increasingly apparent that exposure and risk assessments must also
consider the impact of atmospheric  reactions  on both hazardous and nonhazardous air pollutants.
Haemisegger et al. (2) found a significant  risk from exposure to formaldehyde,  an  air-toxic
compound that is both directly  emitted from combustion sources and is also formed from  the
photochemical oxidation of the many hydrocarbons  present in urban  air.  The a,0-dicarbonyl
compound, glyoxal, is a photooxidation product of  a  wide variety of  important atmospheric
aromatic hydrocarbons  (e.g..toluene) and has also been shown to  be mutagenic (8).  Researchers
have also found that a variety of oxygenated  and nitrogenated species caused mutagenicity in the
Ames test (9). These classes of compounds are  continuously created and removed during the  normal
photooxidation of hydrocarbons. The  atmospheric transformation of many ubiquitous urban air
pollutants may, therefore, contribute significantly to the presence of mutagens in the ambient air,
even though they, themselves, may be harmless.

      Recent studies have shown that human exposure to wood stove and fireplace emissions may
be a cause for public health  concern.  In a study of the Denver "brown cloud," Wolff ef al. (10)
determined that a significant fraction of the organic fine paniculate matter (<2.5 pm) from  Denver
aerosol could be attributed to wood smoke emissions.  In addition,  paniculate extracts from wood
smoke have been tested for mutagenic  activity by using the Ames test (11-13).  These studies have

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generally shown low direct-acting mutagenic activity with Salmonella  typhimurium Strain TA98,
although Dasch (11) reported substantial increases with the addition of S9 metabolic activation.  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 ^Os on the surface of
atmospheric paniculate matter can lead to increases in the mutagenic activity of adsorbed species
such as PAHs (14-19). In addition. Bell and Kamens (20) and Kamens et al. (21) demonstrated that
PAHs adsorbed on wood smoke paniculate matter can photodegrade at significant rates.  Clearly,
atmospheric reactions can have a significant impact on the mutagenic activity of atmospheric
paniculate matter.

      Although there is some evidence (7) of significant concentrations of gas-phase mutagens in
ambient air, there  has been a relatively small effort aimed at identifying mechanisms for the
production  of gas-phase mutagens through atmospheric photochemistry compared to efforts in the
area of particulate-phase mutagenesis. To test for the mutagenic activity of gas-phase species  by
using the standard plate incorporation test  (22), it  is necessary to concentrate the volatile air
pollutants from the air sample into a suitable solvent  for testing.  Some attempts to  measure
gas-phase ambient  mutagenicities have employed a solid sorbent, such  as XAD, to first collect the
species and then to extract them into a solvent.  However, these processes pose a  number  of
potential problems, especially loss of volatile species during the extraction and workup procedures.

      In this report we present a review of the results of experiments we have conducted by using  an
alternative  technique for measuring the mutagenic activity of gas-phase species generated  by
photochemical reactions. For this technique, the Ames assay test plates are dosed continuously by an.
experimental air mass flowing over the uncovered plates, thereby enabling the soluble species to
deposit continuously during the exposure (23).

      Although some preliminary screening  experiments indicated that irradiation of propylene
produced mutagenic products, toluene was the first hydrocarbon we studied in detail. Toluene was
selected because of its predominance  in urban atmospheres.  It represents an important reactive
component in  the  ambient urban hydrocarbon  mix (24).  It is frequently the most abundant
nonmethane hydrocarbon pollutant found  in populated areas, and its concentration at urban and
suburban sites typically  ranges from 5 to  30 ppb  (25,26).  In  addition,  many  of toluene's
photooxidation products (e.g., formaldehyde and peroxyacetyl nitrate [PAN]) are also produced  by
other simpler hydrocarbons.  Finally,  the  photochemistry of toluene had  been  fairly  well
characterized (27).

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      Our experiments with toluene demonstrated that its photooxidation products were indeed
mutagenic,  but the product  distribution proved  to be too  complex to permit  a  convenient
determination of the mutagenic species. We turned our attention back to propylene, for which the
photooxidation had been thoroughly studied and the major products had been identified.  The
chemical mechanisms for the photooxidation process had been characterized in detail (28), and they
appeared to be far simpler than the mechanisms for toluene.  Propylene appeared to be a good
prospect for describing and understanding the formation of  mutagens through photochemical
processes.

      The irradiations of propylene produced mutagenic products, confirming our  preliminary
results.  In an attempt to identify the specific mutagenic compound(s), we began  to change the
experimental conditions.  We  varied the residence time (and, therefore, the product distribution)
and learned that more mutagenicity was observed at long  reaction times.  Because ozone  and
NO3/N2Os are present in the system at long residence times, we studied the reaction of propylene
with these compounds in  separate,  isolated  experiments.  These  major subsystems of the
photooxidation process could  not account for the formation of the observed mutagenicity.  The
results led us to consider whether a major product of the photooxidation process, acetaldehyde,
might react further and produce the mutagenic compound(s).  We therefore conducted a series of
experiments with acetaldehyde and found that it did indeed  produce mutagenic products.  The
implications of this result are far reaching, for acetaldehyde  is produced during  the  normal
photochemical oxidation of many common pollutants and is ubiquitous in urban atmospheres at
levels as high as 10-20 ppb (26,29).

      Although most of the  reactive hydrocarbon concentration  in urban atmospheres  is
represented by organic compounds containing carbon and hydrogen only, a variety  of chlorinated
solvents are also present that are important in assessing urban exposures to air toxics (2).  Many of
these  chlorinated hydrocarbons are considered  hazardous air pollutants (HAPs) and are currently
being considered for regulatory action (30) because of their potential human health effects. During
the normal photochemical processes that occur in polluted atmospheres, the chlorinated HAPs may
be converted to products that are more (or less) mutagenic than the reactant HAP.  The mutagenicity
of the chlorine-containing products can be substantially different from the products of the more
common hydrocarbons.  For example, chloroethylene oxide  (a  possible oxidation product  of vinyl
chloride) is 10,000 to 15,000 times more mutagenic than isethylene oxide (31).

      We conducted  a series of experiments involving  exposure of the Ames  test bacteria
Strain TA100 to the photooxidation products of allyl chloride (3-chloropropene). This  particular HAP

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was chosen both because it is of interest to the regulatory community and because it is the
chlorinated analogue of propylene, which we also studied in detail.  Our experience with propylene,
along with the fact that the chemical mechanism  and reaction kinetics for ally! chloride are fairly
well understood (32), should facilitate the identification of individual mutagenic products for this
particular HAP.

      For the individual hydrocarbons stud led, 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 dynamic/flowing mode, in which reactants are  added continuously, resulting in a  steady-state
product distribution in the chamber.  The product distribution is dependent upon the residence time
T (where T = total volume/flow rate) of the gases into the chamber.  With this method, a bioassay of
the chamber effluent can be performed for  long periods of  time with  a constant  composition
mixture of pollutants.

      In urban photochemical smog systems the air toxics present exist both as gas-phase species and
as paniculate matter. We thought it important, therefore, to examine the distribution of mutagenic
compounds between the gas and particulate phases in complex mixtures. We conducted a series of
experiments  in which wood smoke/NOx mixtures were irradiated, using  the same 22.7-m3 smog
chamber operated in a static mode.  In these experiments the mutagenic activities of both the gas-
and particulate-phase reactants  and products were measured.  Similar experiments  were also
conducted for automobile  exhaust.  Information regarding the extent to which mutagenic
compounds can be produced from irradiation of automobile exhaust/NOx mixtures is clearly needed,
given the importance of automobile exhaust as a source of reactive hydrocarbons that control urban
photochemical smog chemistry.

      It has also recently been  demonstrated that NO3/N2Os  mixtures can react with gas-phase
species such as naphthalene (33), a wood stove emission component, to produce mutagenic products
(i.e., nitronaphthalenes). We also conducted an experiment in which we reacted NOs/I^Os with
propylene and also one in which we reacted NOs/NjOs with a wood smoke mixture. The mutagenic
activity of the gas- and particulate-phase (for wood smoke) reaction products was measured.

      The results of a preliminary laboratory study indicated that PAN is one major mutagenic
product of the photooxidations of toluene, acetaldehyde, and propylene. We therefore conducted a
detailed investigation of the mutagenic activity  of PAN and several other peroxyacyl nitrates,

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specifically, peroxypropionyl nitrate (PPN), peroxybutyryl nitrate (PBN), and peroxybenzoyl nitrate
(PBzIM).

      The results of all experiments described in this report will be discussed in terms of the nature
of the photochemical processes that  produce the mutagenic products and in terms of the specific
photooxidation products that contribute significantly to the observed mutagenic activities. We will
also attempt to determine the relative potential contribution to atmospheric mutagen  production
for each of the  individual hydrocarbons studied and,  to some extent, for the complex  mixtures
studied.

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                                       SECTION 2
                          CONCLUSIONS AND RECOMMENDATIONS


      The experiments described in this report indicate that mutagenic compounds (as determined
by using the Ames test) are produced as a result of atmospheric photochemistry.  For many HC/NOx
systems studied, including propylene an'd toluene, the photooxidation products were much more
mutagenic than the reactant hydrocarbons.  Not all HC/NOx irradiations produced  mutagenic
products, however. The photooxidation products of allyl 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 substantial 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 paniculate 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 biological 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  measurements of mutagenic activities in urban air masses would be helpful in determining
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
photochemistry to the presence of atmospheric mutagens.

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                                       SECTION 3
                               EXPERIMENTAL PROCEDURES
      A schematic diagram of the reaction chamber and exposure chamber system is shown in
Figure 3-1.  The reaction chamber is a cylindrical Teflon bag surrounded by lights to simulate the
solar spectrum at the earth's surface. The chamber can be operated in two distinct modes: static and
dynamic.  In the static mode, the chamber is first brought to the desired initial  pollutant
concentrations.  Then, the lights are turned on, and the concentrations of the pollutants are
monitored as a  function of time.  (Figure 4.1-1 is an example of a concentration vs. time profile
obtained from a static-mode irradiation.) In the dynamic mode, the chamber can be  operated as a
continuously  stirred tank reactor, with reactants being continuously added  to the  irradiated
chamber.  The extent of reaction in the dynamic mode is controlled by the residence time.  The
residence time is the average time that a  molecule remains in the chamber and is  calculated by
dividing the volume of the chamber by the flow rate through the chamber. At short residence time,
the product distribution is similar to that present at short reaction during the comparable
static-mode irradiation.  Similarly, at long residence time, the products are like those found near the
end of the static-mode  experiment.  Because  the product distribution for the  dynamic mode is
determined by the residence time and not by how long the lights have been on,  a consistent,
unchanging product distribution can be maintained for whatever period of time is necessary to
conduct chemical and biological analyses.  (Figure 4.1-2  is an example of a concentration  vs.  time
profile of a dynamic-mode irradiation.)

      Clean air was produced by an AADCO clean air generator supplied with compressed air from a
Quincey Model  325-15 air compressor.  The dilution  air from the AADCO was  controlled in the
0-5ft3/min range with a Teledyne Hastings-Raydist Model NAHL-5P mass flow controller.  For the
toluene experiments the air was humidified by a Sonimist Model 600L ultrasonic spray nozzle.  For
the dynamic-mode experiments the reactants were allowed to flow into the chamber  at  2 to
5ft3/min, and they were mixed with a 60.5-cm  diameter three-blade impeller powered  with a
0.25-hp motor.  The chamber was constructed of a 7.5-m-long cylindrical Teflon  bag  connected on
each end to aluminum end plates,  1.96  m in  diameter and  coated with fluorocarbon paint.
Irradiation of the reactor was provided by a total of 180  GE F-40 blacklight bulbs and 36 sunlamps.
For experiments with the individual hydrocarbon/NOx mixtures, in which the chamber  is operated in
a dynamic mode, the reactants are mixed in a 150-L stainless steel inlet manifold and then
transferred to the chamber through the end plate.   Toluene (Fisher Scientific,  HPLC grade) was

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added to the inlet manifold by bubbling N2 (MG Scientific, prepurified grade) through an impinger
bottle containing ~ 100 ml toluene maintained at 0°C. Flow through the impinger was controlled with
a Tylan Model FC260 (0-20 cm3/min) mass flow controller.  One percent mixtures in nitrogen of
acetaldehyde, propylene, allyl chloride, and nitric oxide (NO) were similarly added (MG Scientific) by
using a mass flow controller.  As shown in Figure 3-1, ^Os was prepared  by mixing 03 and NO2
(1% in N2)  in a 3-L Pyrex mixing bulb. The 03 was produced at ~1% in 02 with a Welsbach
Model T-408 03 generator supplied with zero-grade ©2- The 03 reacted with NO2 in the mixing bulb
to produce NOs, which was in equilibrium with ^Os, as indicated in Reactions 1 and 2. The resultant
         mixture was then diluted with clean dry air in the inlet manifold.
                                                                                          1
For the wood smoke/^Os experiment the initial
propylene + ^Os experiment, it was ~1.8 ppm.
                                                                                       2,-2
                                                    concentration was —2 ppm, and for the
                                  NO
                                  inNj
                              Mass
                              Flow
                            Controllers
                                           Mass /
                                           Flow
                                         Controller
                                                                Paniculate
                                                                  Filter

\J Reaction Chamber
"A 22 7 m3
Mixing
Fan
T \
-1}


Effluent
Bio-
chamber


Effluent
Bio-
chamber

1
Sam
Exhi
i

                                               Lights
                                                       Teflon
 Figure 3-1. Schematic diagram of the reaction chamber apparatus.

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      For the wood smoke experiments, wood combustion was achieved by using an Ashley
Model 71 SOB wood stove. The wood used was a mixture of locally obtained varieties of oak.  To
load the reaction chamber, a portion of the wood smoke from the chimney was continuously drawn
through an 8-cm steel pipe to a dilution tunnel where the wood smoke was diluted with ambient air
and cooled to ambient temperature.  The unfiltered ambient air was drawn through the dilution
tunnel by a 0- to 20-m3/min turbine.  This dilute wood smoke mixture was then added to the reaction
chamber by using a Metal Bellows Corporation Model MB-151 metal bellows pump. Similarly, in the
experiments involving automobile exhaust, the mixture was transferred to the chamber from the tail
pipe with a metal  bellows pump. A 1980 Toyota Corolla (catalyst equipped) operating at idle at
relatively high engine rpm was the source.  The fuel used was a  "super unleaded" grade
([R + M]/2 = 91). For the wood smoke and automobile exhaust experiments  the initial total
hydrocarbon  concentrations were ~20 and  12 ppmC,  respectively. In both cases the initial NOx
concentration was —0.7 ppm.

      The PAN-type  compounds were prepared by several different techniques.  For PAN itself, a
number of experiments  were conducted in which PAN was synthesized in the  gas phase  by
irradiation of C^/NCVCHsCHO mixtures (34), as indicated in Reactions 4-6 below.
                          Cl+CH.CHO -?HCl+CH
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                                                                                       9'*9
      Four 190-L Teflon-coated exposure chambers were used for exposure of the bacteria to the
various air mixtures studied.  The bacteria were exposed to the followi    different types  of air
masses: reactants, filtered or unfiltered products, clean air, and, for the wood smoke experiments,
ambient air.  The gases were transferred to each of the exposure chambers through 3/8-in. Teflon
tubing at a flow rate of 14L/min, which was maintained and  measured with a needle valve and a
calibrated rotameter. All lines were as short as possible to maximize transfer efficiency.

      The experiments for measuring gas-phase mutagenic activities were conducted by exposing
the Ames test bacteria Salmonella typhimurium to the effluent from the smog  chamber.  Strains
TA100 and TA98 were used, both with and without 59 metabolic activation.  The exposures were
conducted by allowing the chamber air to flow (at 14 L/min) through Teflon-coated,  190-L exposure
chambers (essentially glove boxes) loaded with, typically, 50 covered Pyrex Petri plates containing
the bacteria in a nutrient agar. In these experiments, the plates were dosed with the components of
the chamber air by uncovering them for a specific period of  time. This allowed the agar-soluble
species to deposit into 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), as are most photooxidation
products, deposit  into the test plates.  Many polar species, such as formaldehyde,  are completely
removed from the air mixture as it flows through the exposure chambers (see Section 4.1).

