MAY  T'9S7
THE PRODUCTION OF MUTAGENIC COMPOUNDS
  AS A RESULT OF URBAN PHOTOCHEMISTRY
ATMOSPHERIC SCIENCES RESEARCH LABORATORY
   OFFICE OF RESEARCH AND DEVELOPMENT
  U.S. ENVIRONMENTAL PROTECTION AGENCY
     RESEARCH TRIANGLE PARK, NC 27711

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                                          GOOR87057
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. Box12313
       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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                                      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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                                        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 paniculate 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 	     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   Complex 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/H2O/air irradiation  	—	      16
4.1-2 Reaction profile: toluene/NOx irradiation,  10/26,1 = 6.7 h   -.	.	.-      20
4.1-3 PAN concentrations: toluene/NOx irradiation, 10/26, T; = 6.7 h  	      21
4.1-4 Formaldehyde concentrations: toluene/NOx irradiation, 11/30,1; = 3.0 h  	      21
4.1-5 Methylglyoxal profiles: toluene/NOx irradiation, 11/30,1; = 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 forthe 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 fori; = 2.7-h irradiated CsHg/NOx mixture, TA100 	      31
4.2-5 Dose-response curve for T = 7.5-h irradiated CsHg/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, CsHsCI/NOx irradiation   	      43
4.4-2 Experiments: reaction chamber component concentrations and mixture
     mutagenic activity, CaHsCl^Hs/NOx irradiation 	      43
4.4-3 Experiment C: reaction chamber component concentrations and mixture
     mutagenic activity, C2He/NOx irradiation	...	      44
4.4-4 Experiment D: reaction chamber component concentrations and mixture
     mutagenic activity, CsHsCI^Hg/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 particulate 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 (10/26,1 = 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 CaHg/NOx Irradiation
      (Dynamic Mode)  	     28
4.2-2  CaHg/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 (± loJfortheCsHe/NOx Irradiations  	     30
4.2-4  Average Reactant and Product Steady-State Concentrations for the CaHg/^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 (CaHg/N2O5 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/Milligramof 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/N2Os (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                         polycylic aromatic hydrocarbon




PAN                         peroxyacetyl nitrate




HAP                         hazardous air pollutant




PPN                         peroxypropionyl nitrate




PBN                         peroxybutyryl nitrate




PBzN                        peroxybenzoyl nitrate




CPAN                        chloroperoxyacetyl  nitrate




PGDN                        propylene glycol dinitrate




2-HPN                        2-hydroxypropyl nitrate




2-NPA                        2-nitratopropyl alcohol




NPPN                        nitroxyperoxylpropyl nitrate




EAA                         electrical aerosol analyzer




CNC                         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, C.M.  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 particulate 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 a/.  (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,p-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 et a/. (10)
determined that a significant fraction of the  organic fine particulate matter (<2.5 urn) from Denver
aerosol could be attributed to wood smoke emissions.  In  addition, particulate 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 O3 and N2Os on the surface of
atmospheric particulate 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 a/. (21) demonstrated that
PAHs adsorbed on wood smoke particulate matter can photodegrade at significant rates.  Clearly,
atmospheric reactions can have a significant impact on  the mutagenic activity  of atmospheric
particulate 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/N205 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 is ethylene 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 allyl chloride are fairly
well understood (32), should facilitate the identification of individual mutagenic products for this
particular HAP-

      Forthe individual hydrocarbons studied, 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 particufate 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  NOs/^Os 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 NO3/N2O5 with
propylene and also one in which we reacted NO3/N2O5 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
(PBzN).

      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 particulate phase in our experiments is probably the result of adsorption of gas-phase
(low volatility) mutagenic products onto existing  particulate matter, rather than a reflection of
reactions occurring on the surface of the particulate matter.

