United States
                    Environmental Protection
                    Agency
Atmospheric Sciences
Research Laboratory              x
Research Triangle Park NC 27711
                    Research and Development
EPA/600/S3-87/020  Sept. 1987
&EPA          Project Summary
                    The  Production  of Mutagenic
                    Compounds as a  Result of
                    Urban  Photochemistry

                    P. B. Shepson, T. E. Kleindienst, and E. 0. Edney
                      A series of atmospheric simulation
                    experiments was conducted to exam-
                    ine the role of  urban photochemical
                    processes on the  formation  and  re-
                    moval of potentially hazardous  air
                    pollutants. The experiments were con-
                    ducted in  a 22.7-m3 Teflon smog
                    chamber, which  was coupled to bioas-
                    say exposure chambers. The mutagenic
                    activities of the tested mixtures of
                    organic chemicals and nitrogen oxides
                    were measured  before and after irra-
                    diation by direct exposure of Salmo-
                    nella typhimurium  (strains TA98 and
                    TA100) to the smog chamber contents.
                    The mutagenic responses of the start-
                    ing  materials and the transformed
                    products were quantified by using a
                    modified Ames  test.  The chemicals
                    examined  included ubiquitous urban
                    pollutants (e.g., propylene,  toluene,
                    and  acetaldehyde),  a potentially
                    hazardous  chlorinated solvent (ally!
                    chloride), and complex mixtures (wood
                    smoke and auto exhaust) from common
                    urban pollutant  sources. In all cases,
                    the  irradiated products were  more
                    mutagenic than the original chemicals.
                    For the transformed complex mixtures,
                    the bulk of the mutagenicity was found
                    to be associated with the gas-phase
                    products rather than with the aerosol-
                    bound chemicals. Increased mutagen-
                    icity was also observed with increasing
                    photochemical oxidation. The common
                    photochemical pollutant, peroxyacetyl
                    nitrate, was found to contribute signif-
                    icantly to  the  overall vapor-phase
                    mutagenicity in  all of the  chemical
                    systems in which it was formed.
                      This Project Summary was devel-
                    oped by EPA's Atmospheric Sciences
                    Research Laboratory. Research Trian-
gle Park, NC. to announce key findings
of the  research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back).

Introduction
  During the past several years there has
been an increasing awareness that
exposure to polluted urban atmospheres
may pose an ill-defined but significant
human  health  threat, especially with
regard to the incidence of cancer. It has
become increasingly  apparent that an
accurate assessment of the potential
human health impact due to exposure to
atmospheric pollutants requires an
understanding of the influence of atmos-
pheric reactions on both hazardous and
nonhazardous species. The photooxida-
tion products of a wide variety of impor-
tant atmospheric aromatic hydrocarbons
have  been  shown to be mutagenic.
Researchers have  found a variety of
oxygenated  and nitrogenated species to
be mutagenic by using the Ames test,
implying that  the photooxidation of
reactive atmospheric hydrocarbons may
have a significant impact on the presence
of atmospheric mutagens.
  Concerns have also been raised with
regard to the possible  human health
threat of exposure to wood stove and
fireplace emissions. In addition to the
possible presence of  mutagens in the
emissions themselves,  evidence has
appeared recently in the literature
indicating that reactions of species such
as 03 and N2O5 on the surface of
atmospheric particulate matter can lead
to increases in the mutagenic activity of
adsorbed species  such as polycyclic