      Glass Petri dishes containing S. typhimurium were provided  by the Health Effects Research
Laboratory of U.S. EPA. The plates were prepared by adding 0.1 ml of the S. typhimurium culture to
3 ml of an agar overlay at 45°C (with or without 0.5 ml of S9 mix).  This mixture was then poured onto
~45 ml of plate agar in a glass Petri dish. Colony counting was performed with an Artec 880 automatic
colony counter, using previously published guidelines (37). The test procedures used were those of
Ames et al. (22), except for the following modifications: [1] glass Petri dishes were used, [2] 45 ml of
base agar per plate was used, [3] minimal histidine at the same final concentration was placed in the
bottom agar rather than the top agar, and [4] 3 ml of overlay agar with ~ 1 x 108 bacteria were used.
The rat-liver homogenate (59) fraction was prepared from male Charles River CD-1 rats (Wilmington,
MO) induced with Aroclor 1254 (22). In many of the experiments the exposure chambers were also
loaded with several "survivor" plates. For the survivor plates, the bacteria concentration was diluted
by roughly 104 and additional histidine was added.  The magnitude of the dilution was such that, in
the absence of toxicity, —500 colonies per plate were produced. Therefore, these plates could be used
to indicate the possibility of toxicity effects for the test plates.
                                            10

-------
      For several experiments, plates containing either distilled buffered water or water containing
 2 mM NaOH (to measure PAN deposition) were added to the exposure chambers as a model for the
 quantity of gas-phase component that solubilized  into the biotesting medium.  For  the toluene
 experiments the acid and  base/neutral  fractions of the water were extracted with methylene
 chloride after each exposure (38).  The extracts were then analyzed for benzaldehyde and the cresols
 by GC/MS.  For several experiments the water plates were analyzed for formaldehyde by the
 chromotropic acid technique (39),  and for nitrite,  nitrate, formate, and acetate  ions by ion
 chromatography.  PAN deposition was measured by using the NaOH plates. In basic media, PAN
 decomposes as indicated in  Reaction  10, enabling determination of its concentration  by
 measurement of acetate or nitrite ions (40).
                                                                                        10
                 CH.C(O)OONO~+20H- -> Cff,C(O)0-+ArO:+O9+H9O
                    O          Z              O           ft    ft   £t
      For the wood smoke and automobile exhaust experiments, paniculate matter was removed
from the reactants effluent, and ambient air streams by Teflon-impregnated glass-fiber filters
(T60A20 Pallflex, 13.34 cm).  The particulate-phase filter samples were Soxhlet extracted with 250 ml
of pesticide-grade methylene chloride (Fisher Scientific) for 6 h.  The mutagenic activities of these
concentrated extracts were then measured by using the standard plate incorporation test (22).

      For each gas-phase exposure, the gas-phase mixture was allowed to flush completely through
the exposure chamber several times, at which point the plates were uncovered, effectively starting
the exposure.  Then, groups of plates were covered periodically throughout the exposure period,
effectively stopping the dosage of the plates at certain points and enabling construction of dose-
response curves. For these  experiments the plates were exposed for a period of time (typically
2-20 h) necessary to induce a significant increase in the reversion level. After the  exposures, the
plates were incubated at 37°C for 48 h and the number of revertants/plate were counted.

      For some experiments (e.g., wood smoke and automobile exhaust), it was not  feasible to
operate the chamber in a dynamic mode.  In those cases, the exposures were conducted by first
preparing the test  mixture in the reaction chamber and then exposing the bacteria to the  air
sampled from the reaction chamber.  Air withdrawn from the chamber was continuously replaced by
clean dilution air, which diluted the test mixture. For these experiments the effective  exposure times
(i.e., dilution-corrected) for the dose-response curves were obtained by using Expression I:
                                                                                        (I)
                                         ex
                                 t   =    exp(-kt)dt
                                       ' o
                                           11

-------
where t  is the exposure time in real time and k is the dilution rate constant.
       ex
      All experiments began with a conventional static-mode smog chamber irradiation, so that the
temporal variation of reactant and product concentrations could be determined and the desired
extent of reaction for the dynamic experiments could be chosen.  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
products (e.g., organic peroxides) 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.  For several experiments conducted with
individual hydrocarbons, exposures were conducted at short  reaction chamber  residence time
(t = 3 h), where NO is present, and at long residence time (x = 7 h) near the ozone and PAN maxima.
When NO is present, formation of peroxides is inhibited, whereas at long residence time when NO
has been removed, peroxides such as PAN can be present in significant concentrations.

      Throughout the static-mode irradiations and the dynamic-mode exposures, a variety of gas-
and particulate-phase species were measured from both the reaction chamber (at the end opposite
the inlet manifold) and the exposure  chambers.  Toluene was measured by using a Varian
Model 1400 GC containing a glass column packed with 0.1% SP-1000 on Carbopack C, operated at an
He flow rate  of 20cm3/min and a temperature of 200°C. Propylene  was measured by using a
Hewlett-Packard  Model 5840A GC containing a 6.4-mmx2-m stainless steel column packed with
80/100 Porapak QA and operated isothermally at 130°C.  Acetaldehyde was also measured by
operating the Porapak QA  column at 145°C.  Allyl chloride was measured by using the Varian GC
containing a  3.2-mmx2-m glass column packed with 1% SP-1000 or Carbopack B  at 80°C.
Hydrocarbons were injected by using a solenoid-activated Seizcor six-port valve that was switched on
and off with  a Chrontrol Model CD timer.  Calibrations were  performed by preparing ~1-ppm
standards in clean air in 200-LTeflon bags.  NO and NOx were measured by using a CSI Model 1600
oxides-of-nitrogen analyzer calibrated with a certified standard of NO in N2 (MG Scientific).  Ozone
was measured with a Bendix Model 8002 ozone analyzer calibrated by using a Oasibi Environmental
Corporation UV ozone monitor.  Relative humidity in  the chamber was measured with an EG + G
Model 880 dew  point  hygrometer calibrated with saturated salt solutions. Particle number
concentrations were measured with a TSI Model 3020 condensation  nuclei counter.  Aerosol size
distributions (0.01-1 urn) were measured with a Thermo Systems, Inc., Model 3030 electrical aerosol
analyzer (EAA).  For the wood smoke and automobile exhaust  experiments the total hydrocarbon
                                           12

-------
concentration was determined by using a Beckman Model 400 HC analyzer, which was calibrated
with pure samples of propane.  CO was measured  by using a Bendix Model 8501-5CA infrared CO
analyzer.

      The PANs were measured by using a Vako electron capture detector after separation on a
column, which was packed with 10% Carbowax 400 on Gaschrom Z, at 25°C. For several experiments
this chromatograph was calibrated according to the procedure of Lonneman et al. (41). For some
experiments PAN and chloroperoxyacetyl nitrate (CPAN) were measured by bubbling 30-L chamber
samples through 5cm3 of deionized water adjusted to pH 12 with NaOH,  which converts these
species to acetate or chloroacetate ions, respectively (40).  The ions were then separated  and
detected by using 10"3M  HCI eluent with a  Dionex System 10  ion chromatograph containing a
Dionex HPICE AS2 column.  For the series of experiments in which we measured the  mutagenic
activity of several PANs (Section 4.6), the PAN chromatograph was calibrated by preparing samples
of the pure, synthesized PAN (35) in air in Teflon bags. The PAN concentration was measured as the
total NOx concentration  by using the  NOx monitor. We determined that  the  efficiency of the
monitor for converting PAN to NOx is  100%. Methyl nitrate was also measured by using the  PAN
chromatograph, which was calibrated with pure samples synthesized according to the procedure of
Johnson (42).

      The aldehydes, formaldehyde, acetaldehyde, acrolein, glyoxal,  methylglyoxal,  and
benzaldehyde were measured by using the DNPH/HPLC technique of Kuntz et al. (43). Benzaldehyde
and o-,m-, and p-cresol analyses were also conducted by injection of 5-L cryotrapped samples into an
HP 5985 GC/MS containing a 2-mx2-mm glass column  packed  with  0.1%  SP-1000 on
80/100Carbopack C.  Nitric acid was  measured by drawing air through a 25-mm nylon filter
(1-um pore size), extraction of the filter with 10"5 M perchloric acid solution, and subsequent analysis
for nitrate ion by using a Dionex System 12 ion chromatograph with a Dionex Model 60361 anion
separator column.

      For the propylene experiments (Section 4.2) the organic nitrates (PGDN, 2HPN, 2NPA) were
measured on a Varian 1200 GC, employing a 6.4-mm x2-m glass column packed with 10% SP-1000 on
80/100 Supelcoport and operated  isothermally at 155°C.  Detection was achieved with a Vako
Model 1408 electron capture detector.  Samples were injected with a 5-ml glass and Teflon syringe.
These products were identified under  the same  conditions by  using an HP Model  5985 GC/MS.
Details of the product identification and the calibration procedure are in a separate publication  (44).
Weak acids (e.g., HCOOH) were collected by bubbling the effluent through a 1  mM NaOH solution.
                                           13

-------
Ion chromatography employing a 2.5 mM B4O92" eluent was used for separation and detection of the
weak acid anions. Calibrations were made with solutions prepared from the alkali salts.

      The allyl  chloride photooxidation products chlorc  :etaldehyde, 3-chloroacrolein,  and
1,3-dichloroacetone were collected by bubbling 10-L chamber samples through 2cm3 of methanol
cooled to 0°C in an impinger bottle. Collection efficiency for this technique was found to be s 99%,
as determined by using two bubblers in series. These three compounds were separated by a
3.2-mm i.d. glass column, packed with 1% SP-1000 on Carbopack B, that was contained  in a
Varian 1200 GC.  Detection was achieved with a Valco Model 1408 electron capture detector.  The
identification of these species was conducted by GC/MS, as described in a previous publication (32).

      An ethylene/NOx dynamic-mode exposure was conducted to test for the mutagenic activity of
a subset of the  photooxidation products, specifically the inorganic  species HNC»3, 03,  H2C>2,  and
HOaNOa- Ethylene was measured by using a Varian 1400 GC (FID), which contained a 3.2-mm x2-m
stainless steel column packed with 60/80 Carbosieve G and operated at 175°C.

      For the wood smoke and automobile exhaust experiments, methane, acetylene, ethylene, and
ethane were measured with a Varian 1400 GC, using a 3.2-mmx2-m stainless steel column packed
with 60/80 Carbosieve G and operated at 150°C.  Chloromethane, propylene, propane, and 1-butene
were measured  with a Varian  1200 GC, using a 6.4-mmx2-m stainless steel column packed with
80/100 Porapak QS and operated at 130°C. Isoprene, furan, and 2-methyl furan were  measured with
an HP Model 5840 GC, using a 2-mmx2-m glass column packed with 0.1% SP-1000 on 80/100
Carbopack C and temperature  programmed from 40 to 200°C at 20°C/min.  Benzene, toluene,
xylenes, benzaldehyde, styrene, and m-methyl styrene were measured by pumping 25-L samples
through Pyrex tubes packed with Tenax GC.  The Tenax-filled tubes were then thermally desorbed at
2758C with a Nutech 320 thermal  desorption  unit.  The desorbed samples were analyzed with an
HP5985 GC/MS,  which contained a 2-mmx2-m glass column packed with 10% SP-1000 on 80/100
Supelcoport. The column was temperature programmed from 50 to 225°C at 20°C/min.  Calibrations
were performed by desorption of Tenax  tubes containing samples of the pure compounds at the
1-nmol level.

      The particulate-phase filter samples were Soxhlet extracted with 250 ml of pesticide-grade
methylene chloride (Fisher Scientific) for 6 h. The extracts were then concentrated under a stream of
prepurified N2 (MG Scientific) and analyzed for PAH concentration, using the GC/MS, by injection
onto a 2-mm x2-m glass column packed with  3% SP-2250 on 80/100 Supelcoport. The column was
programmed from 200 to 300°C at 10°C/min.  For the compounds measured, the GC/MS peak areas
                                           14

-------
were measured relative to pyrene and quantified as pyrene.  The extracts were prepared for bioassay
by solvent exchanging a portion of the methylene chloride extract with 2-ml samples of dimethyl
sulfoxide (Burdick and Jackson Laboratories, Inc.)- Blank filters were also extracted to serve as both
chemical and bioassay controls.

      To determine the effectiveness of the use of XAD-2  as a collection medium for subsequent
measurement of gas-phase mutagenicities, a wood smoke/NOx irradiation was conducted in which
gas-phase mutagens were collected by pumping the filtered reaction chamber air through an
11-cmx8.5-cm diameter stainless steel cylinder packed with XAD-2 resin. After exiting this XAD-2
trap the air then  passed into an exposure chamber.  After the exposure period the XAD-2  was
Soxhlet extracted  with methylene  chloride,  followed by extraction with methanol.   Each of the
extracts were then concentrated, using a kuderna-danish concentrator, and then solvent exchanged
with DMSO, followed by the standard plate incorporation test.
                                            15

-------
                                        SECTION 4
                                        RESULTS
4.1  IRRADIATED TOLUENE/NOX MIXTURES

      For irradiated toluene/NOx mixtures, the consumption of toluene occurs solely through
reaction with OH radicals.  Static toluene/NOx irradiations were first performed to determine the
product distributions as a function of irradiation time.  Figure 4.1-1 shows the results for a static (i.e.,
no flow) irradiation of 970 ppb toluene and 390 ppb  NO,  along  with the time profiles for several
major components  of  interest.  As can be seen in this figure, a  sharp burst of particle formation
occurred at or near the ozone maximum.  We have found the overall reaction rate to be heavily
dependent on the chamber humidity, possibly due to heterogeneous production (45) of nitrous acid
(MONO), which can photolyze to produce OH  radicals.  We therefore attempted to operate the
chamber at ~50% relative humidity (at 22°C) in all experiments.  The relative humidity for the static
experiment was 57% at 19°C.
                1.0-1
                                  34567
                                   11/16   11/30         10/11
                                          Time, h     u"26
 Figure 4.1-1. Static-mode toluene/NOx/H2O/air irradiation.
                                           16

-------
      For the dynamic runs, we attempted to maximize the compositional differences between the
two sets of exposures.  For the toluene study, duplicate exposures were conducted at both short
extent of reaction (i.e., with  NO present) and  at long extent of reaction (i.e.,  near the ozone
maximum). We were, however, limited on the fast-flow (i.e., small t) end by the maximum output of
the clean air generator (5ft3/min or T = 2.7 h) and on the slow-flow (large x) end by the required
sampling and exposure chamber feed  rates (2ft3/min or i - 6.7 h). For the dynamic exposures,
residence times (T) of 3.0 h and 6.7 h were chosen. Because of the dependence of the reaction rate
on humidity and apparent wall radical sources, the extent of reaction at these times was difficult to
duplicate exactly.  Figure 4.1-1 shows, by experiment date, the extent  of reaction (by the vertical
lines) at which the bioassays were performed, based on the observed distribution of toluene, NO,
NOx-NO, and ozone. The 11/16 and 11/30 and the  10/11 and 10/26 experiments were meant to  be
duplicates of each other.  Each of the four experiments were  conducted at the same nominal
reactant concentrations, as indicated in Figure 4.1-1.

      The results of the biotesting for these four experiments are presented in Table 4.1-1.  The
"spontaneous" samples are a group of plates prepared and counted in the same way as the exposed
plates, but  which remain  in the biotesting laboratory throughout the exposure.  The spontaneous
plates thus  measure the natural reversion rate observed under sterile conditions.  Although the data
for the clean air biochamber are, on the average, larger than the data for the spontaneous plates,
we do not  interpret this  as being a positive response to clean air.  Rather, we attribute this  to
exposure of the clean air plates to a number of environmental factors (such as a brief exposure to
sunlight) that the spontaneous plates do not experience. For the purposes of interpreting the data
for the other exposure chambers, it is reasonable to compare the  numbers to those for the clean air
biochamber.

      In addition, two clean air irradiations were performed at T; = 3.0 h and x = 6.7 h. The bacteria
were exposed to the irradiated clean air, but no significant increase in the revertant level for the
irradiated clean  air, relative to the nonirradiated air, was observed for either residence time.
Therefore, we concluded that irradiation of clean air does not produce gas-phase mutagens.

      The following products were measured in these experiments:  PAN, ozone, formaldehyde,
acetaldehyde, glyoxal, methylglyoxal, benzaldehyde, the three cresol isomers, nitric acid, carbon
monoxide, and total particulate matter. The average effluent concentrations for each species are
presented in Table4.1-2.  These concentrations agreed for the  duplicate experiments to  within
±20%.
                                            17

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    TABLE 4.1-2. AVERAGE (± 20%) PRODUCT CONCENTRATIONS. TOLUENE/NOX IRRADIATIONS

PAN
03
HCHO
CH3CHO
CHOCHO
CH3C(0)CHO
C6H5CHO
o-cresol
m-cresol
p-cresol
HNO3
CO
Particles/cm3
Effluent, ppb
T; » 3.0 i
14
48
48
5
16
20
7
11
3.5
3.5
62
169
0

- 6.7
132
280
82
8
24
34
12
5
1.0
1.5
84
490
400
Test Plates,*
nmol/plate
t-3.0 x = 6.7
91 198
-
367 933
-
34 103
125 333
38 66
111 30
37 6
37 9
-
-
-
* Calculated from Expression II.
      In Figure 4.1-2 we present the reactant profiles for the 10/26 (t = 6.7 h) experiment, which
demonstrate the approach to steady state and the degree to which it was  maintained in  these
experiments.  Once the effluent reactant and product distributions reached steady state, the four
biochambers were loaded with  the covered Petri plates.  The biochamber product concentrations
were then allowed to come back up to their steady-state values. At this point the various product
concentrations were measured  in the  biochambers with the plates covered.  The values obtained
thus represented the transfer  efficiencies between the reaction chamber and the exposure
chambers. These measurements required an ~2-h period before the plates could be uncovered. At
the end of this 2-h period the plates were uncovered and the exposure was begun. During the ~ 18.5-h
exposures the product concentrations  were measured in the chamber effluent and in the
biochambers.  From the differences in biochamber concentrations before and after the plates were
uncovered, we can determine the relative amounts of exposure to each chemical (see Discussion).
                                           19

-------
                O Effluent Toluene  + CNC
                ® Input Toluene    T BR Toluene
                D NO           • BF Toluene
                A NOX-NO        4 BU Toluene
                V03
                                                                                   -6000
                                                                                   -1000
                                                                   40
                                                                          45 PIMM   50
                                                                            CloMd
                    10       15      20       25 PIMM  30       35
                                             Open
                                        Time, h
Figure 4.1-2. Reaction profile: toluene/NOx irradiation, 10/26, t * 6.7 h.
      In Figures 4.1-3 - 4.1-5 we present reaction and exposure chamber measurements for PAN,
formaldehyde, and methylglyoxal, respectively. PAN is only slightly water soluble; only — 15% of the
PAN passing through the exposure chamber deposits into the plates. For very water soluble species
such as formaldehyde and methylglyoxal, essentially all of the gas-phase components deposit into
the plates when they are opened. A similar measurement could not be done for acetaldehyde, since
it is emitted from the plates when they are opened,  possibly as a metabolic product from  the
bacteria.  For all  but glyoxal, the transfer efficiencies of the various products from the reaction
chamber to the biochambers were at least 50%.