      For most of the product mixtures investigated, a significant portion of the mutagenic activity
observed may have been caused by PAN, which has been shown to be mutagenic. The absolute value
of the mutagenic activity of this species is currently uncertain, however, and much more work is
necessary to determine the mutagenic potential of PAN at ambient concentration levels.  Because
PAN is ubiquitously present in polluted urban atmospheres, it would also be desirable to determine
the biological impact of PAN on other 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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                                       SECTIONS
                               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 atO°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, N205 was prepared by mixing O3 and  NO2

(1% in N2) in a 3-L  Pyrex mixing bulb. The 03 was  produced at -1% in  O2 with a Welsbach

Model T-408 O3 generator supplied with zero-grade O2. The 03 reacted with NO2 in the mixing  bulb

to produce NO3( which was in equilibrium with N2O5, as indicated in Reactions 1 and 2.  The resultant

N2Os/N02 mixture was then diluted with clean dry air in the inlet manifold.

                                                                                          1
                                            ^                                          2,-2


 For the wood smoke/NzOs  experiment the initial  N20s concentration was ~2 ppm, and  for the

 propylene + N2Os experiment, it was ~1.8 ppm.
                                   NO
                                  inN2
                              X
                              Mass
                              Flow
                            Controllers
                                           Mass /
                                           Flow
                                          Controller
                                         Mixing
                                           Fan
                                             Aluminum End Plates
                                               Reaction Chamber
                                                   22.
                                             \
'\
                            Samples
                         >•   and
                            Exhaust
                                               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 71 508 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.
                                                                                      4
                          Cl+CH^CHO  -?HCl+CHC(0)0^
                                 o                o      Z
                      CH3C(0)02+N02 ^ CH3C(0)02N02 (PAN)                         ''
The PAN was then collected in a cold trap and vacuum distilled from -63°C to -1 10°C. PAN, PPN, and
PBN were also prepared in dodecane solution by nitration of the corresponding peroxy-acid (35,36).
The PAN-type compounds were also produced photochemically with the reaction chamber operating
in a dynamic mode, using mixtures of 63, NC>2, and the corresponding  aldehyde, as shown  in
Reactions 7-9 below for PBzN.
                                                                                      7


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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 following 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 S9 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

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      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 GGMS.  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
                 CH3C(0)OONO2 + 20IT -» CH f(O)0~ + N0~+ 0 2+ H 20
      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:
                                       ft                                               0)
                                         ex
                                 t   -    exp(-kt)dt
                                  e"   J a
                                            11

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where t is the exposure time in real time and k is the dilution rate constant.

     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 (i = 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 CarbopackC, 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 Dasibi 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

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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 Valco 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-pm pore size), extraction of the filter with 10"5M 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 x 2-m glass column packed with 10% SP-1000  on
80/100 Supelcoport and operated isothermally at 155°C.   Detection was achieved  with a Valco
Model 140B 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

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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 chloroacetaldehyde, 3-chloroacrolein, and
1,3-dichloroacetone were collected by bubbling 10-L chamber samples through 2 cm3 of methanol
cooled to 0°C in an impinger bottle. Collection efficiency for this technique was found to be >99%,
as determined by using two bubblers in series.  These three compounds were separated  by a
3.2-mmi.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, Oj, H2C>2, and
HO2NO2- Ethylene was measured by using a Varian 1400 GC (FID), which contained a 3.2-mm x 2-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
CarbopackC 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
275°C with a  Nutech 320 thermal  desorption unit. The desorbed samples were analyzed with an
HP 5985 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-mmx2-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 GGMS 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-cm x8.5-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

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                                        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-
                            O Toluene
                            O NO
                            O NOX-NO
                            A 03
                            • PAN
                            x CNC
                                  34567
                                   11/16   11/30         lp/11
                                          Time, h     l6'26
 Figure 4.1-1. Static-mode toluene/NOx/H2O/air irradiation.
                                            16

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      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 (5 ft3/min or -c = 2.7 h) and on the  slow-flow (large T) end  by the required
sampling and exposure chamber feed  rates (2ft3/min or t = 6.7h).  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 T; = 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 4.1-1. BIOASSAY RESULTS. TOLUENE/NOX EXPERIMENTS. AVERAGE REVERTANTS/PLATE (S.D.)*
            Expo-
            suret
                                11/17and 11/30; t = 3.0h
                                                                         10/1 land 10/26; i = 6.7 h
          TA100
TA100 + S9
TA98
TA98 + S9
TA100
TA100 + S9
TA98
TA98 + S9
oo
 S        119  (14)    117    (5)     25   (5)     31    (5)