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aromatic hydrocarbons (PAHs). Consid-
erable effort has  been  expended  in
studying reactions that  occur on the
surface of paniculate  matter and  in
studying the impact of particulate-phase
photochemistry on the mutagenic activ-
ities of adsorbed species.
  In contrast, there has been a relatively
smaller effort aimed at identifying mech-
anisms for the production of  gas-phase
mutagens through atmospheric photo-
chemistry. A principal  reason for the
emphasis on particulate-phase muta-
gens is the relative ease of collection and
extraction of paniculate matter. To test
for  the mutagenic activity of  gas-phase
species by  using  the  standard plate
incorporation test, it  is  necessary  to
concentrate  the gas-phase  pollutants
into a suitable solvent in some manner.
Some limited attempts have been made
to measure gas-phase ambient mutagen-
icities by using XAD or other adsorbents
to  concentrate the species present.
However,  this process poses a number
of potential  problems, such  as loss of
volatile species during the extraction and
solvent concentration steps.
  In this report, we present a review of
the results  of  experiments  we have
conducted by using an alternative tech-
nique  for measuring the mutagenic
activity of gas-phase species generated
by photochemical reactions. The product
mixtures to be tested were produced in
a 22.7-m3 Teflon smog chamber. For this
technique, the Ames test plates were
dosed by continuously flowing the reac-
tion chamber air  over the  uncovered
plates, thereby permitting the soluble
species to deposit continuously while the
plates are uncovered.
  Toluene, propylene, and acetaldehyde
were chosen for this study because they
are all important reactive components of
urban air that contribute significantly to
the evolution of photochemical smog.
None of these species are, themselves,
mutagenic in the Ames test.  Allyl chlo-
ride was chosen to investigate whether
chlorinated  hazardous  air  pollutants
(HAPs)  might yield photooxidation pro-
ducts that are more mutagenic than their
nonchlorinated analogues. This particu-
lar HAP was also chosen  because it is
the chlorine-substituted  analogue  of
propylene, one of the hydrocarbons we
have studied in detail.
  In urban photochemical smog systems,
the air  pollutants exist  as a  mixture of
gas-phase species and  organic-laden
particulate matter.  It is therefore impor-
tant to determine the ultimate distribu-
tion of mutagenic compounds between
the gas  and  paniculate phases for
complex mixtures. Thus, we  conducted
irradiations  of  wood  smoke/NOx and
automobile exhaust/NOx mixtures in the
smog  chamber and measured the muta-
genic  activities of the reactants and the
gas- and particulate-phase products.
  Throughout  these experiments, we
have  attempted to describe the nature
of the photochemical  processes that
produce the mutagenic products and the
specific photooxidation products that
contribute significantly to the observed
mutagenic activities. Measurements of
the mutagenic  activity of peroxyacetyl
nitrate (PAN)  and other peroxyacyl
nitrates were  made in an attempt to
identify photochemically derived  muta-
gens.  PAN  is  particularly  important
because it is a  product of the photoox-
idation of a variety of atmospheric
hydrocarbons,  and it is present in sig-
nificant concentrations in urban air.
From  the  results of the studies of the
individual hydrocarbons, we attempted to
determine their relative potential contri-
bution  to   atmospheric  mutagen
production.

Procedures
  For the  individual hydrocarbons stu-
died, we irradiated mixtures of ~ 1 ppm
hydrocarbon in the presence of  ~ 0.5
ppm  NOx in a  22.7-m3 Teflon smog
chamber.  The   smog chamber can  be
operated in  a  flowing  mode  in which
reactants  are   continuously added at
constant  concentration, resulting in a
steady-state product distribution  in the
chamber.  The  product distribution is
dependent  on  the residence time T
(where T = chamber volume/total flow
rate) of the gases on the chamber. With
this  method,  a constant composition
mixture of pollutants can be maintained
for long periods of time. A  schematic
diagram of  the  reaction chamber and
exposure chamber system is shown in
Figure 1. For studies of the simple HC/
NOx mixtures, in which the chamber was
operated in  a dynamic  mode, the  reac-
tants  were mixed and diluted in a 140-
L stainless steel inlet manifold and then
transferred to the chamber through the
end  plate. For the N2Os/N03  experi-
ments, Ns05 was  prepared in a small
mixing bulb  from reaction of N02 with
O3 To study  irradiated wood smoke/NO,
mixtures, we added diluted wood smoke
(from  an oak-burning wood stove) directly
to the reaction chamber by using a metal
bellows  pump.  Automobile  exhaust,
added directly to the chamber from the
tail pipe by using a metal bellows pump,
was obtained from a 1980 Toyota Corolla
(catalyst equipped). In the wood smoke
and  automobile  exhaust experiments,
the initial total hydrocarbon concentra-
tions were ~ 20 and 12 ppmC, respec-
tively.  In  both  cases  the  initial NO,
concentration was ~ 0.7 ppm.
  Four  190-L Teflon-coated  exposure
chambers were used for exposure of the
bacteria to the various air streams. These
four  exposure chambers enabled mea-
surements of the mutagenic activity of
the following types  of air  masses: the
starting materials (reactants),  photooxi-
dation products  (filtered or unfiltered),
clean air,  and  for  the wood smoke
experiments, ambient air. For  the wood
smoke and automobile exhaust experi-
ments, particulate matter was collected
from the reactant, product, and ambient-
air exposure chamber air streams  on
Teflon-impregnated, glass-fiber filters.
  The  experiments  described in this
report were  conducted by exposing the
Ames test bacteria  Salmonella  typhi-
murium to the test gases. Strains TA100
and  TA98 were used, both  with and
without metabolic activation. The expo-
sures were conducted by allowing the air
to flow through the exposure chambers
loaded with, approximately, 50 covered
petri dishes  containing the bacteria in a
nutrient agar. We exposed the bacteria
to the components of the  chamber  air
by uncovering the dishes for a specific
period of time.  This  allowed the agar-
soluble species  to deposit onto the test
plates as the air mass flowed through
the exposure chamber. Because the agar
is mostly water, those species that are
water soluble (i.e., polar) are expected to
deposit to the test plates.
  All experiments began with a conven-
tional static-mode smog chamber irradi-
ation. Once the temporal variation of
reactant and product concentrations
could be  determined and the desired
extent  of reaction  for the  dynamic
experiments chosen,  the dynamic-mode
exposures were  begun. By varying the
reaction chamber residence time  in the
dynamic-mode  experiments,  we were
able to conduct bioassay measurements
for steady-state mixtures having product
distributions  corresponding  to different
regions of a conventional static-mode
irradiation. Because  the concentration
profiles for  many  photooxidation pro-

