United States
Environmental Protection
Agency
Atmospheric Sciences
Research Laboratory x
Research Triangle Park NC 27711
Research and Development
EPA/600/S3-87/020 Sept. 1987
&EPA Project Summary
The Production of Mutagenic
Compounds as a Result of
Urban Photochemistry
P. B. Shepson, T. E. Kleindienst, and E. 0. Edney
A series of atmospheric simulation
experiments was conducted to exam-
ine the role of urban photochemical
processes on the formation and re-
moval of potentially hazardous air
pollutants. The experiments were con-
ducted in a 22.7-m3 Teflon smog
chamber, which was coupled to bioas-
say exposure chambers. The mutagenic
activities of the tested mixtures of
organic chemicals and nitrogen oxides
were measured before and after irra-
diation by direct exposure of Salmo-
nella typhimurium (strains TA98 and
TA100) to the smog chamber contents.
The mutagenic responses of the start-
ing materials and the transformed
products were quantified by using a
modified Ames test. The chemicals
examined included ubiquitous urban
pollutants (e.g., propylene, toluene,
and acetaldehyde), a potentially
hazardous chlorinated solvent (ally!
chloride), and complex mixtures (wood
smoke and auto exhaust) from common
urban pollutant sources. In all cases,
the irradiated products were more
mutagenic than the original chemicals.
For the transformed complex mixtures,
the bulk of the mutagenicity was found
to be associated with the gas-phase
products rather than with the aerosol-
bound chemicals. Increased mutagen-
icity was also observed with increasing
photochemical oxidation. The common
photochemical pollutant, peroxyacetyl
nitrate, was found to contribute signif-
icantly to the overall vapor-phase
mutagenicity in all of the chemical
systems in which it was formed.
This Project Summary was devel-
oped by EPA's Atmospheric Sciences
Research Laboratory. Research Trian-
gle Park, NC. to announce key findings
of the research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
During the past several years there has
been an increasing awareness that
exposure to polluted urban atmospheres
may pose an ill-defined but significant
human health threat, especially with
regard to the incidence of cancer. It has
become increasingly apparent that an
accurate assessment of the potential
human health impact due to exposure to
atmospheric pollutants requires an
understanding of the influence of atmos-
pheric reactions on both hazardous and
nonhazardous species. The photooxida-
tion products of a wide variety of impor-
tant atmospheric aromatic hydrocarbons
have been shown to be mutagenic.
Researchers have found a variety of
oxygenated and nitrogenated species to
be mutagenic by using the Ames test,
implying that the photooxidation of
reactive atmospheric hydrocarbons may
have a significant impact on the presence
of atmospheric mutagens.
Concerns have also been raised with
regard to the possible human health
threat of exposure to wood stove and
fireplace emissions. In addition to the
possible presence of mutagens in the
emissions themselves, evidence has
appeared recently in the literature
indicating that reactions of species such
as 03 and N2O5 on the surface of
atmospheric particulate matter can lead
to increases in the mutagenic activity of
adsorbed species such as polycyclic
-------
aromatic hydrocarbons (PAHs). Consid-
erable effort has been expended in
studying reactions that occur on the
surface of paniculate matter and in
studying the impact of particulate-phase
photochemistry on the mutagenic activ-
ities of adsorbed species.
In contrast, there has been a relatively
smaller effort aimed at identifying mech-
anisms for the production of gas-phase
mutagens through atmospheric photo-
chemistry. A principal reason for the
emphasis on particulate-phase muta-
gens is the relative ease of collection and
extraction of paniculate matter. To test
for the mutagenic activity of gas-phase
species by using the standard plate
incorporation test, it is necessary to
concentrate the gas-phase pollutants
into a suitable solvent in some manner.
Some limited attempts have been made
to measure gas-phase ambient mutagen-
icities by using XAD or other adsorbents
to concentrate the species present.
However, this process poses a number
of potential problems, such as loss of
volatile species during the extraction and
solvent concentration steps.
In this report, we present a review of
the results of experiments we have
conducted by using an alternative tech-
nique for measuring the mutagenic
activity of gas-phase species generated
by photochemical reactions. The product
mixtures to be tested were produced in
a 22.7-m3 Teflon smog chamber. For this
technique, the Ames test plates were
dosed by continuously flowing the reac-
tion chamber air over the uncovered
plates, thereby permitting the soluble
species to deposit continuously while the
plates are uncovered.
Toluene, propylene, and acetaldehyde
were chosen for this study because they
are all important reactive components of
urban air that contribute significantly to
the evolution of photochemical smog.
