PA-600/1-78-021
larch 1978
Environmental Health Effects Research Series
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are.
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5 Socioeconomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-78-021
March 1978
PHYSIOLOGICAL RESPONSE TO ATMOSPHERIC POLLUTANTS
by
Mary 0. Amdur*
Department of Physiology
Harvard University
School of Public Health
665 Huntington Avenue
Boston, Massachusetts 02115
*Present address:
Department of Nutrition and Food Science
Massachusetts Institute of Technology
Cambridge, Massachusetts 02139
Grant No. R-802030
Project Officer
David L. Coffin
Office of the Director
Health Effects Research Laboratory
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects
Research Laboratory, U.S. Environmental Protection Agency,
and approved for publication. Approval does not signify
that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute
endorsement or recommendation for use.
El
11
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FOREWORD
The many benefits of our modern, developing, industrial
society are accompanied by certain hazards. Careful assessment
of the relative risk of existing and new man-made environmental
hazards is necessary for the establishment of sound regulatory
policy. These regulations serve to enhance the quality of our
environment in order to promote the public health and welfare and
the productive capacity of our Nation's population.
The Health Effects Research Laboratory, Research Triangle
Park, conducts a coordinated environmental health research
program in toxicology, epidemiology, and clinical studies using
human volunteer subjects. These studies address problems in air
pollution, non-ionizing radiation, environmental carcinogenesis
and the toxicology of pesticides as well as other chemical
pollutants. The Laboratory develops and revises air quality
criteria documents on pollutants for which national ambient air
quality standards exist or are proposed, provides the data for
registration of new pesticides or proposed suspension of those
already in use, conducts research on hazardous and toxic mate-
rials, and is preparing the health basis for non-ionizing radia-
x tion standards. Direct support to the regulatory function of the
'v Agency is provided in the form of expert testimony and prepara-
tion of affidavits as well as expert advice to the Administrator
to assure the adequacy of health care and surveillance of persons
having suffered imminent and substantial endangerment of their
health.
Previous studies have shown that certain sulfates and
sulfuric acid elicit greater response in the lung than
comparable amounts of sulfur dioxide. There is therefore a
need to develop information which will relate these findings
to the concentration of pollutants found in ambient air.
This project has performed this function.
John H. Knelson, M.D.
Director,
Health Effects Research Laboratory
111
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ABSTRACT
During the period of this grant several materials were
examined as air pollutants of interest for their irritant effects.
These included sulfuric acid, a series of inorganic sulfates, and
a combination of ozone and sulfur dioxide. Some attention was
also given to the effect of various oil mists on the irritant
response to sulfur dioxide. The method used for measuring
irritant response was by simultaneous tracings of intrapleural
pressure, tidal volume, and rate of flow of gas in and out of the
respiratory system. By relating the intrapleural pressure change
to the change in flow rate at points of equal lung volume, it was
possible to calculate the flow resistance; by relating pressure
change to volume at the beginning and end of inspiration, it
was possible to calculate compliance. The concentrations used in
these studies are well within the range of human exposure.
These studies indicate that the irritant response previously
observed at higher concentrations of sulfuric acid is also
observed at concentrations below 1 mg/m^. The failure of alter-
ations in resistance to return promptly to control values
following termination of exposure has been a consistent finding
in the work with various irritant aerosols. The lowest concen-
trations used in these studies (100 jig/m3) are in the range of
concentrations which have been reported as short-term maxima in
urban atmospheres. Data obtained by the methods used in these
studies can be applied in concept (although not by direct
extrapolation) to the response of sensitive individuals to
short-term peaks of pollution.
IV
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CONTENTS
Page
Foreword iii
Abstract iv
I. Scope of Work 1
II. Physiological Methods 1
III. Sulfuric Acid 4
IV. Inorganic Sulfates 13
V. Ozone and Sulfur Dioxide 26
VI. Oil Mists and Sulfur Dioxide .... 33
VII. Preliminary Work on Sulfites .... 34
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I. Scope of Work
During the period of this grant we have examined several
materials of interest as air pollutants for their irritant effects.
These have included sulfuric acid, a series of inorganic sulfates,
and a combination of ozone and sulfur dioxide. Some support was
also given to a project studying the effect of various oil mists
on the irritant response to sulfur dioxide. Preliminary work was
done with sulfite and bisulfite.
