United States
Environment Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-84-002 Mar. 1984
&EPA Project Summary
Interaction of Complex Pollutant
Mixtures and Particulates in
Causation of Pulmonary Disease
Richard Ehrlich
Studies were conducted to determine
the effects of exposure to particulate
and gaseous air pollutants on the
resistance to experimental respiratory
infection. Changes in resistance to
bacterial pneumonia, expressed as in-
creases in mortality rate and decreases
in mean survival time, served as the
most sensitive and consistent indicators
of damage produced by the exposure.
Using these parameters it was possible
to rank the effects of single 3-hr inhala-
tion exposure to various sulfate and
nitrate particulate aerosols. Among the
particulate pollutants included in these
studies, cadmium sulfate and cadmium
nitrate were most toxic. They were
followed in decreasing order of toxicity
by copper, aluminum and magnesium
sulfate or nitrate aerosols. The metallic
cation appeared to be most important
in altering the resistance to infection.
Multiple 3-hr exposures to significantly
lower concentrations of zinc or cupric
sulfate similarly reduced the resistance
to the respiratory infection.
Nitrogen dioxide (IMO2) and ozone (O3)
mixtures reduced the resistance to
bacterial pneumonia after 6-month in-
halation exposure to various regimens
of these photochemical oxidants.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory, Research Triangle Park, NC,
to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering infor-
mation at back).
Introduction
Changes in resistance to respiratory infec-
tion provide a highly sensitive experimental
animal model system for studies of the ef-
fects of air pollutants at concentrations
resembling those found in urban en-
vironments. The model reflects the impair-
ment of the host defense mechanisms by
combined exposures to air pollutants and to
infectious bacterial aerosol.
The objectives of this program were to
determine the effects of single or multiple
short-term exposures to various sulfate and
nitrate particulate aerosols on resistance to
streptococcal pneumonia and to study the
changes in resistance to this infection
resulting from long-term exposures to N02
and 03 mixtures. In the latter experiments
it was of special interest to determine the ef-
fects of short-duration peaking of the N02
and 03 mixture superimposed on a con-
tinuous exposure to low N02 concentrations.
Materials and Methods
The basic experimental protocol used in
the studies called for exposure of a group of
animals to the pollutants and a second group
to clean filtered air. After the exposure the
two groups were combined and within less
than 1 hr challenged by the respiratory route
with an aerosol of infectious bacteria. After
the challenge the animals were kept for 14
days, during which mortality rates and sur-
vival time were observed.
The short-term exposures to the air
pollutants were carried out in 432 liter Plexi-
glas chambers. The pollutants were diluted
in filtered room air and introduced into the
chambers. To assure a homogeneous dis-
tribution of the pollutants, a small fan was
operated continuously in each chamber. For
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long-term exposure, 4420 liter aluminum-
lined walk-in chambers were used. The ran-
domly selected mice were housed in inhala-
tion cages provided with an automatic water-
ing system. To assure unbiased exposure to
the experimental atmospheres the cages
were rotated weekly to various positions on
the cage rack. Ammonia build-up during the
exposures was prevented by the placement
of deotized boards beneath each cage row.
The exposures to the pollutants were inter-
rupted twice weekly for a total of approxi-
mately 3 hr to provide clean cages and fresh
feed.
A high-voltage generator was used to con-
vert oxygen to 03. To provide the desired
concentration, 03 was diluted with filtered
air in a glass mixing chamber and passed into
the exposure chamber. The 03 concentra-
tion was monitored with an 03 chemi-
luminescent analyzer. A 1 or 5% mixture of
N02 in air was diluted with filtered air and
the N02 concentration in the chambers was
monitored continuously by an N0-N02-N0x
chemiluminescent analyzer.
The particulate aerosols were generated
by a nebulizer from aqueous solutions of
crystalline materials. The estimated mass
median aerodynamic diameter of the
evaporated particles was 0.63 ^m and the
median diameter, approximately 0.31 ^m.
For particle size analysis, aerosol samples
were collected through a 7 ^m single-stage
preimpactor onto 25 mm glass discs cleaned
with methanol and coated with a 50%
glycerol-water solution. Microscopic ex-
amination indicated that approximately 90%
of the airborne particles were <3 pm in
diameter.