          117  (16)    117   (12)     48   (4)     50    (4)


BCA      162  (23)    160   (35)     39   (6)     52    (5)

          263  (39)    280   (47)     81  (15)     88   (13)


 BR       181  (26)    171   (26)     30   (4)     51    (6)

          222  (40)    250   (45)        -        72   (12)


 BF       242  (23)    209   (37)     28   (6)     44    (9)

          308  (50)    286   (31)     75  (11)     81   (10)
                                        124    (19)     119   (16)

                                        154    (15)     146   (19)    31    (5)     40


                                        213    (23)     219   (44)

                                        158    (23)     179   (18)    21    (9)     16   (4)


                                        321    (40)     380   (24)

                                        242    (44)     249   (31)    24   (10)     49   (7)
                                                                           689    (27)    639   (106)

                                                                           751   (109)    548   (177)     63  (22)
            BU      211   (30)     189  (24)    31    (5)     45    (6)      705    (44)    583    (52)

                     285   (41)     274  (37)    67    (7)     78  (12)      863   (112)    636    (81)
                                                                                  91  (10)
                                                                                            76    (8)     80  (11)
          *  First and second lines show 11 /17 (or 10/11) and 11 /30 (or 10/26) results, respectively.
          t  S = spontaneous; BCA = clean air; BR = reactants; BF = filtered effluent; BU = unfiltered effluent.

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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
i = 3.0 i
14
48
48
5
16
20
7
11
3.5
3.5
62
169
0
Test Plates,*
nmol/plate
= 6.7 i = 3.0 i = 6.7
132 91 198
280
82 367 933
8 - -
24 34 103
34 125 333
12 38 66
5 111 30
1.0 37 6
1.5 37 9
84 - -
490
400
* 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 this2-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

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O Effluent Toluene
® Input Toluene
O NO
A NOX-NO
V03
   •f
                                CNC
                                BR Toluene
                                BF Toluene
                                BU Toluene
                                                                                   -6000
                                                                                   -5000
                                                                   -4000
                                                                                        g
                                                                                        O
                                                                                        •o
                                                                                        a
                                                                                         '
                                                                                   -3000 o
                                                                                   -2000
                                                                                   -1000
                                            25 Plates  30
                                              Open
                                                                   40
                                                          45 Plates  50
                                                             Closed
             5       10      15      20
                                        Time, h
Figure 4.1-2. Reaction profile: toluene/NOx irradiation, 10/26, i = 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, x = 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

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       150-
       100-
    -D
     a
     a.
    2?
    <
    a.
        50-
                         O Effluent
                         O BU
                         D BF
                         10
                                15
                                        20
                                               25 PI M  30
                                                 Op«n
                                              Time, h
                                                              35
                                                                     40
45  Plain   50
   Closed
Figure 4.1-3. PAN concentrations: toluene/NOx irradiation, 10/26, t = 6.7 h.
                                             Time, h
Figure 4.1-4. Formaldehyde concentrations: toluene/NOx irradiation, 11/30, i = 3.0 h.
                                               21

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                                             Time, h
                                                     30 Plain  35
                                                       Open
                                                                   40
45   Plato 50
    Cloied
  Figure 4.1-5. Methylglyoxal profiles: toluene/NOx irradiation, 11/30, T; = 3.0 h.
             TABLE 4.1-3. WATER PLATE CONCENTRATIONS, NANOMOLES PER PLATE
                         (10/26, T; a 6.7 h,TOLUENE/NOx IRRADIATION)
Exposure QH5CHO o-Cresol m-Cresol p-Cresol
BCA <1 <1 <1 <1
BR <1 <1 <1 <1
BU 5 17 6 8
BF 5 11 3 4
HCHO
43
130
600
613
N02-
87
456
348
261
N03-
32
16
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 T = 6.7 h (roughly 500 induced revertants per plate for TA100,
                                            22