None of these species are, themselves,
mutagenic in the Ames test. Allyl chlo-
ride was chosen to investigate whether
chlorinated hazardous air pollutants
(HAPs) might yield photooxidation pro-
ducts that are more mutagenic than their
nonchlorinated analogues. This particu-
lar HAP was also chosen because it is
the chlorine-substituted analogue of
propylene, one of the hydrocarbons we
have studied in detail.
In urban photochemical smog systems,
the air pollutants exist as a mixture of
gas-phase species and organic-laden
particulate matter. It is therefore impor-
tant to determine the ultimate distribu-
tion of mutagenic compounds between
the gas and paniculate phases for
complex mixtures. Thus, we conducted
irradiations of wood smoke/NOx and
automobile exhaust/NOx mixtures in the
smog chamber and measured the muta-
genic activities of the reactants and the
gas- and particulate-phase products.
Throughout these experiments, we
have attempted to describe the nature
of the photochemical processes that
produce the mutagenic products and the
specific photooxidation products that
contribute significantly to the observed
mutagenic activities. Measurements of
the mutagenic activity of peroxyacetyl
nitrate (PAN) and other peroxyacyl
nitrates were made in an attempt to
identify photochemically derived muta-
gens. PAN is particularly important
because it is a product of the photoox-
idation of a variety of atmospheric
hydrocarbons, and it is present in sig-
nificant concentrations in urban air.
From the results of the studies of the
individual hydrocarbons, we attempted to
determine their relative potential contri-
bution to atmospheric mutagen
production.
Procedures
For the individual hydrocarbons stu-
died, we irradiated mixtures of ~ 1 ppm
hydrocarbon in the presence of ~ 0.5
ppm NOx in a 22.7-m3 Teflon smog
chamber. The smog chamber can be
operated in a flowing mode in which
reactants are continuously added at
constant concentration, resulting in a
steady-state product distribution in the
chamber. The product distribution is
dependent on the residence time T
(where T = chamber volume/total flow
rate) of the gases on the chamber. With
this method, a constant composition
mixture of pollutants can be maintained
for long periods of time. A schematic
diagram of the reaction chamber and
exposure chamber system is shown in
Figure 1. For studies of the simple HC/
NOx mixtures, in which the chamber was
operated in a dynamic mode, the reac-
tants were mixed and diluted in a 140-
L stainless steel inlet manifold and then
transferred to the chamber through the
end plate. For the N2Os/N03 experi-
ments, Ns05 was prepared in a small
mixing bulb from reaction of N02 with
O3 To study irradiated wood smoke/NO,
mixtures, we added diluted wood smoke
(from an oak-burning wood stove) directly
to the reaction chamber by using a metal
bellows pump. Automobile exhaust,
added directly to the chamber from the
tail pipe by using a metal bellows pump,
was obtained from a 1980 Toyota Corolla
(catalyst equipped). In the wood smoke
and automobile exhaust experiments,
the initial total hydrocarbon concentra-
tions were ~ 20 and 12 ppmC, respec-
tively. In both cases the initial NO,
concentration was ~ 0.7 ppm.
Four 190-L Teflon-coated exposure
chambers were used for exposure of the
bacteria to the various air streams. These
four exposure chambers enabled mea-
surements of the mutagenic activity of
the following types of air masses: the
starting materials (reactants), photooxi-
dation products (filtered or unfiltered),
clean air, and for the wood smoke
experiments, ambient air. For the wood
smoke and automobile exhaust experi-
ments, particulate matter was collected
from the reactant, product, and ambient-
air exposure chamber air streams on
Teflon-impregnated, glass-fiber filters.
The experiments described in this
report were conducted by exposing the
Ames test bacteria Salmonella typhi-
murium to the test gases. Strains TA100
and TA98 were used, both with and
without metabolic activation. The expo-
sures were conducted by allowing the air
to flow through the exposure chambers
loaded with, approximately, 50 covered
petri dishes containing the bacteria in a
nutrient agar. We exposed the bacteria
to the components of the chamber air
by uncovering the dishes for a specific
period of time. This allowed the agar-
soluble species to deposit onto the test
plates as the air mass flowed through
the exposure chamber. Because the agar
is mostly water, those species that are
water soluble (i.e., polar) are expected to
deposit to the test plates.