Infectious challenge with Streptococcus
sp. (Group C) was performed in a 400-liter
exposure chamber housed within a
microbiological safety cabinet. A continuous
flow nebulizer (DeVilbiss Model 841) was
used to produce the bacterial aerosol. For
infectious challenge, mice were housed in in-
dividual compartments of stainless steel wire
cages. After the challenge the mice were
removed from the chamber and housed for
a 14-day observation period in an isolated
clean-air animal room or were returned to the
pollutant exposure chambers.
Effects of the exposures were measured
in terms of changes in mortality rates and
mean survival time. Free pulmonary cells
were obtained by tracheobronchial lavage
and total cell counts were made on fixed cell
smears. Viability of the alveolar macrophages
was determined by dye exclusion, and the
cellular ATP concentration in the lavaged
cells was monitored. Pulmonary bactericidal
activity was determined in individual mice,
whereby the ratio of viable count to the
radioactive count of Klebsiella pneumoniae
in the lungs provided the rate at which the
bacteria were killed by the lung in a given
time after infection. The clearance of inhaled
viable bacteria from lungs was determined
and expressed as the time in hours required
for 50% of the inhaled bacteria to clear the
lungs.
The significance of the differences in mor-
tality rates was determined by the Chi-
square (X2) test with a 2 x 2 contingency
table. The Student's t test, analysis of vari-
•ance, and least square linear regression were
applied to the data, as appropriate.
Conclusions
Results of the studies reconfirmed
previous observations that an increase in
mortality rates resulting from bacterial
pneumonia is a reproducible and sensitive in-
dicator of adverse effects of air pollutants.
A single 3-hr exposure to cadmium, cupric
or aluminum sulfate or nitrate resulted in
significant increase in mortalities and
decrease in mean survival time in mice in-
fected with a streptococcus aerosol. These
effects were seen at concentrations of ap-
proximately 0.40 mg/m3 cadmium, 0.85
mg/m3 cupric or 1.80 mg/m3 aluminum
sulfate or nitrate. In all exposures the metallic
cation appeared to be the fraction of the
pollutant that was effective in altering the
resistance to the infection. Several sulfate,
nitrate and nitrite pollutants used in the ex-
periments had no effect on the mortality
rates. Thus, the results of this phase of the
program confirmed the utility of this animal
model in ranking the toxic effects of inhala-
tion of particulate pollutants.
A 6-month continuous (24 hr/day, 7
days/week) exposure to 0.19 mg/m3 N02
with superimposed 3 hr/day, 5 days/week
peaks of 0.94 mg/m3 N02 and 0.2 mg/m3 03
mixture resulted in a significant increase in
mortality of mice infected with streptococ-
cus aerosol. A similar effect was seen after
9 months of continuous exposure to 0.38
mg/m3 N02 with superimposed twice daily
peaks occurring 5 days/week. The morning
exposure peak was 1.88 mg/m3 N02 for 1
hr, and the afternoon peak consisted of 0.2
mg/m3 03 for 3 hr mixed with 1.88 mg/m3
N02 for 1 hr. The changes in the resistance,
however, were not seen after 4 or 8 month
exposure to the same experimental regimen.
Superimposition of a single 3-hr exposure
to 0.3 mg/m3 cupric sulfate at the end of the
9-month exposure to the photochemical ox-
idant pollutant mixture resulted in further
enhancement of the mortality due to the
streptococcal pneumonia.
Recommendations
Studies should continue to define more
precisely the causal relationship between in-
halation of air pollutants and acute
respiratory infections in laboratory animals.
To simulate environmental pollution condi-
tions, studies should include the use of mix-
tures of gaseous and particulate pollutants.
Exposure to additional stresses such as ex-
ercise or extremes of temperature should
also be included in such investigations. Since
humans are usually exposed to low concen-
trations of pollutants with superimposed
cyclical higher concentrations, similar ex-
posure regimens should be used during long-
term exposure studies. This is especially im-
portant in studies of pollutants such as
nitrogen dioxide, where within a given ex-
posure regimen the concentration is of
greater importance than the duration of
exposure.
Richard Ehrlich is with 117 Research Institute, Chicago. IL 60616.
Donald E. Gardner is the EPA Project Officer (see below).
The complete report, entitled "Interaction of Complex Pollutant Mixtures and
Particulates in Causation of Pulmonary Disease," (Order No. PB 84-138 965;
Cost: $8.50, 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:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC27711
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