      As a model for determining  the amount of vapor-phase material deposited into the plates,
four plates containing buffered water (phosphate/biphosphate buffer, pH = 7.4) and one with pure
deionized water were placed in each biochamber during the exposure period. The buffered water is
used as the best model of the agar that can also be easily analyzed.  Deionized water is used to
circumvent ion chromatographic analysis problems. The concentrations found in the water plates
can then be compared with the calculated deposition into the bioassay medium (see Discussion).
This was done for the 10/26, i = 6.7-h run for the cresols, benzaldehyde, formaldehyde, nitrite,  and
nitrate.  The cresols, benzaldehyde, and formaldehyde were measured in the buffered water plates,
and nitrite and nitrate were measured in the deionized water plates.  The results are presented in
Table 4.1-3.
                                            20

-------
       150-
                                                                          45 PIMM  50

                                                                             aowd
Figure 4.1-3. PAN concentrations: toluene/NOx irradiation, 10/26, t - 6.7 h.
       80-
       70-
       60-
       50-
   o
   x

   i
                       10
                                           Time, h
                                                                          45  PI. n 50

                                                                             Qowd
Figure 4.1-4. Formaldehyde concentrations: toluene/NOx irradiation, 11/30, i = 3.0 h.
                                             21

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          80-
                                                   X
                                            Time, h
                                                    30 PI t«  35
                                                       Op«n
                                            40     45   Ptiui 50
                                                       Closed
 Figure 4.1-5. Methylglyoxal profiles: toluene/NOx irradiation, 11/30, i » 3.0 h.

            TABLE 4.1-3. WATER PLATE CONCENTRATIONS, NANOMOLES PER PLATE
                        (10/26,1 « 6.7 h,TOLUENE/NOx IRRADIATION)

       Exposure   CgHsCHO   o-Cresol   m-Cresol   p-Cresol   HCHO    NO2'
       BCA            <1       <1       <1        <1        43      87       32
       BR
<1
<1
<1
<1
130
456
16
BU
BF
5
5
17
11
6
3
8
4
600
613
348
261
210
32
      It is clear from inspection of the data in Table 4.1-1 that there is a significant difference in
results between the two residence times for the effluent biochambers.  Whereas there appears to be
no measurable increase in revertants for the effluent-exposed biochambers at T = 3.0 h (relative to
the clean  air biochambers [BCA]), there  is a definitely significant increase for the filtered and
unfiltered biochambers (BF and BU) at x = 6.7 h (roughly 500 induced revertants per plate for TA100,
                                            22

-------
 relative to the clean air plates). The effect appears to be of the same relative order of magnitude for
 both TA100 and TA98, indicating the possibility of more than one type of mutagen being present at
 this residence time. (Mutations occur by different mechanisms in the two strains. [22]) In addition,
 the number of revertants appears to be the same with and without S9 activation, indicating that the
 mutagens present are direct acting.  Although there is  a  measurable concentration of aerosol
 present at T = 6.7 h, the fact that the BF and  BU responses were  comparable  indicates that
 paniculate matter does not contribute significantly to the observed response. It is quite likely that
 the extent of deposition of paniculate matter into the plates in BU is very low.

      After these  initial toluene experiments, a problem with the clean  air bioassay plates was
 identified.  The clean air plates were being exposed to sunlight during transport of the plates to the
 NSI-ES facility, resulting  in an elevated level of mutagenicity.  In addition, we found  that
 dose-response curves could be obtained by covering groups of plates throughout  the exposure.
 Therefore,  in 8/86, an additional toluene/NOx dynamic experiment was  conducted at shorter
 residence time (i = 2.7 h) for an irradiated mixture of 5.1 ppm toluene/0.9 ppm NOX.  Because the
 HC/NOx ratio was much higher for this experiment, the light intensity was lowered by a factor of five
 to decrease the extent of reaction.  This experiment was conducted both to obtain a dose-response
 curve and  to determine the magnitude of the mutagenic activity at  an extent  of reaction
 intermediate between those of the previous experiments.  In Table 4.1-4 we present the steady-state
 reactant and product concentrations  measured during  the  20-h exposure (TA100 only,  -59).  The
 indicated product distribution corresponds to that in Figure 4.1-1  near the beginning  of the increase
 in OB concentration.  In Figure 4.1-6  we present the dose-response curve obtained for this
 experiment. As witnessed by decreased survivor counts at 20 h, toxicity effects began  to appear near
 10 h.  Because the  change in toluene  concentration, A toluene, for this experiment  is only slightly
 larger than that for the  previous t =  6.7-h experiments, it appears that the response observed for
this experiment is even larger than that observed at longer extent of reaction.  It is possible that in
the i = 6.7-h exposures, toxicity effects resulted in smaller apparent mutagenic activities. It is clear,
 however, that the reaction of OH with toluene produces products that are significantly mutagenic,
as determined by  using  the Ames test.  The  possible  mutagenic products will be discussed in
Section 5.
                                            23

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               TABLE 4.1-4. REACTANTAND PRODUCT CONCENTRATIONS, PPB
                           (TOLUENE/NOx IRRADIATION, 8/86)
                              Inlet
                       Effluent
                     % Yield (C/C)
 Toluene
 NO
 NOX
 03
 HNO3
 CH3COOH
 PAN
 HCHO
 CH3CHO
 C6H5CHO
 (CHO)2
 CH3C(O)CHO
5145(1310)
    896
    942
4403 (±144)
     2
    394
    153
    160
     47
     85
     50
     6
     37
    150
    134
                                               1.8
                                               3.3
                                               0.1
                                               0.2
                                               5.0
                                               5.8
                                               7.7
          800-
          700-
          100-
                      8/12/86 Toluene/NOx
                                       10 11  12  13  14 15 16  17  18  19  20
                                   Exposure Time, h
Figure 4.1-6. TA100 dose-response curve for toluene photooxidation products: 8/86, t * 6.7 h.
                                         24

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4.2 IRRADIATED PROPYLENE/NOX MIXTURES

      At the time of the initial toluene study, none of the identified products, including
formaldehyde, benzaldehyde, glyoxal, and methylglyoxal, could account for the observed response
(see Discussion).  We were, at that point, faced with a difficult task in terms of accounting for the
observed response.  The photooxidation of toluene occurs through a very complex  mechanism
producing a large  number of products with small yields, the majority of the products being
unavailable commercially (46,47). The fact that the photooxidation of toluene led to mutagenic
products was a very interesting result, but the identification of all the mutagenic products appeared
to be a formidable task. We turned our attention to a photochemically simpler system and focused
our efforts, once again, on determining the potential for the production of mutagenic products
from the photooxidation of propylene. We present here the results of exposures of TA100 to the
products of irradiated propylene/NOx mixtures at both short and  long extents of reaction.  We also
sought to isolate major subsystems of the photooxidation process and, therefore, examined the dark
reactions of 03 with propylene and of NOa/^Og with propylene.  For the CaHe/NOx irradiations, the
reaction chamber was operated at flows of 50 and 140 Lpm, yielding average residence times of 7.5
and 2.7 h, respectively.  In the CsHe/^Os exposure, the average residence time was 2.7 h.
      For the CaHe/NOx irradiations the effluent from the reactor flowed through two exposure
chambers in parallel. One chamber contained plates with TA100 and the other with TA100 plus 59.
Surrogate plates for chemical analyses were placed in both chambers.  The flow through the
exposure chambers was 14 Lpm, yielding an exposure chamber residence time of 13.5 min. The
exposures were conducted at 25°C for 20 h, and the total  gas volume through each chamber was
17 m3.  Two additional exposure  chambers were employed. Clean air  was passed through one
chamber and the reactant mixture through the second.  Each chamber contained TA100, with and
without metabolic activation, and two sets of surrogate plates.
      For the CsHe/^Os exposure the two effluent exposure chambers contained 25 plates each of
TA100 with and without  S9.  To one of these exposure chambers we added 0.7  ppm NO
(continuously) to test for the presence of mutagenic peroxy nitrates (which are removed due to the
presence of NO). For the CsHe/^Og exposure, a reactants exposure chamber was not employed since
NzOs would react with the  water evaporated from the plates to produce gas-phase HNOa, which is
toxic to the bacteria.

      Each of the exposures was conducted by adjusting the reactant concentrations to the  desired
levels  in the inlet manifold and allowing the product  distribution to reach a  steady state in the
chamber.  The exposure chambers were then loaded with the covered test plates.  The exposure
                                           25

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chambers were resealed, and the product concentrations in the exposure chambers were brought
back to their steady-state levels. At this point the plates were uncovered, starting the exposures.
      Figure 4.2-1 shows the time profiles of 03, NO, NOx-NO, CjHe, and PAN measured  in the
         static run, which had the same initial conditions as employed in the dynamic experiments.
The NO2 photolysis rate constant under these conditions was ~0.3 min"1, and the dilution rate was
0.037 h"1 . The relative humidity was maintained at ~50%. In Figure 4.2-1, the vertical lines at 2.7 and
7.5 h indicate the product distributions predicted for dynamic experiments with residence times of
2.7 and 7.5 h, respectively, and performed under the same initial conditions as the static experiment.
At 2.7 h, the reaction is still at a relatively early stage since there is approximately 20% (100 ppb) of
the initial NO remaining.  At 7.5 h, the reaction is near the ©3 and PAN maxima.
                                                            r=7.5 hrs
          800-
          700-
          600-
          500
       JD
       Q.
       Q.
       C
       O
       u
                                                                         10
                                         Time, h
 Figure 4.2-1. Time profiles for the major products in the CsHg/NOx irradiation (static mode). The
 concentrations intersecting the vertical lines at 2.7 and 7.5 h represent the nominal product
 distributions for the two residence times.

      The time profiles of HCHO, CH3CHO, HNO3, and HCOOH are given in Figure 4.2-2.  The
aldehydic concentration difference between the two residence times is minimal.  Nitrate extracted
from nylon filters is generally identified with  HNOs. In this experiment, the measured HCOO" is
associated with HCOOH.  For conditions under which the sampling was performed, the detection
limit for HCOOH is 15 ppb. Figure 4.2-3 shows the formation of three organic nitrates produced
                                           26

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during the photooxidation:  propylene glycol dinitrate (PGDN), 2-hydroxypropyl nitrate (2-HPN),
and 2-nitratopropyl alcohol (2-NPA).  The reaction of OH with C^ in the presence of 62 and NO
forms 2-HPN and 2-NPA as stable products.
                       • HCHO
                       D CHjCHO
                       A HCOOH
                       O HNO,
                                                                  10
                                         Time, h
 Figure 4.2-2. Time profiles for the aldehydes and nitric and formic acids
 irradiation.
      The dynamic experiments had the
same initial concentrations as the static ex-
periment, within experimental uncertainty.
The  inlet concentrations, total flow, and
irradiation intensity remained constant
throughout the dynamic experiments and
yielded steady-state product  distributions.
The average values for the observed product
concentrations in the reaction chamber for
each of the two dynamic experiments are
given in Table 4.2-1.  These concentrations
can be compared with those of the static
experiment by examining  Figures 4.2-1,
4.2-2, and 4.2-3 at the appropriate extent of
reaction.  A nominal correspondence be-
tween the two experiments can be seen.
Figure 4.2-3. Time profiles for nitrates in the
         irradiation.
                                           27

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         TABLE 4.2-1. AVERAGE REACTANT AND PRODUCT CONCENTRATIONS (ppb) FOR
                                   IRRADIATION (DYNAMIC MODE)
Compound
C3H6
NO
NOx-NO
03
HCHO
CH3CHO
PAN
HNO3
CH3ONO2
PGDN
2-HPN
2-NPA
CO
RH* (%)
HCOOH
Effluent
Input Concentration
Concentration (T • 2.7 h)
826 ± 33 548 ± 24
505 ± 24 155 ± 29
200 ± 39 521 ± 38
18 ± 7
219 ± 32
118 ± 8
6 ± 3
40 ± 10
1.2 ±0.2
BDL
1.8 ± 0.4
5.6 ± 1.1
81 163
52
BDL
Effluent
Concentration
(i = 7.5 h)
100 ± 13
BDt
347 ± 21
451 ± 29
247 ± 71
90 ± 4
181 ± 12
73 ± 17
1.4 ± 0.2
0.8 ± 0.2
3.0 ± 1.1
10.4 ± 2.6
-
65
40
       * RH - relative humidity in units of percent.
       t BDL-below detection limit.
      The gas-phase products  in the exposure chambers were measured before and during the
exposure  of the biological assay.  These values are used as one means of estimating the amount of
material deposited into each plate.   The net deposition of material into the plates during the
exposure  is tabulated in Table4.2-2 under the  heading "Chamber Decrease."  These values are
calculated from the relationship given in Expression II:
                   X.
plate
RT
                                                   N
                                                                                      (ID
                                           28

-------
 where Xj is the plate concentration of species i; PfiR) and PfiE) are the partial pressures (patm) of
 species i in the reaction and exposure chambers, respectively; V^ is the volume of effluent passing
 through  the exposure chamber; and Np is the total number of plates.  The amounts of material
 presented in Table 4.2-2 represent upper limits since some of the material may also be lost to the
 exposure chamber walls.
              TABLE 4.2-2. C3H6/NOx IRRADIATIONS: CONCENTRATIONS OF SPECIES
               DETECTED FROM EXPOSURE CHAMBER DECREASE (Exp. II) AND/OR
                         APPEARANCE IN THE SURROGATE PLATES *
Compound
HCHO
PAN
HNO3
CH3ONO2
PGDN
2-HPN
2-NPA
HCOO-
X
Chamber
Decrease
1.62
0.048
_t
0.001
0
0.017
0.047
-
* 2.7 h
Surrogate
Plates
1.15
-
0.085
-
-
-
-
0.21
t
Chamber
Decrease
2.28
0.89
0.78
0.004
0.0024
0.034
0.10
-
= 7.5 h
Surrogate
Plates
1.65
1.48*
0.83
-
-
-
-
1.29
      * Concentration given as iimol/plate.
      f Not measured.
      * From nitrite measurement.

      A second method for determining the amount of material deposited into the biological assay
is from the analysis of the surrogate plates. The results for each measured compound are given in
Table 4.2-2.  A comparison of the results for the two methods can be made for HCHO, PAN,  and
HNOa.  'n each case, the agreement is within 50%. This enhances the confidence for a species that
can be measured using only one of the techniques.
      The mutagenic activities observed for the CsHe/NOx photooxidation mixture are presented in
Table 4.2-3. Three sets of experiments are presented, each with its own set of laboratory controls
and  background measurements.  The first three rows in Table 4.2-3  represent laboratory
measurements of the characteristics of the particular assay used in the experiment.   The
"spontaneous" plates measure the natural reversion rate observed under sterile conditions in the
laboratory.  These serve as a check to establish the viability of the bacteria.  The strain sensitivity is
                                           29

-------
determined by adding 1.0 and 0.5 pg, respectively,  of known mutagens,  sodium azide and
2-aminoanthracene, to TA100 and TA100 + S9,  respectively.  A clean air control chamber was
employed to measure the background reversion  rate in the ambient environment under which the
photochemical experiments take place. In addition, a reactants exposure chamber, which samples
the reactant gases directly from the inlet manifold, was used to check for mutagenic activity of the
reactants. The clean air irradiation (Table 4.2-3) is a separate experiment that serves as a check to
ensure that the observed revertant level is due to the photochemical effluent rather than a chamber
artifact.  The revertant levels at both residence times show increased values over the clean air
control.  However, the mutagenic activity for the long residence time is substantially greater than
that for the short residence time.  The addition of 59 metabolic activation does not statistically
increase the mutagenic activity, relative to those plates without S9 mix, indicating that the mutagens
present are direct acting.
         TABLE 4.2-3. MEASURED MUTAGENIC ACTIVITY FOR EXPOSURE OF TA100 TO THE
   EXPERIMENTAL GAS STREAMS IN REVERTANTS/PLATE ( ± 10) FOR THE C3H6/NOX IRRADIATIONS
     Exposure
     Condition
                       Clean Air
                      Irradiation
                                2.7 h
                                                                         7.5 h
w/oS9
                            w/S9
W/OS9
w/S9
w/oS9
w/S9
Spontaneous    164+12   178 + 11
15.4 nmol
sodium azide    909 + 73
                                            142±10   131 ±8
                                             919
                                                  184 + 29   131 + 18
                                                   1388
2.6 nmol
2-a mi no-
anthracene - 1 525
Clean air
chamber* 361 ±60 379 ±70
Reactants
chamber* 356 ±48 363 + 67
Effluent
chambert 374 + 59 404 ± 69
421 - 536
194±15 225±32 278±92 290 + 58
261 ±41 293 ±54 294 ±58 268 ±44
356 + 69 373 + 48 903 ±69 968+101
  * Numbers are averages for-25 plates.
  * Numbers are averages for —50 plates.
      The t = 2.7-h and x = 7.5-h exposures were repeated in 1/86 (roughly  1.5 years later) in
experiments in which groups of plates were covered throughout  the exposures.  The  product
distributions were nearly identical to those previously observed.  The dose-response curves obtained
for the i; = 2.7-h and T; = 7.5-h experiments are presented in Figures 4.2-4 and 4.2-5, respectively. In
both cases it can be seen that the initial part of the curve is reasonably linear, but that toxic effects
begin to  occur near 10 h. For the t = 7.5-h exposure the survivor counts were substantially lower
                                           30

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                                                                          C)
beyond a 10-h exposure time.  In agreement with the earlier experiments, the response at the
T = 7.5-h residence time was at least twice as large as that observed for the T; = 2.7-h residence time. It
is clear from these experiments that mutagenic products are produced from the  photooxidation of
propylene.