All experiments began with a conven-
tional static-mode smog chamber irradi-
ation. Once the temporal variation of
reactant and product concentrations
could be determined and the desired
extent of reaction for the dynamic
experiments chosen, the dynamic-mode
exposures were begun. By varying the
reaction chamber residence time in the
dynamic-mode experiments, we were
able to conduct bioassay measurements
for steady-state mixtures having product
distributions corresponding to different
regions of a conventional static-mode
irradiation. Because the concentration
profiles for many photooxidation pro-
-------
Mass
3L ^ Flow
Mixing] Controller
Bulb
Reaction Chamber
22.7m3
Samples
and
Exhaust
Lights
Teflon
Figure 1. Schematic diagram of the reaction chamber apparatus.
ducts are highly dependent on the extent
of reaction (e.g., presence or absence of
NO), the change in the mutagenic activity
of the mixture as the reaction proceeds
can be interpreted in terms of changes
in the product distribution.
In these experiments, plates were
exposed for varying periods of time
(typically 2 to 20 h), depending on the
sensitivity of the bioassay test. After the
exposures, the plates were incubated at
37°C for 48 h, and the number of
revertants/plate were counted. The
particulate-phase filter samples obtained
in the wood smoke and automobile
exhaust experiments were soxhlet
extracted with methylene chloride, and
the extracts were tested for mutagenic
activity by using the standard plate
incorporation test. For the wood smoke
and automobile exhaust experiments,
the smog chamber was operated in a
static mode, in which the gas-phase
exposures were conducted after the
chamber lights were turned off.
To improve our understanding of the
nature of the photochemical processes
occurring in the smog chamber that lead
to the production of mutagenic products,
we quantitatively measured a wide
variety of reactant and product species
during the chamber irradiations. These
measurements were made by using a
wide variety of techniques, including
continuous gas monitors, gas, liquid, and
ion chromatography, and gas chromatog-
raphy/mass spectrometry. From infor-
mation about changes in the mutagenic
activity of the product mixture with
respect to changes in product concen-
tration (such as PAN), it is possible to
discover the possible causes of observed
increases in mutagenic activity.
Results and Discussion
A typical static-mode smog chamber
HC/NOx irradiation for toluene is pres-
ented in Figure 2. For all hydrocarbons
studied, the removal of the hydrocarbon
occurred through OH radical reaction, as
long as NO was present. After the NO
was removed, O3 began to accumulate.
For propylene and ally! chloride, signif-
icant reaction with Oa occurred at long
extent of reaction. The static mode plots
for propylene, acetaldehyde, and ally!
chloride are similar to that in Figure 2.
We conducted Ames test exposures at
short extent of reaction and at long extent
of reaction, near the ozone maximum. In
all cases, a relatively small increase in
the number of revertants/plate was
observed for the short extent of reaction,
whereas at long extent of reaction, a
much larger response was observed. In
the case of toluene, roughly 500 induced
revertants/plate (i.e., in excess above the
clean air or reactant controls) were
observed with Strain TA100. Similar
results were observed for propylene and
acetaldehyde. In all cases, the mutagenic
activities determined with Strain TA100
were considerably larger than with TA98.
For many of the exposures conducted,
groups of plates were covered during the
exposure period, effectively stopping the
dosage of those plates at that point. This
method allowed construction of a dose-
response curve for each exposure. A
typical dose-response curve (with
TA100) is shown in Figure 3 for the
products of the photooxidation of propyl-
ene at the long extent of reaction. The
curve ultimately bends over because of
-------
1.0 -\
0.9-
Dilution
-8000
O Toluene
ONO
D /VO.-A/O
A 03
• PAN
x CNC
23456
Time, h
Figure 2. Static-mode toluene/NO*/HiQ/air irradiation.
700-
0 5 W 15 20
Exposure Time, h
Figure 3. Dose-response curve for T= 7.5-h irradiated Ctft/NO, mixtures, TA 100.
toxicity effects. The observation (using
the Ames test) that these relatively low
molecular weight, nonmutagenic,
organic pollutants are converted to
mutagenic products in the course of their
photooxidation should be regarded as
significant given their important rotes in
urban photochemistry. Thus, it is impor-
tant to attempt to determine the cause
of the observed mutagenic activities.
To determine which chemical species
may have caused the mutagenic activity
resulting from exposure of the Ames
assay plates to these product mixtures,
it is necessary to know the dose of each
product in the plates and the mutagenic
activity of each product. It is possible to
estimate the dose of each chemical by
measuring its gas-phase concentration
in the exposure chambers before and
after the plates are uncovered.
If we know the dosage and the product
mutagenic activities in revertants/yumol,
then the contribution of each product to
the total observed mutagenic activity can
be determined. This type of calculation
was conducted for all the known products
of the photooxidation of toluene and
propylene we were able to measure.
Although some products (e.g., HCHO,
H202, glyoxal, methylglyoxal) have been
found to be mutagenic, none were
mutagenic enough to account for more
than 5 to 10% of the total observed
response.