II. Physiological Methods
The method we use of measuring irritant response has been
described in the literature (Amdur and Mead, Am. J. Phys. 192:364,
1958). Simultaneous tracings are needed of intrapleural pressure,
tidal volume, and rate of flow of gas in and out of the respiratory
system. Intrapleural pressure is measured by recording the pressure
changes in a fluid-filled catheter which is inserted under ether
anesthesia of brief duration. Tidal volume is measured by record-
ing the pressure changes produced in a body plethysmograph. Rate
of flow is measured by electrical differentiation of the volume
signal with respect to time. By relating the intrapleural pressure
change to the change in flow rate at points of equal lung volume,
it is possible to calculate the flow resistance. By relating pres-
sure change to volume at the beginning and end of inspiration, it
is possible to calculate compliance.
The method has certain advantages. One of these is its simplic-
ity, which permits routine toxicological use of unanesthetized
animals. The fact that each animal serves as its own control per-
-------
mits the use of paired data for statistical evaluation. The in-
crease in flow-resistance is related to the concentration of irri-
tant; this permits the development of dose-response curves. These
curves yield information on such factors as the effect of particle
size on the potency of an irritant material, on the relative irri-
tant potency of different materials, and on the effect of inert
particulate material on the degree of response to irritant gases.
Data such as these dose-response curves resulting from a single ex-
posure of many animals over a wide range of concentrations provide
a tool for the demonstration of potentiation effects and other sub-
tle toxicological phenomena which cannot readily be detected by
experiments on human subjects. Experimentation with human subjects
is by practical necessity limited both as to number of subjects and
conditions of exposure.
The method also has certain limitations which should be kept
clearly in mind. The standard exposure time is one hour; at best,
the preparation can only be used for five or six hours. Thus, the
results obtained cannot be used to predict effects of chronic expo-
sure to the compounds studied. Although the animals are unanesthe-
tized during the experimental period, an intrapleural catheter has
been inserted under anesthesia. That this surgical procedure per
se causes an increase in resistance was demonstrated by measurements
made by another method before and after the insertion of the catheter
(Mead, J. Appl. Physiol. 15^:325, 1960). The physiological tech-
nique thus constitutes a stress which might render such animals more
sensitive to irritant exposure. There is experimental evidence to
support this suggestion. Some unpublished experiments in which the
-2-
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response to sulfur dioxide was measured by another method that
did not involve the use of an intrapleural catheter indicated that
the two methods were of equivalent sensitivity at high concentrations
but that at concentrations of 2 ppm and less the intrapleural cath-
eter method was more sensitive. Thus the stress of the technique
used has rendered the animals more sensitive to low-grade irritant
exposure. This might be considered an advantage rather than a
limitation, as it is the sensitive segment of the human population
that is most affected by air pollution. In any effort, to draw
meaningful extrapolations from these data, it should be kept in
mind that if these guinea pigs are analogous to anything at all, it
is to the sensitive individual and not to the normal healthy indi-
vidual.
Random-bred guinea pigs weighing 200-300 g were used in these
experiments. The plethysmograph was clamped so that the animal's
head projected into the exposure chamber. Respiratory measurements
were made every five minutes during a half-hour control period. The
material or materials being studied were then added to the entering
air stream of the exposure chamber. Respiratory measurements were
again made every five minutes during an exposure period of one hour
(for sulfuric acid or sulfate salts) or two hours (for the ozone-
sulfur dioxide studies). Each animal thus served as its own con-
trol. The chamber was cleared of irritant material and measure-
ments were made during a post-exposure period of one-half to one
hour.
-3-
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III. Sulfuric Acid
A. Method of Aerosol Generation and Measurement.
The sulfuric acid aerosol is produced by a Rappaport-Wein-
stock condensation type aerosol generator (Rappaport and Weinstock,
Experientia LI: 363, 1955). The sulfuric acid is first, heated and
nebulized to a heterogeneous aerosol. The larger particles are
removed by impaction and the smaller particles are carried upwards
through a heating column in which they are vaporized. Upon emerging
from the heating column, the vapor cools and condenses into drop-
lets of uniform size. The size of the aerosol can be controlled
by appropriate adjustments of the amount of sulfuric acid nebulized, of
the temperature of the reservoir, and the temperature of the heating
column. The mass concentrations can be controlled by regulating
the amount of dilution air. All air is pre-dried and filtered to
remove foreign particles. Relative humidity of the chamber was
50% and temperature was 70°F.