              500-
              480-
              460-
              440-
              420-
              400-
           jo  380-
           Q.
           |  360-
           nj
           5  340-
           I  320-
              300-
              280-
              260-
              240-
              220-
              200
                   n   r
                               5  6
                                       8  9  10  11
                                          Exposure t, h
                                                   iT   i   i   i   r   i   ir
                                                   12  13  14 15 16  17  18 19 20
 Figure 4.2-4. Oose-response curve for i » 2.7-h irradiated CaHg/NOx mixture, TA100.
      To determine the extent to which
     reaction  with CsHe  may lead to
mutagenic products that could account for
the large mutagenic activity observed in the
irradiated CsHe/NOx system (at  x = 7.5 h),
we conducted a CsHe/^Os exposure.
                                             700-
      As mentioned previously,  to one
effluent stream we added NO at ~ 0.7 ppm.
It has been reported (48) that the reaction
of CsHe with NOs leads to the production of
large yields of nitroxyperoxypropyl nitrate
(NPPN). Since we have shown that PAN (a
peroxy nitrate) is a mutagen with TA100
(see  Section 4.6),  it is possible that NPPN or
                                                                   10
                                                               Exposure Time, h
                                                                                       20
                                           Figure 4.2-5. Dose-response curve for x = 7.5-h
                                           irradiated CsHg/NOx mixture, TA100.
                                            31

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other peroxy nitrates formed may be mutagenic as well.  Peroxy nitrates such as PAN are in
equilibrium with their respective peroxy radical and  NO2 (49), so they can be removed via NO
addition as shown in Reactions 11 and 12 jelow.
                               RO NO   ^RO +NO                               11'~11
                                 £i   £t       £.     &

                               R02+NO -+RO+N02                                  12
where R is an organic group. The resultant alkoxy radical would then be removed by reaction with
Oz or NO2 or by unimolecular decomposition.  The presence  of mutagenic peroxy nitrates can
therefore be checked by comparison of the results for the two exposure chambers, since these
compounds will not be present in the exposure chamber with added NO.

      The average inlet and effluent reactant concentrations, chamber parameters, and steady-state
concentrations for the products measured in the CaHg/^Os exposure are presented in Table 4.2-4.
This experiment was conducted without added humidity.
               TABLE 4.2-4. AVERAGE REACTANT AND PRODUCT STEADY-STATE
                      CONCENTRATIONS FOR THE C3rVN205 EXPOSURE
          Parameter Measured*	Inlet	Effluent	
         Inlet Reactant Flow, L/min        140 ± 0.085
         NOX                           1.76 + 0.20               1.9410.19
         C3H6                           1.26 ± 0.04               1.00 ± 0.04
         HCHO                                                 0.017 ± 0.004
         CH3CHO                                              0.023 ± 0.007
         a-nitratoacetone                                       0.032 ±0.011
         PGDN                                                 0.002 ± 0.001
         2-HPN                                                 0.008 ± 0.002
         2-NPA                                                0.002 ± 0.001
         PAN                                                     0.020
        * Concentrations in parts per million.
                                          32

-------
      In this experiment, CaHe and N2Os were diluted and mixed together in the inlet manifold.  It
was possible that they could react as shown in Reactions -2 and 13,
                                NO3 + Cff  -» Products                                   1 3
prior to entering the reaction chamber. In a separate CsHe/I^Os static experiment under similar
conditions (44), we observed a value for A^Os/ACsHe of roughly  2:1.  Since AC3He  in this
experiment is 0.26 ppm, the assumption can be made that the initial ^65 concentration was
-0.5 ppm. This then leads to an initial NC>2 concentration of ~0.8 ppm, given the total NOx value of
1.8 ppm listed in Table 4.2-4.  By use of values for l<2 and k-2 of 4.6 x 10"12 cm3 molecule"1 s"1 and
3.7 x 10"1s"1, respectively (50), an equilibrium  NO3 concentration  of 2.2 ppb is estimated. Using the
value ki3 = 4.2x 10"15 cm3 molecule"1*"1 (51), and a residence time in the inlet manifold of 1 min, we
calculate that < 1% CsHg reacted in this time. Therefore, the inlet manifold concentrations can be
taken as the starting concentrations.

      The mechanisms for formation of the  products listed  in  Table 4.2-1  have been presented
elsewhere (44,48).  A mass balance of the product concentrations from Table 4.2-4 indicates that we
cannot account for a large part of the reacted CsHg.  It may be that much of it is present as NPPN,
which we could  not detect.  The  PAN concentration was measured as 0.020 ppm in  a  separate
experiment under equivalent conditions.

     The results of the bioassays performed are presented in Table 4.2-5, along with the laboratory
controls.  The final plate concentrations for each product of the CsHg/^Os reaction are presented in
Table 4.2-6, as calculated from Expression II.

     As shown in  Table 4.2-5, the two sets  of effluent chamber plates exhibit induced revertant
levels of approximately 180 revertants/plate  above the clean air values.  There seems to be no
significant difference between those with  and without metabolic activation,  indicating  that the
mutagens present are direct acting. In addition, no significant difference was observed in the results
for the two effluent exposure chambers, one of which had NO added. Although this may indicate
that  the  peroxy  nitrates present are not mutagenic, their transfer efficiencies to the exposure
chambers may be low,  or they may  be deposited on the reaction chamber walls during the 2.7-h
average residence time. Bandow et al.  (48) observed a significant wall loss rate for NPPN in their
reaction chamber.
                                            33

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        TABLE 4.2-5. OBSERVED MUTAGENIC ACTIVITIES IN REVERTANTS PER PLATE FOR
                                           EXPOSURE
Exposure Condition
Spontaneous
1 5.4 nmol sodium azide
2.6nmol
2-am i noanthracene
clean air chamber*
effluent chamber*
effluent ( + NO) chamber*
TA100
133 ± 13
668

180 ± 31
375 ± 122
323 ± 81
TA100 + S9
122± 21

590
169 ± 33
351 ± 102
369 ± 112
* Numbers are averages for ~25 plates.
                  TABLE 4.2-6. CONCENTRATIONS OF PRODUCTS (C3H6/N->05
              EXPOSURE) DETECTED IN MICROMOLES PER PLATE AS CALCULATED
                                    FROM EXP. II
Product
HCHO
HNO3
PAN
2-HPN
2-NPA
PGDN
CH3ONO2
HCOOH
CH3C(O)CH2ON02
pmol/plate
(max. - Exp. II)
0.2
<0.6
0.2
0.1
0.02
0.02
-
-
0.4
     In this experiment, we showed that chemical mutagens are produced from the reaction of NO3

with C3H$. However, a computer modeling study we conducted of the C3Hg/NOx irradiated system

indicated that NO3 reaction with C3He would have occurred only to the extent of -0.010 ppm at 7.5 h.

Since ACsHg due to NO3 reaction in the CsHg/^Og exposure was 0.26 ppm, we can estimate that
                                        34

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     reactions would have contributed on the order of only 10 revertants/plate in the irradiated
 C3H6/NOX system at 7.5 h.

      Because the mutagenic activity at long residence time, when 03 was present, was substantially
 greater than at short residence time, it seemed reasonable to determine whether the products of
 ozone reaction with  propylene may account for the observed response in the irradiated CaHe/NOx
 system. We conducted exposures of TA100 to the products from the reaction of 5.4 ppm CsHg with
 0.90 ppm 03 in the dark at x = 2.7 h. In addition, an experiment was conducted in which 0.21 ppm
 NC>2 was also added to this mixture to test for the possibility that radicals produced in the ozonolysis
 might react with  NO2 to produce mutagenic  organic nitrates.  For both experiments, ACsHe was
 ~ 1.0 ppm and the overwhelmingly predominant products were acetaldehyde and formaldehyde. The
 observed mutagenic activities for these two exposures are presented in Figure 4.2-6.
                                     O  C3H6/03	
                                     D  C3H6/03/N02*	
                                        * points displaced 0.1 h to the right)
                                       10
 I
15
                                                                20
                                          Time, h
 Figure 4.2-6. Dose-response curve for products of propylene ozonolysis, TA100.

      As shown  in Figure 4.2-6, the  number of revertants per plate increases measurably (but
nonlinearly) with exposure time. The survivor data (not presented here) indicate that toxicity effects
may become important for exposures of  10 h or more.  It is clear from these experiments that
mutagenic organic nitrates are not formed as a result of addition of NO2-  The principal objective of
                                            35

-------
these experiments was to attempt to determine the extent to which CsHe reactions with 03 could
account for the large response (-600 excess revertants/plate) observed in the irradiated CsHg/NOx
system.  Computer simulations of the irradiated CsHe/NOx system, under the conditions of that
exposure, indicated that only ~0.20 ppm of the reacted CaHg had been removed  by reaction with
ozone. Because the ozone plus propylene dark experiment (Figure 4.2-6) involved five times as much
reaction, it appears that the products of the  CsHg/Os reaction could not have caused a significant
response in the CaHe/NOx irradiations.  It will  be shown in the Discussion section that formaldehyde
could account for the entire response observed in these C^H^/O^ experiments.
      An exposure of TA100 to the products of an irradiated ethylene/NOx mixture (t = 2.7 h) was
conducted to determine whether HCHO and the inorganic photooxidation products present might
yield a significant mutagenic activity.  For this experiment the inlet C2hU and NOx concentrations
were  1.16 and 0.28 ppm,  respectively, and ACaHU was 0.54 ppm.  The steady-state 03 and HCHO
concentrations were 0.47 and 0.33 ppm, respectively.  In addition, concentrations of HMOs,
and HO2NO2 should have been comparable to those present in the long residence time
experiments. The results of the ~20-h exposure of TA100 to this mixture indicated only -35 excess
revertants/plate (relative to the clean air and reactant controls).  This response is essentially what
would be expected based on the mutagenic activity of formaldehyde (see Discussion). It is clear from
this experiment that the inorganic photooxidation products did not contribute significantly to the
observed mutagenic activities in the CsHg/NOx (and toluene/NOx) experiments.
4.3 IRRADIATED CH3CHO/NOX MIXTURES

      Our initial attempts at accounting for all the mutagenicity in the CaHe/NOx system indicated
that the two major products found to be mutagenic, PAN and HCHO (see Discussion), were  not
mutagenic enough to account for a significant fraction of the total response.  Because the response
was much larger at longer  residence time, we began to consider whether secondary reactions
involving acetaldehyde could be responsible for the production of the mutagenic products.  To
investigate this possibility, we conducted a series of experiments with acetaldehyde similar to those
conducted with propylene (i.e., 20-h exposures of TA100 to the photooxidation products of
acetaldehyde under conditions of both short [some NO present] and long [near the ozone maximum]
extents of reaction).
                                           36

-------
  800-
  700
O CHjCHO-^2
D NO
• PAN
• HCHO
A CHjONOj *20
      To determine the temporal behavior of the reactants and products  in the irradiated
CH3CHO/NOX system, we conducted a static-mode irradiation.  For this experiment the reaction
chamber operated as a conventional smog chamber; that is, the reactants were added through the
mixing manifold to the desired initial concentrations, the lights were turned on, and dilution air was
added at 10 L/min to account for an equivalent sampling rate at the effluent end of the chamber.
The initial reactant  concentrations were 1440, 418, and 436 ppb for CH3CHO, NO, and  NOX,
respectively. Figure4.3-1 shows the time profiles for CH3CHO, PAN, HCHO, CH3ONO2, NOX-NO
(which is associated with PAN + NO2>, NO, and O3.

      We  present here the results of two
dynamic-mode exposures under conditions
in which the product distributions should
have been considerably different.  One was
conducted with input concentrations of
1237, 406, and 427 ppb of CH3CHO, NO, and
NOx,  respectively, and a residence time of
2.6 h. The product distribution was similar to
that at ~1.5 h in Figure4.3-1.  Under these
conditions, where  considerable  NO  is
present, the product distribution is simpli-
fied because peroxy radical-radical reactions
will not occur  (see Reactions 14-24).  The
other exposure was  conducted  at  inlet
concentrations of 1190, 31, and 212 ppb of
CH3CHO, NO, and NOx, respectively.  The
product distribution at steady state was
similar to that at —6 h in Figure 4.3-1.  (Only
NOx  is  substantially  higher in the  static
experiment at 6 h.  NO2 is the  major component of difference since the PAN  concentrations are
similar for the static and dynamic [t = 4.0 h] experiments.)  Under these conditions, organic peroxides
can be produced and the product mixture is considerably more complex.  The average chamber inlet
and effluent reactant and  product  concentrations for these two exposures  are presented in
Table 4.3-1.
Figure 4.3-1. CH3CHO/NO/NO2 static-mode
irradiation.
 37

-------
   TABLE 4.3-1.  AVERAGE REACTANT AND PRODUCT CONCENTRATIONS (ppb) FOR IRRADIATED
                                 CH3CHO/NOX  EXPOSURES
Compound
CH3CHO
NOX
NO
03
PAN
HCHO
i = 2.6 h
Input Cone. Effluent Cone.
12371124 1064+113
427 ± 26 388 ± 22
406 ±35 40 1 4
40 ±7
102 ±7
72 ±6
t »4.0h
Input Cone. Effluent Cone.
1190126 977158
212113 174110
3113 512
311112
171111
101116
      The results of both exposures are presented as dose-response curves in Figure 4.3-2. For these
experiments the numbers at each  exposure period are averages for ~25 plates.  Throughout the
T; = 2.6-h exposure there was no discernible change in the survivor levels. For the T; = 4.0-h exposure
the survivor counts were 675, 742,  552, and 0, for 2.5, 5.0, 10.0, and 20.0 h, respectively, indicating
the potential for toxicity effects in the latter part of the exposure.  Although there appears to be
some curvature for both dose-response curves, we have drawn straight lines representing the initial
slopes to facilitate comparison of the mutagenic activities of the two mixtures.  As can be seen in
Figure 4.3-2, for a 20-h exposure at long extent of reaction  ( T = 4.0 h) we observed roughly 400
excess revertants/plate, a clearly significant response.  In fact, given the amount of acetaldehyde
consumed under these conditions (210 ppb), and given our estimate of the amount of acetaldehyde
that had reacted under the conditions of the long  residence time propylene experiments, it is clear
that the photooxidation  products of acetaldehyde can account for essentially all  of the mutagenic
activity associated with the propylene system (at long extent of reaction).

      Given the important role propylene and acetaldehyde play in urban photochemistry, there is a
strong motivation to attempt to identify the species that caused the observed mutagenic activity. It
is important to  recognize that there was also a significant response at the short extent of reaction,
under conditions in which NO was present.  In fact, when the responses were normalized to the
amount of CHsCHO consumed, the mutagenic activities  of the two mixtures were  essentially the
                                           38

-------
same. An important point in this regard is that the mechanism for the photooxidation of CHaCHO is
very simple under conditions in which NO is present (52).








^"^
iX .,
/^ ^^^
.^ -I—
^


f ^-1 T
,x^^
^ \ -L

)

r -^^ ~
^
i —

i i i i i i i i i i i i i i i i i i i i
0 1 2 34 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
                                    Exposure Time, h
 Figure 4.3-2. Dose-response curves for irradiated CHaCHO/NOx exposures.
     Under these conditions, the reaction proceeds as shown in Reactions 14-24 below.
                         CHfHO + OH -*2 CH3C(O)O2+H£>
14
                      CH3C(0)02+N02 ?±
                                                                                 15.-15
                        CH3C(O)O2+NO -> CH3C(0)0+NO2
                                                                                    16
                                      °
                             CH3C(0)0
                                                                                    17
                                          39

-------
                                        2                                              18
                         CH^CHO + hv -> CH.O. + CO +HO~                             °
                            o               d  2         £
                             CH302+NO -                                             19
                             CH3O + O2 -                                             20
                                            CH3ON02                                21
                              CH.O+NO -» CHONO                                 22
                                 O             u
                            CH CH,0 + ATO                               23
                               O               O
                                         » OH+NO2                                  24
Because the photolysis of methyl nitrite (CHsONO) is fast (52), it is unlikely that the concentration of
CHaONO reaches significant levels. Thus, the only organic products measured in the presence of NO
are PAN, HCHO, and much smaller amounts of methyl nitrate (CH3ONO2). One of these three species
must account for the majority of the mutagenic activity in the CHsCHO/NOx experiments, and,
therefore, as indicated above, in the CaHg/NOx irradiation as well. As discussed in Section 4.6 and in
the Discussion section of this report, that species is PAN.