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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 59 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
particulate matter does not contribute significantly to the observed response.  It is quite likely that
the extent of deposition of particulate 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 Cc = 2.7 h) for an irradiated  mixture of 5.1 ppm toluene/0.9 ppm IMOx-  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, -S9).   The
indicated product distribution corresponds to that in Figure 4.1-1 near the beginning of the increase
in Os 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

-------
              TABLE 4.1-4. REACTANT AND PRODUCT CONCENTRATIONS, PPB
                           (TOLUENE/NOx IRRADIATION, 8/86)
Inlet Effluent
Toluene 5145(1310) 4403(±144)
NO 896 2
NOX 942 394
03 ' 153
HNO3 160
CH3COOH 47
PAN 85
HCHO 50
CH3CHO 6
C6H5CHO 37
(CHO)2 150
CH3C(O)CHO 134
8/1 2/86 Toluene/NOx
800-

700-
600-
„ 500-
S 400-

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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 63 with propylene and of NCV^Os with propylene. For the CaHg/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 CsHg/^Os exposure, the average residence time was 2.7 h.
      For the CaHg/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 CaHg/^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 CaHg/^Og exposure, a reactants exposure chamber was not employed since
N2©5 would react with the water evaporated from the plates to produce gas-phase HNOs, 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 O3, NO, NOX-NO, C3H6, and PAN measured  in the
C3H6/NOx static run, which had the same initial conditions as employed in the dynamic experiments.
The N02 photolysis rate constant under these conditions was ~0.3 min"1, and the dilution rate was
0.037 h"i  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 O3 and PAN maxima.
                                                            =7.5 hrs
          800-
                                                                         10
 Figure 4.2-1. Time profiles for the major products in the C3H6/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 HNO3.  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 C3H6 in the presence of 02 and NO
forms 2-HPN and 2-NPA as stable products.
              360-
              340-
              320-
              300'
              280-
              260'
              240
• HCHO
Q CHjCHO
A HCOOH
O HNOj
                                                                   10
                                         Time, h
  Figure 4.2-2. Time profiles for the aldehydes and nitric and formic acids in the
  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

-------
         TABLE 4.2-1. AVERAGE REACTANT AND PRODUCT CONCENTRATIONS (ppb) FOR
                         C3H6/NOX  IRRADIATION (DYNAMIC MODE)
Compound
C3H6
NO
NOX-NO
03
HCHO
CH3CHO
PAN
HN03
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
(t = 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 J
                                       RT
                                                                                       (M)
                                           28

-------
where X- is the plate concentration of species i; P^R) and Pj{£) are the partial pressures (uatm) of
species i in the reaction and exposure chambers, respectively; VE 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-
t
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
i
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 nmol/plate.
      t 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
HNO3. In 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 C3H6/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 ng, 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 ( ± 1o) FOR THE C3H6/NOX IRRADIATIONS
                      Clean Air
                      Irradiation                 i a 2.7 h                 i a 7.5 h
     Exposure   __
     Condition      w/oS9      w/S9        w/oS9      w/S9         w/oS9      w/S9
   Spontaneous    164112   178±11          142110   13118         184129   131118
   15.4nmol
   sodium azide    909173      -              919         -           1388
2.6 nmol
2-amino-
anthracene - 1525
Clean air
chamber* 361160 379170
Reactants
chamber* 356148 363167
Effluent
chambert 374159 404169
421 - 536
194115 225132 278192 290158
261141 293154 294158 268 + 44
356169 373148 903169 9681101
  * Numbers are averages for ~25 plates.
  t Numbers are averages for—50 plates.

      The T = 2.7-h and T  = 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 T, = 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 i = 7.5-h exposure the survivor counts were substantially lower
                                           30

-------
beyond a 10-h exposure time.  In agreement with the earlier experiments, the  response at  the
T = 7.5-h residence time was at I east twice as large as that observed for the i = 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-
           
           a-  320-
              300-
              280-
              260-
              240-
              220-
              200
                                                                         C)
                                                          C)
                               5  6
                                          9  10  11
                                          Exposure t, h
                                                    I  I   I   i   I   I   I   i   I
                                                   12  13  14  15  16  17  18  19 20
Figure 4.2-4. Dose-response curve for t = 2.7-h irradiated
      To determine the extent to which
     reaction with  C3H6  may lead  to
mutagenic products that could account for
the large mutagenic activity observed in the
irradiated CsHg/NOx system (at  i = 7.5  h),
we conducted a CsHg/^Os exposure.
                                           700-
                                                              mixture, TA1 00.
      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 NOa 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
                                                              Eiposure Time, h
                                          Figure 4.2-5. Dose-response curve for x = 7.5-h
                                          irradiated CaHg/NOx mixture, TA100.
                                            31