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                                                                                     Mass
                                                                             3L ^   Flow
                                                                            Mixing]  Controller
                                                                             Bulb
                                                           Reaction Chamber
                                                               22.7m3
                                                                 Samples
                                                                   and
                                                                 Exhaust
                                                           Lights
                                                                      Teflon
Figure  1.    Schematic diagram of the reaction chamber apparatus.
ducts are highly dependent on the extent
of reaction (e.g., presence or absence of
NO), the change in the mutagenic activity
of the mixture as the reaction proceeds
can be interpreted in terms of changes
in the product distribution.
  In these experiments, plates were
exposed for  varying  periods of  time
(typically 2 to  20 h), depending on the
sensitivity of the bioassay test. After the
exposures, the plates were incubated at
37°C for 48  h,  and the  number of
revertants/plate were  counted. The
particulate-phase filter samples obtained
in the wood  smoke  and  automobile
exhaust experiments  were  soxhlet
extracted with methylene chloride,  and
the extracts were tested for mutagenic
activity  by using  the standard plate
incorporation test. For the wood smoke
and  automobile exhaust experiments,
the smog chamber  was operated in a
static mode,  in which  the gas-phase
exposures  were  conducted after  the
chamber lights were turned off.
  To improve our understanding of the
nature of the photochemical processes
occurring in the smog chamber that lead
to the production of mutagenic products,
we quantitatively measured a  wide
variety of reactant and product species
during the chamber irradiations.  These
measurements were made by using a
wide  variety of techniques,  including
continuous gas monitors, gas, liquid, and
ion chromatography, and gas chromatog-
raphy/mass spectrometry. From  infor-
mation about changes in the mutagenic
activity of the product  mixture with
respect to changes in product concen-
tration (such as PAN),  it  is possible to
discover the possible causes of observed
increases in mutagenic activity.

Results and Discussion
  A typical static-mode smog chamber
HC/NOx irradiation for toluene is pres-
ented in Figure 2.  For all hydrocarbons
studied, the removal of the hydrocarbon
occurred through OH radical reaction, as
long as NO  was present. After the NO
was removed,  O3 began to accumulate.
For propylene and  ally!  chloride, signif-
icant reaction with Oa occurred at long
extent of reaction. The static mode plots
for propylene, acetaldehyde, and ally!
chloride are similar to that in Figure 2.
We conducted Ames test  exposures at
short extent of reaction and at long extent
of reaction, near the ozone maximum. In
all cases,  a relatively small increase in
the  number  of revertants/plate was
observed for the short extent of reaction,
whereas at long  extent of reaction, a
much larger response was observed. In
the case of toluene, roughly 500 induced
revertants/plate (i.e., in excess above the
clean air  or reactant controls) were
observed  with  Strain TA100. Similar
results were observed for propylene and
acetaldehyde. In all cases, the mutagenic
activities determined with Strain TA100
were considerably larger than with TA98.
For many  of the exposures conducted,
groups of plates were covered during the
exposure period, effectively stopping the
dosage of those plates at that point. This
method allowed construction of a dose-
response  curve for each  exposure.  A
typical  dose-response  curve  (with
TA100)  is  shown in Figure 3 for  the
products of the photooxidation  of propyl-
ene at the long extent of reaction. The
curve ultimately bends over because of