4.4 IRRADIATED ALLYL CHLORIDE/NOX MIXTURES

     To determine whether chlorinated hydrocarbons might yield more mutagenic  products than
their nonchlorinated analogues, we conducted a series of static-mode irradiations and exposures in
which we attempted to measure the mutagenic activity of the products of the photooxidation of
allyl  chloride.  We  report here the results of four  static-mode smog chamber irradiations
(Experiments A-D) of allyl chloride (C3H5CI)/NOx and CaHsCI^He/NOx mixtures in  which TA100 was
periodically  exposed to the chamber effluent during the reaction  profiles.  Because the
photooxidation of allyl chloride proceeds through OH-radical and Cl-atom chain reactions (32), the
experiments with ethane were conducted  to allow measurement of the mutagenic activity of the
allyl chloride  photooxidation products in the presence and absence of Cl-atom  reaction products
                                          40

-------
      is used here as a Cl-atom scavenger). Because of the magnitude of the observed mutagenic
 activity it was possible to conduct these experiments with the chamber operated in a static mode
 (i.e., as a conventional smog chamber).  This enabled us to determine mutagenicity profiles as the
 reaction proceeded.

      For the four experiments conducted, the initial NO and  N02 concentrations were ~350 and
 115 ppb, respectively.  In the first two experiments (A and B), the initial allyl chloride concentrations
 were —725 ppb.  In Experiment B we also added -11 ppm CjHe, which had no significant effect on the
 overall reaction rate. The light intensity was identical for Experiments A and B (we estimate the rate
 constant for NOa photolysis to be  ~0.2 min"1).   For Experiments C and D the initial  ethane
 concentration was — 150 ppm, and the overall reaction rate was therefore dominated by OH reaction
 with ethane. Experiment C contained no allyl chloride, and in Experiment D the initial allyl chloride
 concentration was 850 ppb.  The light intensity was identical  for Experiments C and D,  but was
 0.4times that used  for Experiments A and B (we estimate the NO2  photolysis rate constant to be
 ~0.08min"1).  From the results of these experiments we attempted to determine  the mutagenic
 activity of the products of OH (and 03) reaction with allyl chloride as compared with the mutagenic
 activity of the Cl-atom reaction products.

      The results for Experiments A through D are presented in Figures 4.4-1 - 4.4-4, respectively,
 which include concentration data for the reactants and some of the major products (e.g., PAN) for
 each exposure.   The Ames test  data  (±1 S.D.) are also included in these plots for  the  effluent
 exposure  chamber as revertants/plate  per  15-min exposure (Figures  4.4-1  and 4.4-2), or
 revertants/plate per 30-min exposure (Figures 4.4-3 and 4.4-4). The clean air exposures (which lasted
 the duration of the irradiations) yielded 180 ±21,212 ± 14, 220 ± 10, and 194 ± 17 revertants/plate for
 Experiments A-D, respectively.  For Experiment A (irradiated allyl chloride/NOx), the first three
 exposure periods lasted 15 min. However, experiments we performed previously under essentially
 identical reaction conditions indicated that 15-min exposures at longer extents of reaction (i.e., near
the ozone maximum) led  to toxicity effects (as witnessed by decreased survivor levels). Therefore,
for Experiments A and B,  the last three exposure periods were  5 min in duration. For Figures 4.4-1
and 4.4-2 the bioassay data are all  normalized to a 15-min  exposure  period.  This was done for the
 5-min exposures  by subtracting the control (covered) plate revertant counts from the observed test
 plate counts, multiplying the result by three, and then adding to this value the control  plate counts.
Although this correction assumes a linear dose-response curve,  for all exposures the survivor plates
indicated no evidence  of toxicity. As  shown in Figure 4.4-1, the number of revertants/plate for the
allyl chloride/NOx irradiation  increased to ~1700 (nearly  10 times the control level) for 15-min
exposures, at a reaction time of 4.5 h. We note that, from the initial exposure period,  allyl chloride
alone does not yield a response even though it has been reported to be a weak mutagen (53). This is
                                            41

-------
probably due to the fact that allyl chloride, being volatile and relatively nonpolar, does not deposit
into the  plates during the exposure.  Although it appears from Figure 4.4-1 that the mutagenic
activity increases as the extent of reaction increases, the mutagenic activity normalized  to the
amount of allyl chloride  consumed is nearly constant throughout the reaction. In fact, near the end
of the reaction, the mutagenic activity decreased, probably because Cl atoms began reacting with
the products rather than  with allyl chloride (see Discussion).

      In a previous study of the kinetics and mechanism of the photooxidation of allyl chloride (32),
we found that a significant fraction of the allyl chloride removal was due to reaction with Cl atoms
that are produced from OH reaction with allyl chloride.  It is possible, therefore,  that the results of
Experiment A are not representative of the atmospheric photooxidation products of allyl chloride,
since under atmospheric conditions, we would expect very little reaction of the Cl atom with allyl
chloride.   To  test for this possibility, we conducted Experiment B with ethane added as a Cl-atom
scavenger. Ethane represents a good  Cl-atom scavenger for this study because its Cl-atom reaction
rate constant (at 298K) is large, 6.38x  10~11 cm3-molecule"|-s"1 (54) and  its OH radical rate
constant  is much  smaller, 2.74x  lO'^cmS-molecule^-s"1 (55).  We reported a rate constant of
1.7x 10">i cm3-molecule'i-s'1 for  OH reaction with allyl chloride at 298K  (32).   To  estimate the
relative rate  of Cl-atom  reaction with allyl chloride vs. ethane, we recently measured the rate
constant  for Cl-atom reaction with allyl chloride by the relative rate method described by Atkinson
and Aschmann (54), using ethane as the reference compound.   Using the  chromatographic
procedures described in the Experimental section of this report we found k(ci + c3H5ci) = 2.3
(±0.1)x 10"10 cm3-molecule"|-s"1,  using k(ci + C2H6) = 6.38 x  10~11 cm3-molecule~1 -s"1.
Therefore, under the conditions of Experiment B (720 ppb allyl chloride, 11.1 ppm ethane) the initial
rate of OH reaction with  allyl chloride was 4.0 times faster than the rate of OH reaction with ethane.
Under these conditions, then, the presence of 11.1 ppm ethane should _have only a small impact on
the overall reaction rate (e.g., conversion of NO to NOa). As can be seen by comparing the  data in
Figures 4.4-1  and 4.4-2,  the time of the  NO2  maximum increased from 2  to  2.5 h, respectively.
However, the rate of Cl-atom reaction with ethane is 4.3 times faster than with allyl chloride.  We
therefore estimate that  the yield of products due to  Cl-atom addition to  allyl chloride would be
roughly five times smaller in Experiment B relative to Experiment A. From inspection of Figure 4.4-2,
it is clear that the number of revertants per plate in  Experiment B is significantly smaller than in
Experiment A.  In fact, the number of excess revertants (i.e.,  relative to the zero reaction time
exposure) at  4.5 h (~230 revertants/plate) is six to seven times smaller than that observed  in the
absence of ethane.  When normalized to the amount of allyl chloride consumed, the mutagenic
activity of the products is three to four times smaller.  This result suggests that the majority of the
response  observed in the allyl chloride/NOx irradiation (ExperimentA) was due to the presence of
Cl-atom addition products.
                                            42

-------
                    O C3H5CI
                    D NO
                    A NOx-NO
                      o3
                      Revertants/plate
                    X CPAN
Figure 4.4-1. Experiment A:  reaction chamber component concentrations and mixture mutagenic
activity, CaHjCI/NOx irradiation.
                                                O C3H5CI
                                                O NO
                                                  NO,-NO
                                                  03
Figure 4.4-2. Experiment B: reaction chamber component concentrations and mixture mutagenic
activity. CaHsCI/CzHg/NOx irradiation.
                                          43

-------
                                                      • C2H6*200
                                                      Q NO
                                                      A NOX-NO
                                                      v 03
                                                        Revertants/plate
af   600H
Q-fc
0)
                                                                   O CjHsCI
                                                                   O NO
                                                                   a NOx-NO
                                                                   V 03
                                                                   9 Revertants/plate
                                                                           Dilution
                                        Time, h
 Figure 4.4-3. Experiment C: reaction chamber component concentrations and mixture mutagenic
 activity, CzHg/NOx irradiation.
      Since the dominant atmospheric
removal process for allyl chloride is reaction
with OH  radicals, it would be desirable to
measure the mutagenic activity of the
products in the absence of any  Cl-atom
reaction.  As described  previously (32), a
major product of Cl-atom reaction with allyl
chloride is 1,3-dichloroacetone.  In addition
to the reactant and product concentration
data presented in  Figures 4.4-1-4.4-4, we
made periodic  measurements of the allyl
chloride  photooxidation products  chloro-
acetaldehyde, formaldehyde,  glyoxal,
acrolein,  and 1,3-dichloroacetone, and the
ethane photooxidation products  acetalde-
hyde,  formaldehyde,  and  PAN.   The
measurement of these species  was made
                                                               Time, h
                                       Figure 4.4-4. Experiment D: reaction chamber
                                       component concentrations and mixture mutagenic
                                       activity, CaHsClA^He/NOx irradiation.
                                        44

-------
 roughly once each  hour during each  irradiation.  To simplify the presentation, we provide in
 Table4.4-1 the estimated maximum concentrations for these species during the reaction for each
 experiment. These estimates were obtained by plotting the actual data and drawing smooth curves
 through the product profiles.  For Experiments A and B the product maxima occurred near the
 maximum in the Ames test results, and for Experiments C and D the maxima occurred at or near the
 end of the irradiation. Product profiles were presented for an experiment similar to Experiment A in
 a previous publication (32).  From comparison of the 1,3-dichloroacetone data for Experiments A and
 B (and given the A allyl chloride data in Figures 4.4-1 and 4.4-2), it can be observed that the yield of
 Cl-atom addition products for Experiment B decreased by a factor of approximately six, which is in
 reasonably good agreement with our estimate based on the relative rate constants.

     TABLE 4.4-1. REACTANT AND PRODUCT CONCENTRATIONS (ppb) AND BIOASSAY RESULTS
Experiment*
Reactants
allyl chloride
NO
NOX
ethane (ppm)
Products (max. cone.)
03
formaldehyde
chloroacetaldehyde
acetaldehyde
1 ,3-dichloroacetone
acrolein
glyoxal
CPAN
PAN
A

729
316
445
_

925
295
132
-
75
9
29
80
-
B

720
360
472
11.1

978
287
58
101
7
12
29
190
72
C

-
416
483
170

148
40
-
104
-
-
-
-
11
D

820
325
474
136

600
200
117
196
<0.5
8
13
no data
no data
 Mutagenic Activity
     revertants-plate'1-rf1-ppb'1             13            3.4          <0.4t           1.4
 *  For Experiments A and 8, the light intensity was 2.5 times greater than for Experiments C and D.
 *  Using A allyl chloride from Experiment D.
      However, since measurable  amounts of 1,3-dichloroacetone existed under these conditions,
some of the observed  mutagenic activity for Experiment B may still have been attributable to the
Cl-atom chain products.  The fact that the observed mutagenic activity decreased by a factor nearly
equal to the decrease in the 1,3-dichloroacetone yield supports this idea.  We have conducted
standard plate incorporation tests  with 1,3-dichloroacetone, and  found it to be  nonmutagenic for
TA100.  The observed mutagenic  activity was, therefore, probably due to  some other dichloro
reaction product that resulted from Cl-atom addition to allyl chloride. A Cl-atom addition reaction is
                                            45

-------
assumed, since Cl-atom abstraction reactions would result in the same products as OH-radical
abstraction reactions.

      To be certain that all Cl atoms were scavenged by ethane we conducted Experiments C and D;
in both cases the overall reaction was dominated by OH reaction with  ethane, in contrast to
Experiments A and B. (For Experiment D the initial OH reaction rate was 2.6 times greater with
ethane than with ally! chloride.)  For Experiments C and D the total  plate exposure time for all
exposure periods was 30 min.  Under the initial conditions of Experiment D the rate of Cl-atom
reaction with ethane is 45 times greater than with ally! chloride.  Given that at the sixth exposure
period for Experiment D ACsHsCI = 145 ppb, assuming that the  Cl-atom yield from OH (and 03)
reaction with ally! chloride is 20% (32), and given the relative Cl-atom reaction rates, we estimate the
total Cl-atom reaction with allyl chloride would correspond to a product concentration of 0.6 ppb
(total  Cl-atom reaction  product).  This estimate is  consistent with  the fact that the
1,3-dichloroacetone concentrations for this experiment were all below the 0.5-ppb detection limit.

      Since much of the product mixture present in Experiment D represents ethane photooxidation
products (i.e., HCHO, CH3CHO, PAN [55j)( Experiment C was conducted as a control for measurement
of their mutagenic activity. As can be seen in Figure 4.4-3, the  revertant  counts did not increase
significantly throughout the experiment.

      For the sixth exposure period in Experiment 0, at which time 145 ppb  of allyl chloride had
been consumed,  the  observed mutagenic activity increased slightly, corresponding to 100 excess
revertants/plate (Figure 4.4-4). The observed mutagenic activity of the allyl chloride product mixture
corresponds to 1.4 revertants-plate"1-h"1-ppb"1 (h~1 refers to  per hour exposure).  We.note that
under the conditions of the sixth exposure in Experiment D, some 03 reaction with allyl chloride had
occurred (we estimate 40-50% of the total A allyl chloride).  Under these conditions, therefore, the
observed mutagenic activity was  smaller than that observed in Experiment B (in terms of
revertants/ppb allyl chloride  consumed) by a factor of approximately three, as a result of the
decreased extent of Cl-atom reaction with allyl chloride.

      From the data  presented in  Figure 4.4-1  (Experiment A), it can  be seen that the  mutagenic
activity of the product  mixture  (at  long extent  of reaction)  was  approximately
13 revertants-plate"1-h"1 per ppb  of allyl chloride  consumed.   This mixture, then, is  roughly
300 times  more mutagenic  than are the photooxidation products (0.043 revertants-
plate"1-h^-ppb"1) of its nonchlorinated analogue, propylene.  The  majority of the mutagenic
activity of this mixture is due to Cl-atom addition products, however. The mutagenic activity of the
product of  allyl chloride photoxidation in  the absence of Cl-atom  reactions (Experiment  D) is
considerably less  (~1.4 revertants-plate"1-h"1 per ppb), but is still roughly 30-40 times greater than
                                           46

-------
that for the photooxidation products of propylene.  It will be shown in the Discussion section that
under the conditions of Experiment D, the observed response can be accounted for on the basis of
the mutagenic activity of the allyl chloride photoxidation product, chloroacetaldehyde.

4.5 COMPLEX MIXTURE IRRADIATIONS

      To compare the results of the experiments with simple hydrocarbons with those of a more
realistic simulated urban hydrocarbon mixture, and to determine the distribution  of mutagenic
species  between the gas  and particulate phases, we conducted a  series of irradiated  wood
smoke/NOx and automobile exhaust/NOx experiments.  For these experiments the hydrocarbon and
total NOx concentrations in the chamber were brought to the desired initial levels (12-20 ppmC and
—0.7 ppm, respectively), at which point the chamber was sealed off and the lights were turned on. The
reaction was then allowed to continue  until the maximum ozone concentration was reached, at
which time the lights were turned off. Exposures of TA100 and TA98  to the gas-phase pollutants
were conducted for periods up to 10 h, and filter samples of the particulate phase were obtained for
bioassay measurements.

      A set  of duplicate wood smoke/NOx irradiations and exposures (Experiments E and  F) was
conducted in which the initial  total  hydrocarbon concentration was ~17ppmC and the initial NOx
concentration was ~0.62 ppm.   Roughly 0.13 ppm of the NOx was combustion derived, and the rest
was added to the chamber  from a compressed gas cylinder.  NOx was added to better simulate the
hydrocarbon to NOx ratio  found  in populated areas (24) and to increase the extent  of reaction,
thereby facilitating analysis of the  species concentrations and  determination of the mutagenic
activities.  Several additional static  runs were conducted, mainly for chemical analysis, for  which
particulate-phase biological assay data were also obtained.