-------
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 below.
                              R02N02  ^
                               R02+NO
                                                                                     12
where R is an organic group. The resultant alkoxy radical would then be removed by reaction with
02 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 C3H6/N2O5 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 CaHg/NjOg EXPOSURE
Parameter Measured* Inlet
Effluent
Inlet Reactant Flow, L/min 140 ± 0.085
NOX 1.76 ± 0.20 1.94
C3H6 1.26 ± 0.04 1.00
HCHO
CH3CHO
a-nitratoacetone
PGDN
2-HPN
2-NPA
PAN
0.017
0.023
0.032
0.002
0.008
0.002
0
± 0.19
± 0.04
± 0.004
± 0.007
± 0.011
± 0.001
± 0.002
± 0.001
.020
* Concentrations in parts per million.
                                          32

-------
      In this experiment, C3H6 and N2O5 were diluted and mixed together in the inlet manifold.  It
was possible that they could react as shown in Reactions -2 and 1 3,
                                        f-* Products                                   13
prior to entering the reaction chamber.  In a separate CsHe/N^s static experiment under similar
conditions (44), we  observed a value for A^Os/ACaHg of roughly  2:1.  Since ACaHg  in this
experiment is 0.26 ppm,  the assumption can be  made that the initial N2Os concentration was
~0.5 ppm. This then leads to an initial NO2 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 k2 and k-2 of 4.6x 10"12 cm3 molecule"1*"1 and
3.7x 10"1s"1, respectively (50), an equilibrium NOa  concentration  of 2.2 ppb is estimated.  Using the
value k13 = 4.2x 10"15 cm3 molecule'V1 (51), and a residence time in the inlet manifold of 1 min, we
calculate that < 1% C3Hg 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 C^H^.  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 C^H^/N^O^ 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 eta/. (48) observed a significant wall loss rate for NPPN in  their
reaction chamber.
                                            33

-------
        TABLE 4.2-5. OBSERVED MUTAGENIC ACTIVITIES IN REVERTANTS PER PLATE FOR
                               THE CHg/^Og EXPOSURE
           Exposure Condition	TA100	TA100 + S9


        Spontaneous                    133 ± 13                122± 21


        15.4 nmol sodium azide              668


        2.6 nmol                                                 590
        2-aminoanthracene


        clean air chamber*               180 + 31                169 ± 33


        effluent chamber*               375 1 122               351 ± 102


        effluent ( + NO) chamber*         323181               3691112

       * Numbers are averages for—25 plates.



                  TABLE 4.2-6.  CONCENTRATIONS OF PRODUCTS (C3H&/N2Og
              EXPOSURE) DETECTED IN MICROMOLES PER PLATE AS CALCULATED
                                     FROM EXP. II
Product
HCHO
HNO3
PAN
2-HPN
2-NPA
PGDN
CH3ONO2
HCOOH
CH3C(0)CH2ONO2
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 C3H6. However, a computer modeling study we conducted of the C3He/NOx irradiated system

indicated that NO3 reaction with C3H6 would have occurred only to the extent of -0.010 ppm at 7.5 h.