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     1.0 -\
     0.9-
                   Dilution
                                                                -8000
                   O Toluene
                   ONO
                   D /VO.-A/O
                   A 03
                   • PAN
                    x CNC
                     23456

                                     Time, h

Figure 2.    Static-mode toluene/NO*/HiQ/air irradiation.
    700-
         0              5               W               15              20

                                  Exposure Time, h

Figure 3.    Dose-response curve for T= 7.5-h irradiated Ctft/NO, mixtures, TA 100.
toxicity effects. The observation (using
the Ames test) that these relatively low
molecular   weight,  nonmutagenic,
organic  pollutants are  converted to
mutagenic products in the course of their
photooxidation  should be regarded as
significant given their important rotes in
urban photochemistry. Thus, it is impor-
tant to attempt to determine the cause
of the observed mutagenic activities.
  To determine which chemical species
may have caused the mutagenic activity
resulting from  exposure of the Ames
assay plates to these product  mixtures,
it is necessary to know the dose of each
product in the plates and the mutagenic
activity of each product.  It is possible to
estimate  the  dose of each chemical by
measuring  its gas-phase concentration
in the exposure chambers before and
after the  plates are uncovered.
  If we know the dosage  and the product
mutagenic activities in revertants/yumol,
then the  contribution of each product to
the total observed mutagenic activity can
be determined. This type of calculation
was conducted for all the known products
of the photooxidation of toluene  and
propylene we were  able to  measure.
Although some  products (e.g., HCHO,
H202, glyoxal, methylglyoxal) have  been
found to be  mutagenic, none were
mutagenic  enough to  account for  more
than  5  to  10% of  the  total  observed
response.
  The observation that a  very significant
mutagenic  activity existed for  the  pro-
ducts of the photooxidation of acetalde-
hyde  at short extent of  reaction, when
NO was present, provided a very impor-
tant clue to the  identity of  a  major
mutagenic  product. Under these condi-
tions, the only organic products present
are PAN, HCHO, and, to a much lesser
extent,  CH3ONOz.  However,  we  had
already  determined  that  HCHO  and
CH3ON02 were not mutagenic enough to
contribute significantly to the observed
response with the Ames test. These
observations  led us to  conduct an in-
depth study of the mutagenic activity of
PAN.  We have subsequently observed
that pure PAN, at concentrations ranging
from ~ 100to 500 ppb, yields a measured
reversion rate (in our test system, using
TA 100) of ~ 14 revertants/h, independ-
ent of PAN  concentration. The   PAN
concentrations measured in the toluene,
propylene,  and acetaldehyde exposures
at long residence time were 198,  181,
and 171 ppb, respectively,  and the
measured reversion rates were 27, 24,
and 20 revertants/h, respectively. There-