      The reactant and  product concentrations measured before and  after the wood smoke
irradiations are  presented in Table 4.5-1.  Concentrations of chemical  species measured from the
particulate extract data are presented  in Table 4.5-2.  In Figure 4.5-1 we  present the reactant and
product profiles for a number  of major species.  As shown in the figure the ozone maximum was
reached in just 2 h, indicating that the photooxidation proceeded  rapidly. As indicated in Table 4.5-1
the major reactive hydrocarbons included alkenes (ethylene, propylene, and  1-butene), aromatics
(benzene, toluene, and  xylenes), and  oxygen-atom  heterocycles (furan, 2-furaldehyde, and
2-methylfuran).  Aldehydes were  a  large component  of the gas-phase emissions from the  wood
stove, the two main species being HCHO and CHsCHO. The aldehydes exist as both reactants and
products in this system.  The oxidation of propylene and toluene leads  to the production of HCHO
and CHsCHO, as was discussed  earlier.  The photooxidation of aromatics (toluene and  xylenes) has
been shown to yield glyoxal (CHO2)  and methylglyoxal [CH3C(O)CHO], and biacetyl [(CH3CO)2l (55),
all of which are also present as reactants.
                                           47

-------
         TABLE 4.5-1. INDIVIDUAL GAS-PHASE INORGANIC AND HYDROCARBON
            CONCENTRATIONS,* ppb, FOR WOOD SMOKE/NOX IRRADIATIONS

Species
Nitric oxide
NOX
Ozone
Carbon monoxide, ppm
Methane
Ethane
Propane
Ethylene
Propylene
1-Butene
Isoprene
Acetylene
Benzene
Toluene
m- + p-Xylene
o-Xylene
Styrene
m-Methylstyrene*
Furan
2-Methylfuran
Formaldehyde
Acetaldehyde
Benzaldehyde
2-Furaldehyde
Glyoxal
Methylglyoxal
Biacetyl
PAN
Chloromethane
HCtt, ppmC
E (March
Initial
454
657
0
38.0
5,060
146
9
537
126
58
_t
177
62
62
40
13
22
9
71
61
269
88
15
31
41
41
-
0
18
16.4
7)
Final
0
252
467
33.4
4,500
135
9
313
8
3
-
155
50
15
19
4
0
0
0
0
365**
109**
12
2**
45**
36**
-
174
18
13.2
F (March
Initial
461
576
0
38.7
4,480
-
-
847
100
39
7
-
102
24
12
2
7
3
59
64
229
57
-
22
26
13
7
0
-•
17.2
28)
Final
0
259
696
35.5
3,920
-
-
439
7
0
0
-
68
10
3
0
0
0
0
0
383
75
-
6
24
16
5
232
-
15.0
 * Other gas-phase species identified: phenol, butadiene, methyl acetate, 2-methyl vinylacetylene, methyl vinyl
   ketone, methyl ethyl ketone, and benzofuran.
 t Not measured.
 * Tentatively identified based on mass spectrum.
** At 1,5-h irradiation time.
 ft Corrected for dilution.
                                          48

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       TABLE 4.5-2. PARTICULATE EXTRACT DATA FOR WOOD SMOKE/NOX IRRADIATIONS.
                   NANOGRAM OF PAH/MILLIGRAM OF PARTICULATE MASS
                                    E (March 7)
                       F (March 28)
Species*
Fluoranthene
Pyrene
Anthracene +
Phenanthrene
Acenaphthene
Fluorene
Chrysene +
Triphenylene
CNC(x 109
parti cles/m3)
EAA (nl_/m3)
Initial
721
672
457
371
214
-
33
286
Final
70
50
100
30
20
-
28
591
Initial
611
560
132
103
224
100
13
72
Final
203
218
89
<100
<100
<100
10
265
       Other particulate-phase species identified: 4-hydroxy, 3,5-dimethoxybenzaldehyde; 4-hydroxy-3-
       methylbenzoate; 4-hydroxy-5-methoxybenzoicacid; benzo(a)pyrene.
      Physical  and chemical characteri-
zations of the particulate phase were
conducted  both  before and following
irradiation.  The number distribution of the
aerosols, although not shown, was similar to
that observed by other researchers (11,15);
that is, the vast  majority  of unreacted
particulates were less than 1 iim in size with
the maximum in the number distribution
occurring  at approximately  0.1 um.  The
maximum  in the number distributions
shifted to —0.2 um after irradiation.  The
volume distribution of the particulate
reactants and products as measured by the
EAA for Experiment F  is presented in Figure
4.5-2.  Although the absolute number of
particles following irradiation is smaller than
      600-
                 Million.    * ' •<•«"•
      100-
                   60    90
                Irradiation Time, min
                             120
                                   150
Figure 4.5-1. Wood smoke static irradiation with
500 ppb of additional NOX. Selected product and
reactant profiles.
                                            49

-------
before (Table 4.5-2), the contribution to the
volume (and  thus  the  mass)  of the
paniculate matter increases as r3, giving the
volume distribution for products shown in
Figure 4.5-2 with a peak at ~0.5 urn.  This
increase in volume following irradiation has
been  observed in all  of our photooxidation
experiments with wood smoke.  It would
seem  likely that the  increase in the total
volume of paniculate is the result  of polar
gas-phase photooxidation  products adsor-
bing  onto the  paniculate matter already
present. In the absence of irradiation, the
number and volume  distribution maximum
of the panicles changed slowly with  time
while the particles were in the chamber.  As
shown in Table 4.5-2, for each of the PAHs
measured in the paniculate phase, signifi-
                  0.05  o.i   0.2
                  Particle Diameter (|jm)
Figure 4.5-2. EAA volume distribution of diluted
wood smoke before and following irradiation in
Experiment F.
cant degradation was observed as a result of the irradiation.  Because the PAH concentrations are
expressed as nanograms of chemical per milligram of paniculate mass, part of the apparent
degradation occurred solely as a result of the diluting effect of increasing the paniculate mass (due
to adsorption from the gas phase).  However, there appeared to be some reaction of the PAHs,
although no photolysis or reaction products from these compounds were observed.

      The mutagenic activities exhibited by Strains TA100 and TA98 from the exposure to the gas-
phase species are presented in Table 4.5-3. The values represent the average of the slopes of dose-
response curves obtained for  Experiments E and F. Sample dose-response curves obtained with
strains TA100 for Experiments E and F are presented in Figure 4.5-3. The exposure times indicated in
this figure are corrected as indicated by Expression I (see Experimental section). In all cases, the clean
air and ambient air revertant  levels were (within experimental error) identical to the spontaneous
laboratory controls (TA100:202; TA98:36).  In Experiment E, data for an exposure time of  10 h
(uncorrected) were also obtained.  However, the irradiated mixture was toxic, as indicated by
depressed revertant levels in the survivor plates.  For all other exposure times, no other toxicity
effects were observed.
                                            50

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     TABLE 4.5-3. MEASURED MUTAGENIC ACTIVITIES? OF THE GAS AND PARTICULATE PHASES
                              OF IRRADIATED WOOD SMOKE*
Gas
(revertants/h)

Reactants
Products
TA100
(1.6± 2.7)
240 ± 22
TA98
(-0.4 ±
45 ±
1.2)
6
Partial late
(revertants/ng)
TA100
0.30 ±0.10
0.2710.15
TA98
0.22 ±0.06
0.94 ±0.30
    * Values in parentheses are zero within the uncertainty.
    t Corrected for collection efficiency.
   1400-
   1200-
   1000-
Experiment E
• Reactants
• Products
Experiment F
O Reactants
D Products
                    1.0
                            3.0
                    Exposure Time, h
                         I
                        4.0
5.0
6.0
 Figure 4.5-3. Dose-response curve for the mutagenic activity (TA100) of the gas-ph
 components of wood smoke (+ 500 ppb NOx) before and following irradiation.
                                                          ase
      The mutagenic activities exhibited by strains TA100 and TA98 for the particulate organic phase
are also presented in Table 4.5-3.  For TA100 the extracts from the products show no increased
activity over those of the reactants. Only for TA98 do the product extracts show significantly higher
mutagenicity than the reactants (approximately a factor of 3), as shown in Figure 4.5-4.   These
particulate data are a compilation of filter extracts for all wood smoke/NOx (added NOx) irradiations
performed during this study. The straight lines drawn through the data represent a least-squares fit
to all the data.
                                           51

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                  500 H
                                • Reactants
                                O Products
                                                         Reactants
                         100
                              200
                                   300
                                       400
                                             500
                                                  600
                                                       700
                                                            800
                                                                 900
                                         jig/plate
 Figure 4.5-4. Dose-response curve for the mutagenic activity (TA98) of the
 wood smoke (+ 500 ppb of NOX) before and following irradiation.
participate extracts of
      We also performed exposures of the gas-phase reactants and  products  and  plate
incorporation tests for extracts using TA100 and TA98 with metabolic activation (59). For exposures
up to 10 h and extract doses up to 850 ug/plate, no significant difference was observed for the
addition of 59 for either reactants or products in both strains when compared to the data without 59.
In the interest of presenting results in the most succinct fashion, these data have not been presented.

      It is apparent from the results of Experiments E and F that irradiation of wood  smoke/NOx
mixtures dramatically increases the mutagenic activity over that of the initial reaction mixture,
particularly for gas-phase species.

      Another objective of this study was to attempt to estimate the fraction of mutagenic activity
(revertants/microgram or revertants/cubic meter) present in the gas vs. paniculate phase. The most
convenient way to do this is to compare  the  mutagenicities on the basis of revertants per cubic
meter. One major difficulty with this determination was obtaining an accurate measurement of the
dose of gas-phase mutagens in the test plates.  The number of revertants produced per cubic meter
of effluent could  be easily calculated, but we were not confident that all of the mutagens  were
removed by the bioassay chambers. We estimated the exposure chamber removal efficiency  in an
experiment that was a duplicate of Experiments E and F, in which the gas-phase exposures  were
conducted by using two exposure chambers in series.  From comparison of the dose-response curves
obtained from each exposure  chamber,  we calculated  the  collection efficiency of gas-phase
mutagens for the first exposure chamber.  From  the slopes of the dose-response curves in this
                                            52

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experiment, the carryover into the second exposure chamber was found to represent 28 and 33% of
the mutagenic activity observed in the  first exposure chamber for strains TA100 and TA98,
respectively. Assuming that the carryover from the second exposure chamber is represented by the
same fractional values, the collection efficiency for the first exposure  chamber is then 72 and 67%
(TA100 and TA98,  respectively).  It should  be noted that this assumes that all mutagenic species
present deposit to some extent.  The observed reversion rates for this experiment (corrected for
collection efficiency) were 185 and 36 revertants/h for TA100 and TA98, respectively, in reasonably
good agreement with those found for Experiments E and F.  These collection efficiency values can
now be used to accurately define the mutagenic activity (revertants/m3) of the gas-phase product
mixtures (see Discussion).

      To understand potential  reactions that may occur with wood  smoke at night, we reacted
20.2 ppmC wood smoke (starved  air conditions) with, nominally, 2 ppm ^Os, followed by a 7-h
exposure of TA100 and TA98 to the resultant product mixture (Experiment G).  The reactant and
product concentrations, as well as the measured mutagenic activities of the gas- and particulate-
phase products, are presented in Table 4.5-4.  For this experiment, two product exposure chambers
were  operated in series, and therefore  the gas-phase mutagenicity  data take into  account the
observed collection efficiencies for each strain (63 and 55%, respectively, for TA100 and TA98).  The
extent of reaction can be related to the observed change in the total hydrocarbon concentration,
2.4ppmC, which is somewhat less than that observed for the wood smoke/NOx irradiations.  The
data in Table 4.5-4 show small increases in concentration for species expected to be products, such as
HCHO and CHsCHO, and some decreases for species reactive to NOs, such as acrolein (56).
      The most apparent and dramatic effect of the addition of I^Os was an observed increase in
paniculate volume.  In Figure 4.5-5 the EAA data for the wood smoke mixture before and after
addition of ^Os indicate  that the total aerosol volume increased by a factor of 21, and  the
maximum in the particle size distribution increased from roughly 0.15 to 0.30 urn. This increase in
particle size and concentration is similar to that observed for the irradiated wood smoke/NOx
mixtures, implying that significant amounts of gas-phase oxidation  products (the equivalent of
1.7 ppmC) are adsorbed onto existing paniculate matter. As indicated in Table 4.5-4 the gas-phase
products exhibited mutagenic activities comparable to (and perhaps greater  for  TA100) those
observed for the irradiated wood smoke/NOx mixtures, even though the extent of conversion of
reactants  to products was somewhat less.  In addition,  the particulate-phase products were
considerably more mutagenic, especially as measured with TA100, than the particulate  matter
produced from  the irradiated mixtures.  As observed with the irradiated mixtures, much of the
decrease in PAH concentrations (ng/mg) can be related to the increase in the total particulate-phase
mass. It is clear from this experiment that reactions of NOs and I^Os with the gas- and particulate-
phase components can lead to substantial increases  in the mutagenic activity of the wood smoke
mixture.
                                           53

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TABLE 4.5-4. WOOD SMOKE/N2O5 (EXPERIMENT G) REACTANT AND PRODUCT CONCENTRATIONS
                    (ppb) AND PRODUCT MUTAGENIC ACTIVITIES

Gases
Total hydrocarbons, ppmC
NO
NOX
03
CO, ppm
PAN
C2H4
HCHO
CH3CHO
Acrolein
Benzaldehyde
Glyoxal
Methylglyoxal
Particulate (ng/mg)
Pyrene
Anthracene + phenanthrene
Fluoranthene
Total participate, nl_/m3
Mutagenic Activities
TA 100, gases (rev/h)
TA98, gases (rev/h)
TA100 paniculate (rev/pg)
TA98 paniculate (rev/pg)
Initial

20.2
18
94
0
19.6
5
545
343
158
173
5
24
70

161
495
205
20





Final (trcn = 20 min)

17.8
0
1,656
85
19.6
16
563
394
182
123
4
25
87

<20
<20
26
420

148
20
1.2
1.0
                                     54

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Volume,
 nL/m3
250-
200-
150-
e,
100-
50-










r§SHB(



Final
jr
Initial
KW99H
      In  our attempt  to  examine other
complex  sources found  in populated areas,
we also conducted an experiment for
automobile exhaust (Experiment H) that was
similar to the irradiated wood smoke/NOx
experiments described earlier. It was not
possible to duplicate  the initial hydro-
carbon/NOx ratios of the wood smoke
experiments, however, because of the
relatively high NOx emissions in  the auto-
mobile exhaust.  Experiment  H  was con-
ducted at a hydrocarbon/NOx ratio of 16.5:1,
which is closer to the typical urban HC/NOx
ratio of -10:1 (24). The ratio of particulate
matter to total  hydrocarbon in the vehicle
exhaust was  0.005  nL-m'S  ppmd.   Figure4.5.5. EAAvo|umedistribution,wood
Therefore, as indicated  in Table 4.5-5, there   smoke/N205.
was very little particulate matter in the chamber at the start of the irradiation.  In Table 4.5-5 we
present the gas- and particulate-phase  reactant and product concentrations and measured
mutagenic activities for Experiment H. The reactant mutagenic activity for the particulate phase was
determined from a filter sample obtained at the tailpipe. As in previous experiments, two exposure
chambers were operated in series, so the gas-phase  mutagenicity  data in Table 4.5-5 reflect the
measured collection efficiencies (TA100: 74%; TA98: 50%).

      The irradiation of the automobile exhaust/NOx mixture was continued until the 63 and PAN
maxima were reached at 5.25 h. In contrast, the Os maximum for the wood smoke Experiment E was
reached at 90 min, even though the HC/NOX ratio was nearly identical to that for  Experiment H.
Therefore, it appears that the "reactivity" of the automobile exhaust mixture is somewhat less than
that of wood smoke.
       0.01
                      0.1  .178  .316  .562  1.0
                Particle Diameter, |im)
55

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TABLE 4.5-5. IRRADIATED AUTOMOBILE EXHAUST/NOX (EXPERIMENT H) REACTANT AND
         PRODUCT CONCENTRATIONS (ppb) AND MUTAGENIC ACTIVITIES

Gases
Total hydrocarbon, ppmC
03
CO, ppm
PAN
NOX
NO
Benzene
Toluene
C2H4
CH4
C3H6
HCHO
CH3CHO
Acrolein
Glyoxal
Methylglyoxal
Particulate (ng/mg)
Pyrene
Fluoranthene
Anthracene + phenanthrene
Total participate, nL/m3
Mutagenic Activities
TA 100, gases (rev/h)
TA98, gases (rev/h)
TA100, participate (rev/pg)
TA98, paniculate (rev/jig)
Initial

11.9
0
19.6
0
716
668
45
71
237
580
50
30
25
0
<4
<6

6,042
4,364
11,571
0.6

3
<2
0.58
1.0
Final

6.8
833
15.2
149
376
0
38
52
107
470
2
213
79
62
19
55

<200
<200
<200
48

91
18
0.18
0.40
                                 56

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      In contrast with the wood smoke irradiation, in which the total number of particles decreased
throughout the irradiation, a sharp burst of particle formation occurred in the automobile exhaust
experiment shortly after the irradiation began (see Figure 4.5-6). Also shown are the NO, NOx-NO,
03, PAN, and total hydrocarbon concentrations measured as the reaction proceeded.  The dramatic
increase in particle concentration may be a reflection of the absence of particulate matter at the
start of the reaction. That is, in the wood smoke irradiations the nonvolatile products condense onto
existing particulate matter, while for the automotive emissions, new particles are formed.
                                                            • THCxIOO, ppmC
                                                            D NO
                                                            V NOx-NO
                                                            A 03
                                                            O CNC-5-104
                                                            « PAN               l~45
                                        T, hours
 Figure 4.5-6.  Automobile exhaust/NOx reactant and product concentrations.