Since AC3H6 due to NO3 reaction in the CaHe/^Os 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 0-$ 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 CsHe/NOx
system. We conducted exposures of TA100 to the products from the reaction of 5.4 ppm C3H6 with
0.90 ppm 63 in the dark at i = 2.7 h.  In addition, an experiment was conducted in  which 0.21 ppm
NO2 was also added to this mixture to test for the possibility that radicals produced in the ozonolysis
might react with NC>2 to  produce mutagenic organic nitrates.  For both experiments, ACsHg 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
                                        C3H6/03/NO;	
                                        (* points displaced 0.1 h to the right)
                                       10
                                                    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 C^H6 reactions with 03 could
account for the large response (~600 excess revertants/plate) observed in the irradiated CsHe/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 C^Q 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  CaHe/Os  reaction could not have caused a significant
response in the CsHg/NOx irradiations.  It will  be shown in the Discussion section that formaldehyde
could account for the entire response observed in these CsHe/Os 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 €2^)4 and NOx concentrations
were  1.16 and 0.28 ppm, respectively, and  AC2H4 was 0.54 ppm.  The steady-state 63 and  HCHO
concentrations were 0.47 and 0.33 ppm, respectively.  In addition, concentrations  of HNOs, H2O2,
and HO2NC>2 should  have been comparable to those present in the long residence time CsHe/NOx
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 CsHe/NOx (and toluene/NOx) experiments.
4.3 IRRADIATED CH3CHO/NOX MIXTURES

      Our initial attempts at accounting for all the mutagenicity in the CsHe/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

-------
      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.  Figure 4.3-1  shows the time profiles for CH3CHO, PAN,  HCHO,  CH3ON02, NOX-NO
(which is associated with PAN + NOz), NO, and O3.
                                            800-
• PAN
• HCHO
A CHjONOj x20
                                          '=. 400-
      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.
                                          Q 300 H
                                            100-
                                          Figure 4.3-1.  CH3CHO/NO/NO2 static-mode
                                          irradiation.
                                           37

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   TABLE 4.3-1. AVERAGE REACTANT AND PRODUCT CONCENTRATIONS (ppb) FOR IRRADIATED
                                 CH3CHO/NOX EXPOSURES
Compound
CH3CHO
NOX
NO
03
PAN
HCHO.
T; = 2.6 h
Input Cone. Effluent Cone.
12371124 1064±113
427 ± 26 388 ± 22
406 ±35 40 ± 4
40 ±7
102 ±7
72 ±6
T = 4.0 h
Input Cone. Effluent Cone.
1190 ±26 977 ±58
212±13 174±10
31±3 5±2
311 + 12
171±11
101 + 16
      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 Ch^CHO is


very simple under conditions in which NO is present (52).
        I
         C
         CO
                              O T = 2.6 h (displaced 0.2 h to the right)

                              Q T = 4.0h
                             \I   I
                       34567
 i   l  l   i   I   I   I   I  l   i

10  11  12  13 14 15  16  17  18  19
                                      8   9


                                      Exposure Time, h


  Figure 4.3-2. Dose-response curves for irradiated CHsCHO/NOx exposures.
20
      Under these conditions, the reaction proceeds as shown in Reactions 14-24 below.



                          CHfHO + OH -»2 CH3C(0)02 + H^O
                                            14
                       CH3C(0)02+N02 ?±
                                                                                    15.-15
                         CH3C(O)02+NO -* CH3C(0)0+N02
                                                                                       16
                                        °
                              CH3C(0)0
                                                                                       17
                                           39

-------
                                      °2                                              18
                                      -» CH302+CO+H02
                            CH^O^+NO -» CH.O + NO.
                               o  z           J       *•
                                                                                     20
                                         HCHO+H02
                                           CH3ON02
                                            CH.ONO
                            CH^ONO + hv
                               J
                              HO2+NO -»• OH+N02                                   24
Because the photolysis of methyl nitrite (CH3ONO) 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 (CHsONOa). 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 C3He/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 (C3H5Cl)/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 NC>2 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 C2Hg, 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 NC>2 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 OB) 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"i1 cnn3-molecule~1-s~1  (54) and  its OH  radical rate
constant is much  smaller, 2.74x  10"13 cm3-molecule"|-s~1 (55).  We reported a rate constant  of
1.7x 10~11 cms-molecule"1 -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(cuc3Hsci) = 2-3
(±0.1)x 10~10 cm3Tnolecule~1-s~1.  using  k«;i + c2H6) = 6.38  x 1 O"11 cmS-rnolecule'1 -s~i
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 NO2).  As can be seen by comparing the data in
Figures 4.4-1 and 4.4-2, the  time of the NOa 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 (Experiment A) was due to the presence of
Cl-atom addition products.
                                            42