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fore, it seems likely that PAN accounts
for a large fraction of the total observed
mutagenic  activity of the products in
these experiments.
  We found that the mutagenic activity
of the photooxidation products of ally!
chloride was highly  dependent on the
extent of Cl-atom reaction  with ally!
chloride. Under conditions in which Cl-
atom reactions  were  significant,  the
mutagenic  activity of the products was
extremely high. When ally! chloride/NO,
irradiations were  conducted in  the
presence  of a  Cl-atom  scavenger to
simulate better its actual tropospheric
chemistry, the mutagenic activity of the
products was substantially less. Even so,
the  mutagenic  activity of this  product
mixture is roughly 30 times greater than
that produced from the photooxidation of
propylene. The mutagenicity in this latter
case was accounted for by  the major
photooxidation product, chloroacetalde-
hyde. Therefore, this particular chlori-
nated HAP yields much more mutagenic
photooxidation  products  than  does its
nonchlorinated analogue.
  We conducted a series of wood smoke/
NOx and automobile exhaust/NO, irra-
diations to determine the extent to which
mutagenic products can be produced in
more complex HC/NOX mixtures. We also
sought to determine the phase distribu-
tion of the mutagenic products that were
produced. The mutagenic activities of the
gas- and particulate-phase species were
measured before  irradiation of  the
mixtures and after the ozone maximum
was reached in the irradiations. For both
wood smoke and automobile exhaust, the
mutagenic  activities of the  gas-phase
species  increased  dramatically as  a
result of the irradiation. The measured
reversion  rates after  the  irradiation,
using TA100, were 174  and  70 rever-
tants/h for wood smoke and automobile
exhaust, respectively. PAN therefore can
account for 10-20% of the total observed
(gas phase) mutagenic activities of these
two product mixtures. For both wood
smoke and automobile exhaust, irradia-
tion of the mixtures resulted in a dramatic
          increase in the total particulate-phase
          mass.  The increase in  the  paniculate
          phase mass was the result of nonvolatile
          photooxidation products adsorbing onto
          existing paniculate matter.
            To compare the mutagenic activities of
          the gas- and particulate-phase species
          in these complex  mixtures,  the muta-
          genic activities for  both phases must be
          expressed in a common set of units. The
          most convenient set of units  is rever-
          tants/m3.  The gas-phase  mutagenic
          activities can be converted to these units
          if the collection efficiencies of the test
          plates for the mutagenic gases that pass
          through the exposure chambers are
          known. These collection efficiencies
          were  determined  in  experiments in
          which the exposures were conducted by
          using two exposure chambers in series.
          Typical collection efficiencies were found
          to be ~ 60 to 70% for both strains. Once
          mutagenic activities for both the gas- and
          particulate-phase species are obtained in
          revertants/m3,  they can also be con-
          verted to revertants/jug using the density
          (fjg/m3) of the gas- or particulate-phase
          species in the reaction  chamber. The
          results of  these  calculations  for irra-
          diated wood smoke/NOx mixtures are
          presented  in Table 1  and  are shown
          graphically in Figure 4. Similar results
          were  obtained for  the automobile
          exhaust/NO, mixtures. The data suggest
          that, on  a revertants/m3  basis, the
          overwhelming majority of the mutagenic
          activity of these product mixtures resides
          in the gas phase. It is therefore clear that
          in any assessment of the production of
          mutagenic species from irradiated com-
          plex HC/NO, mixtures, it is very impor-
          tant to consider gas-phase mutagens, as
          well as those that are particle bound.
            The extent to which mutagens can be
          produced from NOs reactions with reac-
          tive atmospheric hydrocarbons was also
          assessed.  We studied the reactions of
          propylene  and wood smoke with  NO3/
          N205. For the C3He/N205 experiment, the
          mutagenic activity increased  substan-
          tially.  It was unclear,  however, which
          products caused the observed mutagenic
                                     activity. In the wood smoke/NzOs exper-
                                     iment, the mutagenic activity increased
                                     substantially for both the  gas- and
                                     particulate-phase species  after the
                                     reaction, as measured with both TA100
                                     and TA98. It is very interesting to note
                                     that, even for reactions with NgOs, both
                                     strains  indicated at least  as  much
                                     mutagenic activity (in revertants/m3) in
                                     the gas phase as in the paniculate phase.
                                     These measurements indicate that night-
                                     time chemistry involving N03 and N205
                                     can also lead to the production of both
                                     gas- and particulate-phase mutagenic
                                     products in the atmosphere.
                                       In addition to our studies  of  these
                                     irradiated mixtures, we also have con-
                                     ducted measurements of the mutagenic
                                     activities  of a  series of peroxyacyl
                                     nitrates, such as PAN. In this study, it
                                     was found that  peroxypropionyl nitrate
                                     (PPN), peroxybutyryl nitrate (PBN), and
                                     peroxybenzoyl nitrate (PBzN)  were all
                                     mutagenic as determined with Strain
                                     TA100. In addition, PBzN  was found to
                                     be considerably more mutagenic (on a
                                     revertants-h"1-ppb"1 basis) than  PAN.
                                     Both PPN and PBzN have been measured
                                     in ambient air.