The mass concentration was measured by collecting a sample of
the aerosol on a type G.S. Millipore Filter, which was then soaked
in 10 ml demineralized water. The electrical conductivity of the
resulting solution was then measured and compared with a standard
curve. All samples were collected throughout the animal exposure
period at a flow rate of 3.5 1pm. Concentrations were reproducible
within ± 10% or better.
The particle size of the larger aerosol was determined before
and after each animal exposure with an ultra-microscope by which
the time of free fall of the aerosol particles across a calibrated
grid could be measured. The size was then determined by appropri-
-4-
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ately inserting the free fall time into the Stokes-Cunningham
equation. Average sizes were based on counts of 50-100 particles.
The standard deviation was consistently less than 10% of the mean
diameter. The particle size of the smaller aerosol was determined
by light scattering at each set of experimental conditions.
B. Background for Present Studies
Sulfuric acid is the most irritant of the particulate sulfur
species formed by the oxidation of sulfur dioxide in the atmosphere.
Acute exposure of guinea pigs has demonstrated that the irritant
potency of a given amount of sulfur, as evaluated by changes in
respiratory mechanics, may be increased three- to five-fold when
given as sulfuric acid rather than as sulfur dioxide (Amdur, Am.
Indust. Hyg. Assoc. J. _3_5_:489, 1974). In two-year exposures of
monkeys to sulfuric acid, pathological changes were produced which
were not observed when corresponding levels of sulfur were given
as sulfur dioxide (Alarie et al., Arch. Environ. Health 27:16,
1973). The greater irritant potency of sulfuric acid is thus
indicated in the acute response of a rodent species and the chronic
response of a primate species. Aerosols of metal salts, which
promoted the conversion of sulfur dioxide to sulfuric acid, poten-
tiated the response to sulfur dioxide three- to four-fold (Amdur
and Underhill, Arch. Environ. Health 16;460, 1968).
Our previously-published study of the respiratory response of
guinea pigs to sulfuric acid (Amdur, Arch. Indust. Health 18:407,
1958) did not examine concentrations below 2 mg/m3 nor particle
-5-
-------
sizes below 0.8 ym (MMD). The studies reported here were performed
to obtain similar data on concentrations of 1 mg/m and below at
particle sizes of 1 and 0.3 ym.
C. Results
Table 1 presents our data on the effect of sulfuric acid on
pulmonary flow resistance and pulmonary compliance. All exposures
produced a statistically significant increase in resistance. The
degree of response was dose-related as shown in Figure 1. The 0.3 ym
particles produced a greater response at a given concentration than
did the 1 ym particles. This difference appears greatest at the
lowest concentration of 0.1 mg/m .
In animals exposed to 0.1 mg/m3 at the 1 ym particle size, the
resistance values had returned to pre-exposure values by the end of
the half-hour post-exposure period. In all other exposures, the
post-exposure value was less than the response at the end of expo-
sure but was still elevated above control values. The time course
of the post-exposure resistance increases, expressed as percent
change from control, is shown in Figure 2 for the 0.3 ym particles
at concentrations of 0.1 and 1 mg/m3. In both cases, the values
increased slightly during the first five minutes of the post-expo-
sure period then declined to an essentially constant Level by
fifteen minutes after the end of exposure.
A decrease in pulmonary compliance was also produced by these
low level exposures to sulfuric acid. In the exposure to 1 ym par-
ticles, the decrease in compliance was not statistically significant
at concentrations of 0.1 or 0.4 mg/m3. A decrease in compliance
was produced by the two higher concentrations of 1 ym particles.
-6-
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At a particle size of 0.3 ym, a decrease in compliance was observed
at all concentrations tested. At corresponding concentrations, the
0.3 ym particles produced a greater decrease in compliance than the
1 ym particles. When the exposure produced a decrease in compli-
ance, the values were still below control values at the end of the
thirty-minute post-exposure period, although less depressed than
at the end of exposure.
Detailed data are not presented for tidal volume, respiratory
frequency, or minute volume, as the low concentrations used in
these studies produced no alterations in any of these factors.
D. Discussion
The concentrations used in these studies are well within the
range of human exposure. Concentrations above 0.1 mg/m^ have been
reported as hourly averages in urban pollution. A concentration
of 1 mg/m3 is the currently recommended standard for occupational
exposure.