      As indicated in Table 4.5-5, the mutagenic activities of the gas-phase products of irradiated
automobile exhaust are similar to those measured for wood smoke.  This seems fairly reasonable
because, although the initial gas-phase concentrations are relatively lower for some species such as
the aldehydes, the final concentrations of these species are similar. The particulate-phase mutagenic
activities before and after irradiation are,  however,  significantly different from those observed for
wood smoke.  As shown in Table  4.5-5, the mutagenic activity (revertants/ug) of the  automobile
exhaust (reactant) particulate  matter is considerably  greater than  that of the wood smoke
particulate matter. In contrast to what has been consistently observed for wood smoke (measured
by TA98), the mutagenic activities of the particulate-phase species decrease upon irradiation of the
                                            57

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mixture.  Because essentially all of the particulate-phase mass represents condensed photooxidation
products, these low-volatility products from the photooxidation of automobile exhaust appear to be
less mutagenic than those produced from irradiated wood smoke mixtures.  It is important to note
that the total mass of particulate matter in the reactant and product automobile exhaust mixtures is
much lower than for the wood smoke mixtures,  and therefore the mutagenic activities of the
automobile exhaust mixture on the basis of revertants per cubic meter will be correspondingly lower
than those of wood smoke (see Discussion).

      For our method, determination of the plate dosage presents a serious difficulty in quantifying
gas-phase mutagenic activities.  One alternative to our procedure is to collect the gas-phase
components on a solid sorbent, followed by solvent extraction and  utilization of the standard plate
incorporation test (57).  To compare the two methods,  we conducted an  additional wood
smoke/NOx irradiation in which the filtered effluent was drawn through a cartridge containing
~500 g XAD-2 and then into an exposure chamber loaded with the same number of TA98 and TA100
test plates used in the other experiments. In this way we could use the exposure chamber to measure
any mutagenic activity that was not  collected on the XAD-2.  The experiment was essentially a
duplicate of Experiment F described above. The initial total hydrocarbon and NOX concentrations
were 19.5 ppmC and 0.667 ppm, respectively, and the total reaction time was 65 min.  The observed
product concentrations were very similar to those  indicated in Table 4.5-1 for Experiments E and F.
After the irradiation, the exposure  and the XAD-2 sample collection were conducted for  10 h at a
flow rate of 14 Lymin. By comparing the observed mutagenic activities from the exposure chamber
plates with those expected according to similar wood smoke/NOx irradiations, we found that XAD-2
removed  83 and 87% (TA100 and TA98, respectively) of the total mutagenic activity  from the gas-
phase airstream. Unfortunately, we  estimate that roughly 60%  of the gas-phase hydrocarbon
removed  from the airstream was lost  in the collection and/or subsequent extraction and
concentration procedures.  The measured mutagenic activities (data not presented)  of the XAD-2
extracts correspond to gas-phase mutagenic activities (revertants/m3) that were roughly only 5 and
10% (TA100 and TA98, respectively) as large as the mutagenic activities measured with the direct
gas-phase exposure technique.  Therefore, although the XAD-2 collects or removes most of the
mutagens, they appear to be lost in the sample workup procedures.

4.6 PEROXYACYL NITRATES

      For the irradiation experiments with toluene,  propylene,  acetaldehyde, wood smoke,  and
automobile exhaust, PAN was a principal product near the ozone maximum where large mutagenic
activities  (TA100) were observed. It is important, therefore, to estimate the contribution of PAN to
                                           58

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the mutagenic activity of the complete system. We conducted a number of experiments by exposing
TA100 to pure PAN in air, using the exposure chamber technique.  Our  first experiment involved
preparation of PAN in the gas phase via irradiation of a CI2/CH3CHO/NO2 mixture, followed by
purification of the PAN  by vacuum distillation.  Two separate exposures were conducted, using
mixtures of 1 ppm (Exposure #1) and 3 ppm (Exposure #2) pure PAN in air.  From the fraction of PAN
that deposited into the plates, the total flow rate of the air mass, and the total exposure time, we
calculated the total mass of PAN deposited. These numbers compared very well to those measured
(as acetate and nitrite) in surrogate basic water plates. We used these values to construct  a dose-
response curve (58).  The slope of this curve yielded a mutagenic activity for PAN of 34 ±5
revertants/umol.  This value  is such that, for example, if applied to  the i = 7.5-h C3He/NOx
experiments, PAN could account for no more than 10% of the total observed mutagenic activity of
the product mixture.  If  this mutagenic activity were correct, we would be faced with a  serious
problem in interpreting the T; = 2.7-h CH3CHO/NOX experiments, because PAN and HCHO are the only
mutagens present and HCHO  is  a very weak mutagen (see Discussion).  We therefore  began to
consider whether the observed mutagenic activity in these experiments could have been caused by
synergistic effects involving the  components present under the conditions of the short residence
time CH3CHO/NOX experiment (i.e., CH3CHO, O3, NO2, HCHO, CH3ONO2, and PAN).

      To test for this possibility, we conducted a 10-h exposure of TA100 to a  mixture of these
products (at concentrations equivalent to those in the CH3CHO/NOx experiments) contained in the
dark reaction chamber.  The result of this experiment was that, for a 10-h exposure, there were
roughly 200 excess revertants/plate, or a significant fraction of that observed for the irradiated
CH3CHO/NOx mixtures. This experiment was then repeated in a series of experiments in which one
of those species was removed in each successive experiment. In each case, a similarly large response
was observed, as long as  PAN was present.  Our final experiment was a 10-h exposure of TA100 to
~200 ppbpure PAN in air. The results of this experiment are shown in Figure 4.6-1, where significant
response can be seen (i.e., the equivalent of ~300 excess revertants/plate for a 20-h exposure).
Therefore, according to this experiment,  PAN could account for most of the observed mutagenic
activity for the acetaldehyde and  propylene experiments.

      Given the quantity  of PAN that was deposited into the plates in this experiment, we calculated
a mutagenic activity for  PAN  of ~350 revertants/umol (TA100), making it a moderately strong
mutagen.  However,  as  indicated above, in laboratory measurements using purified PAN we
obtained a mutagenic activity  of roughly  a factor of 10 smaller. The only difference between this
latter experiment and the first two  pure PAN exposures (with regard  to procedures) was the
                                           59

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considerably lower PAN concentration.  From a series of exposures of TA100 to pure PAN at
concentrations ranging from —100 to 500  ppb, we observed that the observed reversion rate in
revertants per hour is, within the experimental uncertainty, independent of the PAN  exposure
concentration.  Specifically, for a number exposures in this range, the observed reversion rate was
14 ± 5 revertants/h. Therefore, the apparent mutagenic activity in either revertants per hour per ppb
or in revertants  per  micromole calculated from  any experiment depends on the exposure
concentration.  It is not, at this point, clear as to why this should be the case, because one would
expect the calculated mutagenic activity to be independent of exposure concentration. For all these
exposures, there was no evidence of serious toxicity problems (the dose-response curves were linear)
or of Henry's Law equilibrium conditions being approached.  However, in order to interpret the
results from the irradiated HC/NOx mixtures, the observed response can be compared with PAN's
expected contribution of 14 revertants/h to the total reversion rate.
   350-
   340-
   330-
   320-
   310-
   300-
   290-
o>
^  280-
Q.
|  270-

-------
0.097 revertants-h~1-ppb~1, respectively.  It should be noted that these mutagenic activities are
expressed per ppb of PAN compound in the inlet to the exposure chamber.  For PAN, PPN, and PBN,
the transfer efficiencies (to the exposure chamber) are all 65 to 70%, and the fraction that deposits is
— 15%.  For PBzN, the transfer efficiency to the exposure chamber was 27%, and the fraction
depositing into the plates was 7%. Therefore, the actual dose for PBzN was -5 times less than for PPN
and PBN, indicating that the mutagenic activity for PBzN is considerably greater than for the other
PANs.  The inlet concentration for PPN, PBN, and PBzN was 500 ppb in each case.  The apparent
mutagenic activity of PAN at this concentration is —0.028 revertants-h"1-ppb"1. Because it is unclear
how the measured reversion rates for these other PAN-type compounds vary  with the  exposure
concentration, it is not possible at this point to calculate a mutagenic activity for them in terms of
revertants per micromole. It does appear, however, that the four peroxyacyl nitrates are mutagenic
(as determined by using the Ames test, Strain TA100) to some extent.  This is  a significant result,
given the fact they should all be produced under urban smog conditions.  PAN  and PPN have  been
routinely detected  in urban atmospheres (59).  These species are  produced in the atmosphere
primarily via OH reaction with the corresponding aldehyde (i.e., acetaldehyde, propionaldehyde,
butyraldehyde, and benzaldehyde).
                                           61

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                                        SECTION 5
                                       DISCUSSION
      In the Results section of this report it is shown that irradiation of the simple hydrocarbons
acetaldehyde, propylene, and toluene in the presence of NOx yields products that are significantly
mutagenic, as determined  by  using the Ames test.  It should be noted that none of these three
hydrocarbon/NOx mixtures are mutagenic prior to irradiation.  This result suggests that common
urban pollutants, which are  themselves nonmutagenic, can be  converted into significantly
mutagenic products in the course of atmospheric photooxidation.  All three of these species are
important reactive components of urban air, so it is important to attempt to determine the cause of
the observed mutagenic activities.

      Throughout this discussion we will consider only the data for TA100 without 59.  In all cases
the reversion rate was higher with TA100  (and therefore easier  to accurately measure), and no
difference was seen between the data with and without S9 metabolic activation.

      To determine which species may have caused the observed mutagenic  activities for the
irradiated mixtures it is necessary to know the dose of each product in the test  plates and the
mutagenic activity (in  revertants-plate~1-pmor1) of each product.  The quantity of each species in
the test plates can be determined from Expression II (see Section  4.2).  To quantify the mutagenic
activity of individual species, we conducted either standard plate  incorporation tests with the pure
chemical or single component gas-phase exposures in which the quantity of the species deposited
into the plates was measured.  Table 5-1 shows the mutagenic activities measured for all product
species found to be mutagenic in this study.
      For the irradiations of toluene and NOx, the reaction products to be considered are PAN,
ozone, formaldehyde, acetaldehyde, methylglyoxal, glyoxal, benzaldehyde, the cresols, and nitric
acid.  The chemical mechanisms leading to these  products have  been  described in detail
elsewhere (28,47,60,61).  The oxidation of toluene  in this type of system occurs via an OH-radical
chain mechanism. The OH radicals can either abstract an H atom from the substituent methyl group
(e.g., to produce benzaldehyde) or add to the aromatic ring, leading to production of the cresols or a
wide variety of ring fragmentation products including formaldehyde, glyoxal, and methylglyoxal.
These ring fragmentation processes are quite complex, and a large degree of uncertainty  exists
regarding the predominant operative reaction pathways.
                                            62

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                   TABLE 5-1.  MUTAGENIC ACTIVITIES MEASURED BY TA100
                                                           Mutagenic Activity
                    Species                                  (revertants/pmol)
                    HCHO                                          12
                    Glyoxal                                         18
                Methylglyoxal                                     200
                     H202                                          10
                     PAN                                   (14revertants/h)*
                    2-HPN                                          3
                    2-NPA                                          3
               CH3C(0)CH2ON02                                    55
                   CH3OOH                                       (100)t
                  CH2CICHO                                       400
 *  Independent of concentration between 100 and 500 ppb. At 200 ppb this corresponds to—680 revertants/iimol.
 t  Value found for (CH3)3OOH.
      As suggested in the reaction profile (Figure 4.1-1) for the irradiation of toluene and NOx, the
product distributions for the two sets of dynamic experiments exhibit significant differences. At
T = 3.0 h, NO in the system converts RO2 radicals to RO radicals, thereby promoting the production of
aldehydes and allowing for regeneration of OH radicals. At T; = 6.7 h, the NO has been removed and
NO2 photolysis leads to buildup of 03.  Under these conditions, various radical-radical reactions can
occur, leading to production of H2O2, organic peroxides, and peroxy nitrates.  In addition, photolysis
of the initially formed aldehydes will occur to a greater extent, and secondary reactions of the
products with OH, 0-3, and NO3 can occur (e.g., cresols can be converted to nitrocresols).

      These differences lead us to believe that at long extent of reaction the observed response for
the toluene experiments may be caused by either peroxide-type compounds or aromatic nitro-
compounds. We have found NO2, O3, acetaldehyde, benzaldehyde, the cresols, and nitric acid to be
nonmutagenic.  The test plate concentrations of HCHO, glyoxal, and methylglyoxal at x = 6.7 h were
0.93, 0.10, and 0.33 umol/plate at the end  of the 18.5-h exposure period (Table 4.1-2).  Using the
mutagenic activities of these three species listed in Table 5-1, we calculate that they contribute 11,2,
and 66 revertants/plate, respectively. Thus, methylglyoxal contributes roughly 13%  of the total
observed response  at i = 6.7  h for TA100.  Measurements were not made for H2O2. However,  a
reasonable upper  limit concentration for this species would be 50 ppb, corresponding to
~0.64 pmol/plate if the  entire mass deposited.   Therefore, H2O2 contributes, at most,
                                            63

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6 revertants/plate, a negligible amount. As indicated in Section 4.6, exposures of TA100 to pure PAN
in the concentration range of this experiment yields a reversion rate of 14revertants/h.  Dividing
500 excess revertants/plate by the total exposure time yields a  reversion rate for  the i; = 6.7-h
experiment of 27 revertants/h, assuming a linear dose-response curve.  Therefore, PAN could account
forupto~50% of the total observed response. It should be noted that no survivor plates were used in
the first toluene experiments, so it is unclear whether there may have been toxic effects for the
i = 6.7-h experiments. The  results of the subsequent 5 ppm toluene/0.9 ppm  NOx experiment
indicate that this is certainly a possibility.  That experiment yielded a considerably larger reversion
rate (50 revertants/h), although it was conducted at shorter extent of reaction.  In this latter case,
PAN contributed roughly 28% of the total mutagenic activity, assuming 14 revertants/h for PAN's
contribution to the reversion rate. Although we have accounted for a large part of  the observed
mutagenic activity for toluene, it appears that  unidentified mutagens are present.  It should be
noted that several potentially mutagenic unsaturated dicarbonyl compounds (e.g., c/s-butenedial,
6-oxo-2,4-heptadienal) have been found to  be ring fragmentation products (46,47). Several terminal
vinyl carbonyl compounds such as 1-pentene-3,4-dione and 5-oxo-1,3-hexadiene have also been
found to be products of OH reaction with toluene (46,47).  Vinyl carbonyl compounds can act as
mutagens, presumably through an epoxidation  mechanism  occurring at the vinyl group (62,63).
Finally, we note that in  the absence of NO, hydroperoxides such as CH^OOH are expected to be
produced in small yields.  As noted in the (CHaJsOOH entry in Table 5-1, these hydroperoxides may be
mutagenic.
      In the irradiated CaHe/NOx mixtures, the mutagens HCHO, 2-HPN, and 2-NPA are present at
both residence times. However, calculations similar to those conducted for the toluene system
indicate that these mutagens do not contribute significantly to the observed response. At T = 7.5 h ,
the measured reversion rates were ~24 revertants/h. Assuming a value of 14 revertants/h for the
contribution from PAN, ~58% of the total observed mutagenic activity of the products of the
photooxidation of propylene at long extent of reaction can be attributed to PAN.  From the results
of the CaHg/Oa and CsHg/^Os experiments it is clear that ozonolysis products and the NOs reaction
products do not contribute significantly to the observed mutagenic activity of the irradiated mixture.
Thus, if other mutagens are present in this system, their identities are unknown. Again, it is possible
that species such as CHsOOH are present and are mutagenic.  It should be noted that CsHg/NOx gave
a measurable response at t = 2.7 h. Although PAN is present at 6 ppb, we are currently uncertain as
to whether it may have contributed to the observed mutagenic activity under those conditions.
                                           64

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      It is interesting to note that products of the reaction of NOa with CsHe were found to be
mutagenic.  It can be shown that CH3C(O)CH20NO2 is the only product measured that should
contribute significantly to the observed mutagenic activity.  Based on the calculated deposition for
this species (0.4pmol/plate), CH3C(0)CH2ONO2 contributes 22 revertants/plate out of the total
observed response of 180 excess revertants/plate, or ~12% of the total. It should be noted PAN is
present at -20 ppb. Unfortunately, we have not conducted exposures with pure PAN at levels this
low, so it is unclear what its contribution might be.  It is quite possible, however,  that PAN
contributes, to some extent, to the observed mutagenic activity of the CsHg/NaOs mixture.  It is
known that organic hydroperoxides such as 2-HPPN (shown below) may be present and would be
produced as shown in Reactions 25 and  26.  These organic hydroperoxides may be significantly
mutagenic, as is (CH3)3OOH.
                                    °2                                                 25
                          CgHg+tfOg -* CH3CH(00)CH2ON02


                 CH.CfKOOKH-ONO.+HO- -» CH
-------
activity of —3000 revertants/umol for these products. The actual plate dose of products was probably
smaller than that calculated, since some of the products (e.g.,  1,3-dichloroacetone, CPAN) probably
were not completely deposited, which would make the actual mutagenic activity of the mixture
greater than that estimated above. In Experiment B the extent of Cl reaction with allyl chloride was
decreased by a factor of 4-5 relative to that in Experiment A, and the mutagenic  activity of the
mixture decreased by a factor of 3-4. This  suggests that the mutagen(s) that caused the majority of
the response resulted from the reaction of Cl atoms with allyl  chloride. Since the yield  of Cl atoms
from OH reaction with allyl chloride is —0.2 (32), the dose of  the Cl-atom reaction products in the
plates is then ~5 times smaller than that used in the calculation above. The actual mutagenic activity
of the products that caused the response is, therefore, at  least 15,000 revertants/umol.  This
represents a high mutagenic activity compared with other  known strong mutagens (e.g., ~4000
revertants/umol for 0-propiolactone [9]). In addition, many of  the products that do dissolve are not
mutagenic, or are only very weakly mutagenic, such as formaldehyde and 1,3-dichloroacetone. This
fact leads us to believe that the mutagenic activity of the  product (or products) that caused the
observed response is very large indeed.  (We note that we are assuming in this discussion that the
large response is not caused by synergistic effects.) The major product of Cl-atom reaction with allyl
chloride, 1,3-dichloroacetone, is nonmutagenic, according to our standard plate incorporation tests.
There are, however, other Cl-atom addition products that could be mutagenic and that are likely to
be present, such as 1,3-dichloro-2-propyl nitrate, which is produced as shown in Reactions 27 and 28.
It is reasonable to expect this product to be mutagenic, since 2-propyl nitrate is mutagenic according
to our standard plate incorporation tests.
                                          °2                                            27
                      Cl+CH2ClCH=CH2 -*CH2CICH(OO)CH2CI

                                                                                        28
                   CH2CICH(OO)CH2CI + NO -+CH 2CICH(ONO JCH fl

      Although it would be desirable to determine which species caused the observed  response in
Experiment A, the product distribution  under these conditions is not representative of allyl chloride
atmospheric photooxidation products. In the atmosphere the majority of Cl atoms are scavenged by
other hydrocarbons. As indicated  in  Section 4.4, the  mutagenic activity  of the allyl chloride
photooxidation products  is nearly  10 times smaller in the  absence of Cl-atom reactions (i.e.,
l^revertants-plate'T-h^-ppb"1 allyl  chloride consumed).  A calculation similar to that described
above (for Experiment A) yields —320 revertants/umol for the product mixture (Experiment D) at the
sixth exposure period.
                                            66

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      Chloroacetaldehyde, one of the major products of OH reaction with allyl chloride, has been
shown to be mutagenic (31). We conducted both standard plate incorporation tests and gas-phase
exposures of TA100 to Chloroacetaldehyde (in which the Chloroacetaldehyde plate dose was
quantitatively measured), and found values of 330 and 460revertants/nmol, respectively.  It would
therefore appear that essentially all of the observed response for Experiment D can be accounted for
by the presence of Chloroacetaldehyde, whose concentration is  roughly  equal to ACsHsCI.
(Interestingly, acetaldehyde is nonmutagenic when tested with TA100.)