-------
                   O C3H5CI
                   D NO
                      NOX-NO
                   
-------
  -a o
   c a
   c m
   I
        900-,
800-
        700-
        600-
500-
                                                         D NO
                                                         a NOX-NO
                                                         tf O3
                                                         8 Revertants/plate
   S~   400 -
   i|
   u «B
    I   300H
        200-
        100-
                                                                11   12
 Figure 4.4-3. Experiment C: reaction chamber component concentrations and mixture mutagenic
 activity, CzH^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
                                                              • C2H6-=-200
                                                              O C3H5CI
                                                              O NO
                                                              & NO,-NO
                                                              7 O3
                                                              9 Revertants/plate
                                                                      Dilution
                                                          Time, h
                                  Figure 4.4-4. Experiment D: reaction chamber
                                  component concentrations and mixture mutagenic
                                  activity,
                                           44

-------
roughly once each hour during each irradiation.   To simplify the presentation,  we provide  in
Table 4.4-1 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^-h^-ppb-1             13            3.4           <0.4t          1.4
 *  For Experiments A and B, the light intensity was 2.5 times greater than for Experiments C and D.
 t  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 allyl 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 allyl chloride.  Given that at the sixth exposure
period for Experiment D  A C3H5CI = 145 ppb, assuming that the Cl-atom yield from OH (and O3)
reaction with allyl 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, CHsCHO, PAN [55]), 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 D, 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^-h^-ppb"1 (h"1 refers to per hour exposure).  We note that
under the conditions of the sixth exposure in Experiment D, some O3 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'T-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'1-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"i-h"i 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 Ch^CHO.  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)2] (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.
 f 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
particles/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
                                          500-
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 participate  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 pm in size with
the maximum  in the number distribution
occurring at approximately 0.1 jam.  The
maximum  in the  number  distributions
shifted to —0.2 pm 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
                                          300
                                          200
                                                     Million.     ~   ~• •
                                                       60     90
                                                     Irradiation Time, mm
                                    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 particulate is the result  of polar
gas-phase photooxidation  products  adsor-
bing  onto the  particulate matter already
present.   In the  absence of irradiation, the
number and volume distribution maximum
of the particles 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 particulate phase, signifi-
            8*for«irradiatioi
            Aft«r Irradiation
                  0.05   01    02
                  Pamclt Oiam«t*r tym)
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  particulate mass, part of the apparent
degradation occurred solely as a result  of the diluting effect of increasing the particulate 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 ACTIVITlESt 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
Paniculate
(revertants/ng)
TA100
0.30±0.10
0.27 + 0.15
TA98
0.22 ±0.06
0.94 ±0.30
   * Values in parentheses are zero within the uncertainty.
   t Corrected for collection efficiency.
  1400 —
  1200-
Experiment E
• Reactants
• Products
Experiment F
O Reactants
D Products
                    ].Q          2.0          3.0          4.0          5.0          6.0
                                    Exposure Time, h
 Figure 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.

      The mutagenic activities exhibited by strains TA100 and TA98forthe 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-
                 400-
                 300-
              °=  200-
                                                         Reactants
                        TOO   200
                                  300
                                       400
                                            500
                                                 600
                                                      700
                                                           800
                                                                 900
 Figure 4.5-4. Dose-response curve for the mutagenic activity (TA98) of the particulate extracts of
 wood smoke (+ 500 ppb of NOX) before and following irradiation.

      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  pg/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. particulate 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  N2Os, 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,
2.4 ppmC, 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 N2Os was an observed increase in
particulate volume.  In Figure 4.5-5 the EAA data for the wood smoke mixture before and after
addition of N2Os 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 um. 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 particulate 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 NOa and N2O5 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
Methyl glyoxal
Participate (ng/mg)
Pyrene
Anthracene + phenanthrene
Fluoranthene
Total particulate, nL/m3
Mutagenic Activities
TA 100, gases (rev/h)
TA98, gases (rev/h)
TA100 particulate (rev/jig)
TA98 particulate (rev/jig)
Initial

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

161
495
205
20





Final (trcn = 20 mm)