These studies indicate that the irritant response previously
observed at higher concentrations of sulfuric acid is also observed
3 3
at concentrations below 1 mg/m . The response to 0.1 mg/m at the
1 ym particle size is slight and rapidly reversible. The response
to 0.1 mg/m at the 0.3 ym size is greater in magnitude and is not
rapidly reversible.
A concentration of 1 mg/m sulfuric acid contains 0.3 mg/m^
of sulfur. The percent increase in resistance produced is on the
order of 80% for 0.3 ym particles and 60% for 1 ym particles. The
same order of magnitude of sulfur (0.2 mg/m^) given as sulfur
-7-
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dioxide (0.16 ppm) produces a slight resistance increase in the
order of 10%. Thus, the same amount of sulfur, when given as
sulfuric acid, produces 6 to 8 times the response observed when
given as sulfur dioxide. Given as 1 ym sulfuric acid, 0.03 mg
S/m produces a response of the same order of magnitude as 0.2
3 3
mg S/m given as sulfur dioxide. The response to 0.03 mg S/m
as 0.3 ym sulfuric acid is four times the response to 0.2 mg S/m-^
given as sulfur dioxide.
The failure of alterations in resistance to return promptly
to control values following termination of exposure has been a
consistent finding in our work with various irritant aerosols.
The post-exposure resistance values of animals exposed to sulfur
dioxide and sodium chloride or to formaldehyde and sodium chloride
remained elevated and were related to the total does of aerosol.
This was one of the earlier findings which suggested that the
potentiation of the response to these gases was mediated by for-
mation of an irritant aerosol rather than by simple transfer of
additional gas as such to the lung (Amdur, Inhaled Particles and
Vap_or_s_ 1:281, 1961; Amdur and Underhill, Arch. Environ. Health
1^:460, 1968). The response to sulfur dioxide or formaldehyde
alone was readily reversible until extremely high concentrations
were reached. The only exception to the slow reversibility of
the response to irritant aerosols was histamine, with which even
very major responses were almost immediately reversible (Amdur,
Arch. Environ. Health 13:29, 1966).
-8-
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The lowest concentrations used in these studies (100 ug/m )
are in the range of concentrations which have been reported as
short-term maxima in urban atmospheres. Data obtained by the
methods used in these studies can be applied in concept (though
obviously not by direct extrapolation) to the response of sen-
sitive individuals to short-term peaks of pollution. Alteration
of pulmonary mechanics is obviously only one manifestation of
irritant response. Another manifestation is alteration in regional
deposition or clearance of aerosols. It is of interest to note
that alterations in regional deposition patterns have been demon-
strated in guinea pigs exposed to concentrations as low as
30 yg/m (size 0.25 urn) for one hour (Fairchild et a_l. , Amer.
Indust. Hyg. Assoc. J. 3J7:584, 1975). More recently, Dr. Morton
Lippmann's group at New York University has found that a one hour
exposure to < 200 ug/m (size 0.3 ym) caused a significant
transient slowing of tracheobronchial clearance of ferric oxide
aerosol in donkeys. Thus, these levels of sulfuric acid produce
irritant effects other than the alterations in pulmonary mechanics
reported in the present studies.
-9-
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o
I
Table 1
Respiratory Response to Sulfuric Acid
0.70
0.59
0.72
Particle Size HMD
o
Concentration mg/m
Number of Animals
Resistance cm l^O/ml/sec
Control
Exposure3
x (E-C)
?*<
% Change
b
Post Exposure
5 (P-C)
g_
PX<
% Change
Compliance ml/cm 1^0
Control
o
Exposure
x (E-C)
S-
PX<
% Change
b
Post Exposure
x (P-C)
<^_
PX<
% Change
a"Exposure": Average of readings at 55 and 60 min.
"Post Exposure": Average of readings at 25 and 30 min.
0.11
20
1
0.40
20
pm
0.69
•20
0.85
20
0.
0.10
23
3 urn
0.51
20
1.