                                     Conclusions  and
                                     Recommendations
                                       The experiments described in this
                                     report  indicate  that mutagenic  com-
                                     pounds  (as  determined by using  the
                                     Ames test) are produced as a result of
                                     atmospheric  photochemistry.  For  many
                                     HC/NO, systems studied,  including
                                     propylene and toluene, the photooxida-
                                     tion  products were much  more  muta-
                                     genic than the  reactant hydrocarbons.
                                     Not  all  HC/NO, irradiations produced
                                     mutagenic products, however. The pho-
                                     tooxidation products of ally! chloride, a
                                     chlorinated  HAP, were shown  to be
                                     extremely mutagenic. Some HAPs may
                                     be important in terms  of atmospheric
                                     mutagenesis, even though their ambient
                                     concentrations are relatively  low.  For
                                     complex reactive mixtures such as wood
                                     smoke,  irradiation can produce substan-
Table 1.    Comparison of the Gas- and Paniculate-Phase Mutagenic Activities for Wood Smoke Before and After Irradiation, Strains TA100
           and TA98
                                    TAJ 00
                                                                                          TA98
                        Gas
                   Paniculate
                                   Gas
                                                                                                     Paniculate
               Rev/m3
Rev/ug
Rev/m3
Rev/ug
Rev/m3
                                                                                  Rev/ug
Rev/m3
                                                                   Rev/ug
Reactants
Products
<230
1 7.300
<0.4
1.6
100
180
0.30
0.27
<100
3,230
<0.17
0.30
80
730
0.22
0.94

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tial increases in the mutagenic activities
of  both  gas- and particulate-phase
species. It was found that the majority
of the mutagenic activity of the product
mixture of wood smoke is associated with
gas-phase species. Much of the increase
in mutagenic activity for the particulate
phase in our experiments is probably the
result of adsorption of gas-phase (low
volatility) mutagenic products  onto
existing particulate matter,  rather than
a reflection of reactions occurring on the
surface of the particulate matter.
  For most  of the product mixtures
investigated, a significant portion of the
mutagenic activity observed may have
been caused by  PAN, which has been
shown to be mutagenic. The  absolute
value of the mutagenic activity of this
species is currently uncertain, however,
and much more  work is necessary  to
determine the  mutagenic potential  of
PAN at ambient concentration  levels.
Because PAN is  ubiquitously present in
polluted urban atmospheres,  it  would
also be  desirable to determine the
biological impact of PAN on other bio-
logical systems by using  additional
bioassay techniques.
  At this point,  laboratory experiments
have shown that  pollutants commonly
found in  urban air can be transformed
into mutagenic products  as  a  result  of
typical urban photochemical processes.
These results suggest the need to extend
this effort in both laboratory and field
measurement areas. Ambient measure-
ments  of mutagenic activities  in urban
air masses would be helpful in determin-
ing the possible correlation of mutagenic
activity of the air  mass with the presence
of photochemical oxidants such as PAN,
and may aid in the overall  assessment
of the contribution of urban photochem-
istry to the presence of atmospheric
mutagens.
     775-
    150-
     125-
    100-
 I
     50H
     25-
      0~\
                  TA 100
                                        |    |  Gas Phase

                                        f"':"\  Particulate Phase
                                                                TA98
                Before
               Irradiation
                After
              Irradiation
 Before
Irradiation
  After
Irradiation
Figure 4.
Comparison of the gas- and particulate-phase mutagenicity of dilute wood smoke
in air.
  P. B. Shepson,  T. E. Kleindienst, and E. O. Edney are with Northrup Services,
    Inc.-Environmental Sciences. Research Triangle Park. NC 27709.
  L. T. Cupitt is the EPA Project Officer (see below).
  The complete report entitled "The Production of Mutagenic Compounds as a
    Result of Urban Photochemistry." (Order No. PB 87-199 675/AS; Cost:
    $13.95, subject to change)
  will be available only from:
          National Technical Information Service
          5285 Port Royal Road
          Springfield. VA 22161
          Telephone: 703-487-4650
  The EPA Project Officer can be contacted at:
          Atmospheric Sciences Research Laboratory
          U.S.  Environmental Protection Agency
          Research Triangle Park, NC 27711

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