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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     250-
     200-
     150-
 Volume.
 nL/m3
     100-
     50-
                                   Final
                              Initial
      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"3 ppmC"1
Therefore, as indicated in Table 4.5-5, there
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 63 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
                 Particle Diameter, iim)
                                      1.0
_.     .„.-,...   i     .• ^ -.  ^       j
Figure 4.5-5. EAA volume distribution, wood
 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
CK4
C3H6
HCHO
CH3CHO
Acrolein
Glyoxal
Methylglyoxal
Particulate (ng/mg)
Pyrene
Fluoranthene
Anthracene + phenanthrene
Total particulate, nUrr\3
Mutagenic Activities
TA 100, gases (rev/h)
TA98, gases (rev/h)
TA100, particulate (rev/pg)
TA98, particulate (rev/pg)
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-=-104
                                                            «  PAN
                                                                                  H63
-54
-45
                                                                                       n
                                        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 ail 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 Umin.  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/NC>2  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 T = 7.5-h C3H6/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 ppb pure 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 HONOX 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-
280-
270-
260-
250-
240-
230-
220-
210-
200-
190-
180
                               O Clean Air
                               O Effluent (displaced 0.2h to the right)
                                                                        10
                                     Effective Exposure Time, h
 Figure 4.6-1. Dose-response curve for exposure of TA100 to pure PAN (220 ppb).

      Measurements of the mutagenic activity of PPN, PBN, and PBzN have also been conducted.
PPN and PBN were prepared according to the procedures of Gaffney ef al. (36). PBzN was prepared
in a dynamic-mode exposure involving a CeH5CHO/O3/NOx mixture that produces PBzN and HNOs as
the primary products. For these three PANsthe mutagenic activities observed were 0.022, 0.016, and
                                            60

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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 S9.  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^-ymol"1) 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, NC>2,
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/nmol)
                    HCHO                                          12
                   Glyoxal                                         18
                Methylglyoxal                                     200
                    H202                                          10
                     PAN                                    (14revertants/h)*
                    2-HPN                                          3
                    2-NPA                                          3
              CH3C(O)CH2ONO2                                     55
                   CH3OOH                                       (100)t
                  CH2C!CHO                                       400
* Independent of concentration between 100 and 500 ppb. At 200 ppb this corresponds to -680 revertants/u,mol.
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, 03, 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, 03, acetaldehyde, benzaldehyde,  the cresols, and nitric acid to be
nonmutagenic.  The test plate concentrations of HCHO, glyoxal, and methylglyoxal at T = 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 x = 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 umol/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
x = 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 CHsOOH are expected to be
produced in small yields.  As  noted in the (CHa^OOH 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 CsHe/Oa and C3H6/N2O5 experiments it is clear that ozonolysis products and the NO3 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 CH3OOH are present and are mutagenic. It should be noted that CsHe/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 NO? with C3H6 were found to be
mutagenic.  It can be shown that CH3C(O)CH2ONO2 is the only product measured that  should
contribute significantly to the observed mutagenic activity. Based on the calculated deposition for
this species (0.4nmol/plate),  CH3C(O)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 C3H6/N2Os 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.
                         C3HB+N03 -*2 CH3CH(00)CH2ON02                            25


                 CH.dKOOKH-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 {5-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 + CH.CICH=CH.
                                                                                         28
                  CH2CICH(00)CH2CI + NO -+CH £1CH(ONO JCH £1

      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.,
1.4revertants-plate"i-h"i-ppb"i 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 460 revertants/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  A CsHsCI.
(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/umol), 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"1-h"1-ppb"1  vs.
0.043 revertants-plate"|-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                        (lll)
                     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 PARTICULATE-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/pig
0.30
0.27
TA98
Gas
Rev/m3
<100
3,230
Rev/pig
<0.17
0.30
Particulate
Rev/m3
80
730
Rev/pg
0.22
0.94
                                            68

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1/b

150-
b
X
1 100-
£
c
£ 75-
IV

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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 unconnected 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 allyl 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"1 ppb"1
Urban cone., ppb
k(OH)-1011 cm3 molecule-is'1
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
C3H5C!
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-tV-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  CK^CHO 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 HGNOx 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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