      It is likely, however, that there were also other mutagens in this mixture.  For example, since
PAN has been shown to be a mutagen when tested with TA100, it would seem reasonable to expect
that CPAN is a mutagen as well.  In addition, CPAN would be expected to be more soluble in the test
medium than is PAN. However,  under the conditions of these experiments, for a 15-min exposure
and  a 100-ppb PAN concentration, we would expect  PAN to contribute no more than five excess
revertants.  Therefore, for CPAN to contribute significantly in these experiments it would have to be
considerably more mutagenic than PAN.  Preliminary experiments we  have done with CPAN indicate
that this is not the case. Other products we have identified, such as 3-chloroacrolein (32), may be
mutagenic as well, although they  have not yet been tested because of the unavailability of
standards.  Although glyoxal is a  weak mutagen  using  this strain (—18 revertants/pmol), its
concentration in this experiment  is much too small to contribute significantly.

      The Salmonella bioassay has been reported as being ineffective  in the detection of chlorinated
carcinogens (64).  In a recent analysis of mutagenesis data, Claxton et al. (65) showed that the
sensitivity with respect to detection of carcinogens using Salmonella for chlorinated organics is low
(63%). However, specificity (measure of the fraction  of noncarcinogens that yield a negative test
result) has a quite high value of 91%. Therefore, a positive result with chlorinated  organics is very
meaningful since more than 90% of the Sa/mone//a-positive compounds are animal carcinogens.

      It is clear from these experiments that, although the photooxidation products of allyl chloride
are much less mutagenic in the absence  of Cl-atom reactions, they are much more mutagenic  than
are  the  photooxidation  products of propylene (1.4 revertants  plate^-h"1-ppb"1  vs.
0.043 revertants-plate~1-h~1-ppb'1,  respectively).   It may be that  other,  more abundant,
chlorinated hydrocarbons yield more mutagenic photooxidation products than their nonchlorinated
analogues.  The fact that Chloroacetaldehyde is a strong mutagen (as  determined by using TA100) is
in itself an important  result because Chloroacetaldehyde would  be expected to  be  a common
                                           67

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photooxidation product of a variety of other chlorinated atmospheric hydrocarbons, such as
chloroethene.

      One objective of the wood smoke and automobile exhaust experiments was to determine the
phase distribution of the mutagenic photooxidation products of irradiated complex HC/NOx
mixtures.  From the results of the wood smoke/NOx experiments in which the exposure chamber
mutagen collection efficiencies were measured, it is possible to measure accurately the mutagenic
activity of these mixtures for gas-phase species in terms of revertants/m3, given the  measured
reversion rates (corrected for collection efficiency) in revertants per hour. This is calculated by using
Expression III below, where R is the measured collection-efficiency corrected reversion  rate in
revertants per plate per hour, F is the total exposure chamber flow rate in cubic meters per hour, and
N is the total number of exposed plates.
                                               3       -1    -1                       <"')
                    Mutagenic Activity, revertants/m =R- F  • N
The mutagenic activity of the gas-phase products can also be estimated in units of revertants per
microgram by  dividing the mutagenic activity (revertants/m3) by the gas-phase total hydrocarbon
concentration in micrograms per cubic meter. This total hydrocarbon concentration  can be
calculated from the total hydrocarbon analyzer values in ppmC, using the ideal gas equation with
the value 18.5g/mol carbon for the average reaction product. The results of these calculations are
presented in Table 5-2 for the irradiated wood smoke/NOx mixtures (Experiments E and F). It is clear
from the data in Table 5-2 that the mutagenic activity of the products of the wood smoke irradiation
is much greater than for the reactants, except for the case of TA100 measurements of the particulate
phase.  In addition, gas-phase mutagens appear to be more important, relative to those  in the
particulate phase. On the basis of revertants per cubic meter, ~99 and 82% of the mutagenic activity
was found in  the gas phase (TA100 and TA98, respectively).  The phase distribution of the
mutagenicity is shown graphically in Figure 5-1.

    TABLE 5-2. COMPARISON OF THE GAS- AND PART1CULATE-PHASE MUTAGENIC ACTIVITIES
        FOR WOOD SMOKE BEFORE AND AFTER IRRADIATION, STRAINS TA100 AND TA98
TA100
Gas

Reactants
Products
Rev/m3
<230
17,300
Rev/pg
<0.4
1.6
Particulate
Rev/m3
100
180
Rev/jig
0.30
0.27
TA98
Gas
Rev/m3
<100
3,230
Rev/jig
<0.17
0.30
Particulate
Rev/m3
80
730
Rev/pg
0.22
0.94
                                           68

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175-
150-
b
X
1 100-
Revertants
en »J
O ui
1 1
25-
0-



TA100






I I Gas Phase
[ | Particulate Phase


TA98
— -j
                      Before      After
                    Irradiation  Irradiation
  Before      After
Irradiation  Irradiation
 Figure 5-1. Comparison of gas- and particulate-phase mutagenicity of dilute wood smoke in air.

      For the wood smoke/I^Os experiment the mutagenic activities of the gas-phase products
were 9510 and 1290 revertants/m3 (TA100 and TA98, respectively). The corresponding numbers were
1620 and 1350 revertants/m3 for the particulate-phase wood smoke/NaOs products. The mutagenic
activities of the products of the I^Os/NOs reaction with wood smoke are, then, comparable to or
higher than (for particulate matter) those from the irradiated mixtures.  This  observation seems
reasonable based on the current understanding of these reactions. For gas-phase species, NOs can
react either by abstraction (e.g., for aldehydes to yield PANs) or addition to alkenes to form organic
nitrates.  The abstraction route produces the same products as the OH abstractions occurring in the
irradiated system.  We demonstrated earlier (Section 4.2) that NOs addition to alkenes  such as
propylene yields mutagenic organic nitrates. As discussed, reactions of N2Os with particle-bound
PAHs can yield mutagenic nitro-PAHs.

      For the  automobile exhaust/NOx irradiation the mutagenic activities of the gas-phase
products are 5850 and 1160 revertants/m3 (TA100 and TA98, respectively). The mutagenic activities
of the particulate-phase products are 28 and 62 revertants/m3 (TA100 and TA98, respectively). These
latter two numbers are clearly considerably  smaller because of the much smaller total particulate
mass present.  On the basis of revertants per microgram, the  particulate-phase products from the
                                            69

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simulation experiments with wood smoke and automobile exhaust mixtures are very similar (see
Tables 4.5-3 and 4.5-5). On the basis of revertants per cubic meter, 99 and 95% of the mutagenic
activity from irradiated automobile exhaust mixtures was found in the gas phase (TA100 and TA98,
respectively).

      These experiments  indicate  that irradiation of complex mixtures can produce substantial
increases in the mutagenic activities for both gas- and particulate-phase products and that much of
the observed mutagenic activity is present in gas-phase products. It would be desirable to determine
the identity of the mutagenic products that are produced.  Unfortunately, these mixtures are very
complex  prior to irradiation, containing a wide variety  of reactive species, and are even  more
complex  after irradiation, since each  reactant can yield a number of different  photooxidation
products. Therefore, to identify each species that contributed to the observed mutagenic activities
would be a formidable  task.   However, in the irradiated wood smoke/NOx and automobile
exhaust/NOx experiments, PAN should contribute 14 revertants/h to the measured reversion rates of
174 and 70 revertants/h, respectively,  measured by TA100.  These are uncorrected reversion  rates,
because not all PAN deposits.  PAN therefore accounts for ~8 and 20%  of the  total gas-phase
mutagenic activity observed for these two systems (wood smoke, automobile exhaust, respectively).
This is a reasonably large  contribution for a single compound, given the complexity of the product
mixture.

      At this point, we have demonstrated that a number of atmospheric hydrocarbons can  be
converted to  mutagenic products  (as  determined by using the Ames test) in the course of their
photooxidation.  An important question one might ask is, "What is the significance of this for
ambient conditions?" To summarize the  results of our studies with toluene,  propylene,
acetaldehyde, and ally! chloride,  we can perform  a  simple calculation  indicating the relative
importance of  these  species in terms  of the rate of production of mutagenic activity under
atmospheric reaction conditions.  This calculation is done with the aid of the typical ambient
concentration data (24,59) and the rate constants (32,55) for their reaction with the OH radical that
are presented in Table 5-3. The first row of this table presents the observed mutagenic activities for
the products of the photooxidation of each of these  species, in terms of revertants per hour of
exposure, per ppb of  hydrocarbon consumed, as measured  in our simulation  experiments. The
relative significance of these mutagenic activities depends, therefore, on the rate of reaction of each
of these species under atmospheric conditions. Values for the quantity (in ppb) of each hydrocarbon
that would be consumed per hour of reaction for an OH concentration of 1 x 106 molecules/cm3
(66,67) are  given in the fourth row of Table 5-3. Finally, the  number of revertants per hour of
                                           70

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 exposure of TA100 to these products for these conditions can be obtained by multiplying the values
 in the first row of Table 5-3 by the values in the fourth row. These values are given in the last row of
 the table. It is important to understand that these values are the number of revertants per plate per
 hour of exposure, according to the exposure techniques employed in our experiments. The absolute
 magnitude of these values has little actual significance.  However, the relative magnitudes represent
 their potential relative contribution to the production of atmospheric mutagens  (i.e., bacterial
 mutagens). These values are listed in the table in order of decreasing importance.  Allyl chloride is
 relatively insignificant because of its very low ambient concentrations.
     TABLE 5-3. CALCULATED MUTAGENIC ACTIVITIES OF THE PHOTOOXIDATION PRODUCTS OF
                              ATMOSPHERIC HYDROCARBONS

Revh"i ppb"1
Urban cone., ppb
k(OH)-10Hcm3molecule-is-i
k(OH)-[OH]-[HC] (ppb/h)
Rev/h
CH3CHO
0.094
9
1.62
0.52
0.049
Toluene
0.069
15
0.62
0.34
0.023
Propylene
0.036
4
2.63
0.38
0.014
C3H5CI
1.4
0.005
1.7
0.0003
0.0004
      If the — 14-revertants/h  reversion rate measured for  100 ppb PAN is applicable at ambient
concentrations (typical urban mean concentrations are —0.6 ppb [59]), then the mutagenic activity of
PAN (according to our experimental conditions) is ~0.14 revertants-h~1-ppb~1.  At ambient
concentrations PAN then might contribute as much as 0.08 revertants/h, which is  larger than the
values calculated in Table 5-3. It must be noted, however, that the sensitivity of the  Ames test
according to our procedures is such that.the mutagenic activity of PAN could not  be measured at
these low concentrations.

      For each  compound  in  Table 5-3, it should also be noted that the indicated  mutagenic
activities may not necessarily be contributed independently.  The two largest potential contributors,
CHsCHO  and  PAN,  are photooxidation products of toluene and propylene, and a portion  of the
mutagenic potential from  CHsCHO and  PAN is already accounted for under the toluene and
propylene columns.  Clearly, however, other ubiquitous pollutants in urban atmospheres could give
rise to these and other mutagens in the ambient atmosphere.

      In  conclusion, this work demonstrates that a variety of atmospheric hydrocarbons can be
converted into mutagenic products in the course  of their photooxidation.  It is important  to
understand which  species  caused the observed mutagenic activities, particularly for reactive
atmospheric hydrocarbons such as acetaldehyde.  A few mutagenic products have  been  identified.
                                           71

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such as PAN, but  much more work  is necessary to determine the potential human health
implications. We have shown in laboratory studies that irradiated HC/NOx mixtures result in highly
mutagenic products, and it would be worthwhile to attempt to measure ambient mutagenicities as a
function of the photochemical pollutant concentrations.  These results should be regarded as a first
step in a long-term research effort. Much more work is clearly called for  in assessing the extent to
which hazardous compounds can be produced from the photooxidation of reactive hydrocarbons in
urban air.
                                           72

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"PB87199675*
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INVOICE:
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M17B
548881
1*79595
NONE
02-24-98

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1. REPORT NO.
   EPA/600/3-87/020
                             2.
                                                          13. RECIPIENT'S ACCESSION NO.
 4. TI~i.E AND SUBTITLE
  THE  PRODUCTION OF MUTAGENIC COMPOUNDS AS A
  RESULT  OF  URBAN PHOTOCHEMISTRY
             B. REPORT DATE
                June 1987
             6. PERFORMING ORGANIZATION CODE
 7. AUTHORIS)
  P. B. Shepson,  T.  E.  Kleindlenst, and
  E. 0. Edney
                                                           B. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
  Northrop  Services,  Inc. -  Environmental  Sciences
  P. 0. Box 12313
  Research  Triangle Park, North Carolina 27709
             10. PROGRAM ELEMENT NO.
                A101/C/72/01-6109  (FY-87)
             11. CONTRACT/GRANT NO.
                68-02-4443
 12. SPONSORING AGENCY NAME AND ADDRESS
  Atmospheric  Sciences  Research Laboratory-RTP, NC
  Office  of Research and Development
  U.S. Environmental Protection Agency
  Research  Triangle Park, North Carolina 27711
             13. TYPE OF REPORT AND PERIOD COVERED
                Interim (7/83-2/87)
             14. SPONSORING AGENCY CODE
                EPA/600/09
 15. SUPPLEMENTARY NOTES
  See also  articles published in Environmental Science & Technology:
  ES&T,  19,  p. 249 (1985);  ES&T, 19, p. 620 (1985); ES&T, 20, p. 493  (1986)
 16. ABSTRACT
       A  series  of atmospheric simulation experiments was conducted  to  examine the
  role of urban  photochemical  processes on the formation and removal  of potentially
  hazardous  air  pollutants.   The experiments were conducted in a  22.7-m^ Teflon smog
  chamber which  was coupled  to bioassay exposure chambers.  The mutagenic activities
  of the  tested  mixtures of  organic chemicals and nitrogen oxides were  measured
  before  and after irradiation by direct exposure of Salmonella typhimurium (strains
  TA 98 and  TA 100) to the smog chamber contents.  The mutagenic  responses of the
  starting 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.  The amount of mutagenicity was  also observed to
  increase with  increasing photochemical oxidation.  The common photochemical pollutant,
  peroxyacetyl nitrate (PAN),  was found to contribute significantly  to  the overall
  vapor-phase mutagenicity in all of the chemical systems in which it was formed.
 7.
                               KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Croup
 8. DISTRIBUTION STATEMENT
    RELEASE TO  PUBIC
                                              10. SECURITY CLASS (This Report)
                                                 UNCLASSIFIED
                           21. NO. OF PAGES
                                87
20. SECURITY CLASS (Thispage)
    UNCLASSIFIED
                           22. PRICE
CPA Form 2220-1 (t-73)
                                             1

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                                      DISCLAIMER


     The information in this document has been funded by the United States  Environmental
Protection Agency under Contract Numbers 68-02-4033 and 68-02-4443 to Northrop Services, Inc. -
Environmental Sciences. It has been subject to the Agency's peer and administrative review, and it
has been approved for publication as an EPA document.

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