The observation that a very significant
mutagenic activity existed for the pro-
ducts of the photooxidation of acetalde-
hyde at short extent of reaction, when
NO was present, provided a very impor-
tant clue to the identity of a major
mutagenic product. Under these condi-
tions, the only organic products present
are PAN, HCHO, and, to a much lesser
extent, CH3ONOz. However, we had
already determined that HCHO and
CH3ON02 were not mutagenic enough to
contribute significantly to the observed
response with the Ames test. These
observations led us to conduct an in-
depth study of the mutagenic activity of
PAN. We have subsequently observed
that pure PAN, at concentrations ranging
from ~ 100to 500 ppb, yields a measured
reversion rate (in our test system, using
TA 100) of ~ 14 revertants/h, independ-
ent of PAN concentration. The PAN
concentrations measured in the toluene,
propylene, and acetaldehyde exposures
at long residence time were 198, 181,
and 171 ppb, respectively, and the
measured reversion rates were 27, 24,
and 20 revertants/h, respectively. There-
-------
fore, it seems likely that PAN accounts
for a large fraction of the total observed
mutagenic activity of the products in
these experiments.
We found that the mutagenic activity
of the photooxidation products of ally!
chloride was highly dependent on the
extent of Cl-atom reaction with ally!
chloride. Under conditions in which Cl-
atom reactions were significant, the
mutagenic activity of the products was
extremely high. When ally! chloride/NO,
irradiations were conducted in the
presence of a Cl-atom scavenger to
simulate better its actual tropospheric
chemistry, the mutagenic activity of the
products was substantially less. Even so,
the mutagenic activity of this product
mixture is roughly 30 times greater than
that produced from the photooxidation of
propylene. The mutagenicity in this latter
case was accounted for by the major
photooxidation product, chloroacetalde-
hyde. Therefore, this particular chlori-
nated HAP yields much more mutagenic
photooxidation products than does its
nonchlorinated analogue.
We conducted a series of wood smoke/
NOx and automobile exhaust/NO, irra-
diations to determine the extent to which
mutagenic products can be produced in
more complex HC/NOX mixtures. We also
sought to determine the phase distribu-
tion of the mutagenic products that were
produced. The mutagenic activities of the
gas- and particulate-phase species were
measured before irradiation of the
mixtures and after the ozone maximum
was reached in the irradiations. For both
wood smoke and automobile exhaust, the
mutagenic activities of the gas-phase
species increased dramatically as a
result of the irradiation. The measured
reversion rates after the irradiation,
using TA100, were 174 and 70 rever-
tants/h for wood smoke and automobile
exhaust, respectively. PAN therefore can
account for 10-20% of the total observed
(gas phase) mutagenic activities of these
two product mixtures. For both wood
smoke and automobile exhaust, irradia-
tion of the mixtures resulted in a dramatic
increase in the total particulate-phase
mass. The increase in the paniculate
phase mass was the result of nonvolatile
photooxidation products adsorbing onto
existing paniculate matter.
To compare the mutagenic activities of
the gas- and particulate-phase species
in these complex mixtures, the muta-
genic activities for both phases must be
expressed in a common set of units. The
most convenient set of units is rever-
tants/m3. The gas-phase mutagenic
activities can be converted to these units
if the collection efficiencies of the test
plates for the mutagenic gases that pass
through the exposure chambers are
known. These collection efficiencies
were determined in experiments in
which the exposures were conducted by
using two exposure chambers in series.
Typical collection efficiencies were found
to be ~ 60 to 70% for both strains. Once
mutagenic activities for both the gas- and
particulate-phase species are obtained in
revertants/m3, they can also be con-
verted to revertants/jug using the density
(fjg/m3) of the gas- or particulate-phase
species in the reaction chamber. The
results of these calculations for irra-
diated wood smoke/NOx mixtures are
presented in Table 1 and are shown
graphically in Figure 4. Similar results
were obtained for the automobile
exhaust/NO, mixtures. The data suggest
that, on a revertants/m3 basis, the
overwhelming majority of the mutagenic
activity of these product mixtures resides
in the gas phase. It is therefore clear that
in any assessment of the production of
mutagenic species from irradiated com-
plex HC/NO, mixtures, it is very impor-
tant to consider gas-phase mutagens, as
well as those that are particle bound.
The extent to which mutagens can be
produced from NOs reactions with reac-
tive atmospheric hydrocarbons was also
assessed. We studied the reactions of
propylene and wood smoke with NO3/
N205. For the C3He/N205 experiment, the
mutagenic activity increased substan-
tially. It was unclear, however, which
products caused the observed mutagenic
activity. In the wood smoke/NzOs exper-
iment, the mutagenic activity increased
substantially for both the gas- and
particulate-phase species after the
reaction, as measured with both TA100
and TA98. It is very interesting to note
that, even for reactions with NgOs, both
strains indicated at least as much
mutagenic activity (in revertants/m3) in
the gas phase as in the paniculate phase.
These measurements indicate that night-
time chemistry involving N03 and N205
can also lead to the production of both
gas- and particulate-phase mutagenic
products in the atmosphere.
In addition to our studies of these
irradiated mixtures, we also have con-
ducted measurements of the mutagenic
activities of a series of peroxyacyl
nitrates, such as PAN. In this study, it
was found that peroxypropionyl nitrate
(PPN), peroxybutyryl nitrate (PBN), and
peroxybenzoyl nitrate (PBzN) were all
mutagenic as determined with Strain
TA100. In addition, PBzN was found to
be considerably more mutagenic (on a
revertants-h"1-ppb"1 basis) than PAN.
Both PPN and PBzN have been measured
in ambient air.
Conclusions and
Recommendations
The experiments described in this
report indicate that mutagenic com-
pounds (as determined by using the
Ames test) are produced as a result of
atmospheric photochemistry. For many
HC/NO, systems studied, including
propylene and toluene, the photooxida-
tion products were much more muta-
genic than the reactant hydrocarbons.
Not all HC/NO, irradiations produced
mutagenic products, however. The pho-
tooxidation products of ally! chloride, a
chlorinated HAP, were shown to be
extremely mutagenic. Some HAPs may
be important in terms of atmospheric
mutagenesis, even though their ambient
concentrations are relatively low. For
complex reactive mixtures such as wood
smoke, irradiation can produce substan-
Table 1. Comparison of the Gas- and Paniculate-Phase Mutagenic Activities for Wood Smoke Before and After Irradiation, Strains TA100
and TA98
TAJ 00
TA98
Gas
Paniculate
Gas
Paniculate
Rev/m3
Rev/ug
Rev/m3
Rev/ug
Rev/m3
Rev/ug
Rev/m3
Rev/ug
Reactants
Products
<230
1 7.300
<0.4
1.6
100
180
0.30
0.27
<100
3,230
<0.17
0.30
80
730
0.22
0.94
-------
tial increases in the mutagenic activities
of both gas- and particulate-phase
species. It was found that the majority
of the mutagenic activity of the product
mixture of wood smoke is associated with
gas-phase species. Much of the increase
in mutagenic activity for the particulate
phase in our experiments is probably the
result of adsorption of gas-phase (low
volatility) mutagenic products onto
existing particulate matter, rather than
a reflection of reactions occurring on the
surface of the particulate matter.
For most of the product mixtures
investigated, a significant portion of the
mutagenic activity observed may have
been caused by PAN, which has been
shown to be mutagenic. The absolute
value of the mutagenic activity of this
species is currently uncertain, however,
and much more work is necessary to
determine the mutagenic potential of
PAN at ambient concentration levels.
Because PAN is ubiquitously present in
polluted urban atmospheres, it would
also be desirable to determine the
biological impact of PAN on other bio-
logical systems by using additional
bioassay techniques.
At this point, laboratory experiments
have shown that pollutants commonly
found in urban air can be transformed
into mutagenic products as a result of
typical urban photochemical processes.
These results suggest the need to extend
this effort in both laboratory and field
measurement areas. Ambient measure-
ments of mutagenic activities in urban
air masses would be helpful in determin-
ing the possible correlation of mutagenic
activity of the air mass with the presence
of photochemical oxidants such as PAN,
and may aid in the overall assessment
of the contribution of urban photochem-
istry to the presence of atmospheric
mutagens.
775-
150-
125-
100-
I
50H
25-
0~\
TA 100
| | Gas Phase
f"':"\ Particulate Phase
TA98
Before
Irradiation
After
Irradiation
Before
Irradiation
After
Irradiation
Figure 4.
Comparison of the gas- and particulate-phase mutagenicity of dilute wood smoke
in air.
P. B. Shepson, T. E. Kleindienst, and E. O. Edney are with Northrup Services,
Inc.-Environmental Sciences. Research Triangle Park. NC 27709.
L. T. Cupitt is the EPA Project Officer (see below).
The complete report entitled "The Production of Mutagenic Compounds as a
Result of Urban Photochemistry." (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
-------
United States Center for Environmental Research
Environmental Protection Information
Agency Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S3-87/020
9Q0G329 PS
U S SNVIR PROTfCTION AGfiNCT
REGION 5 LIBRARY
230 S DfARSCRN STREET ^,
CHICAGO IL 60604
------- |