25
0
0.68
0.80
0.72
0.23
0.24
0.20
0.21
0.22
0.24
0.74
0.80
0.10
0.039
0.02
+14
0.77
0.07
0.041
NS
+ 10
0.77
0.18
0.033
0.001
+30
0.71
0.12
0.051
0.05
+20
1.06
0.34
0.042
0.001
+47
O.S9
0.17
0.047
0.001
+24
1.09
0.41
0.051
0.001
+60
0.86
0.18
0.053
0.01
+26
1.13
0.33 '
0.048
0.001
+41
1.01
0.21
0.059
0.01
+26
1.15
0.43
0.052
0.001
+60
1.09
0.37
0.058
0.001
+51
1.32
0.58
0.063
0.001
+78
1.20
0.46
0.067
0.001
+62
0.20
0.20
•0.03
0.015
NS
-13
0.21
0.02
0.018
NS
-9
0.22
-0.02
0.016
NS
-8
0.23
-0.01
0.012
NS
-4
0.15
-0.05
0.014
0.01
-25
0.17
-0.03
0.011
0.02
-15
0.15
-0.06
0.017
0.01
-28
0.17
-0.04
0.013
0.01
-19
0.16
-0.06
0.012
0.001
-27
0.17
-0.05
0.013
0.01
-22
0.16
-0.08 '
0.017
0.001
-33
0.18
-0.06
0.016
0.01
-25
0.12
-0.08
0.019
0.01
-40
0.14
-0.06
0.021
0.01
-30
-------
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ro
P
CO
g
P
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P
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-ii-
% Change in Resistance
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W td
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a G
ro
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Jd
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tn
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-------
-2T-
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H
m —
x o
o
m
i
a
3 o
FO
en
CM
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% INCREASE IN RESISTANCE
ro
O
CO
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I Hour EXPOSURE
x ©
- o
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330
ui 01 ^1
ro _,
HI ii! 3
-------
IV. Inorganic Sulfates
A. Methods of Aerosol Generation and Measurement
These aerosols were produced with Dautrebande D~Q aerosol
generators. These produce a heterogeneous aerosol of sub-micron
size. The several models we have have slightly different charac-
teristics in terms of mean size produced with a given concentra-
tion of solution. To vary size, we utilized this fact in combina-
tion with the use of O.I/ 0.3 and 1% solutions of the various
salts. The size was measured by collecting a sample on a
carbon-coated EM grid by electrostatic precipitation. The size
was measured from electron micrographs.
The mass concentration of the sulfate salts was measured by
various methods following collection on a Millipore filter. For
the copper sulfate, copper was measured by atomic absorption.
Ammonium sulfate and ammonium bisulfate were measured with an
ammonium ion electrode, by direct weighing on a Cahn electro-
balance or by increase in electrical conductivity.
Sulfur dioxide was generated by metering gas from a tank
containing 0.1% sulfur dioxide in air. The concentration was
measured by collecting a sample in dilute sulfuric acid-hydrogen
peroxide solution and measuring the increase in conductivity.
The collecting bubbler was preceded by a membrane filter to
remove the aerosol, which would have also altered the conductivity.
-13-
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The main air stream entering the exposure chamber was
filtered to remove extraneous particles and dried. The relative
humidity of the exposure chamber atmosphere was 50%.
B. Background
Data on the comparative toxicity of sulfate salts are of
importance because sulfur dioxide in urban atmospheres is con-
verted into particulate sulfur species. The rather meager ex-
isting toxicological data indicate that some, but not all, sul-
fate salts are respiratory irritants and that the irritant
potency of a given sulfate increases with decreasing particle
size.
Zinc ammonium sulfate causes an increase in pulmonary
flow resistance in guinea pigs (Amdur and Corn, Amer. Indust.
Hyg. Assoc. J. 2_4_:326, 1963). Over the size range studied
(0.29 to 1.4 ym, mean size by weight) the change in flow resis-
tance increased as the particle size decreased. In cats, zinc
ammonium sulfate also caused an increase in flow resistance
and a decrease in pulmonary compliance (Nadel et al., Inhaled
Particles and Vapors 11:55, 1965). These changes were similar
to the response to an aerosol of histamine, but were of lesser
magnitude.
Zinc sulfate and ammonium sulfate (0.3 pm in size) produced
an increase in flow resistance in guinea pigs (Amdur and Corn,
1963) as did ferric sulfate (Amdur and Underhill, Arch. Environ.
-14-
-------
Healthy 16_:460, 1968). Neither ferrous sulfate nor manganous
sulfate was irritant (Ibid.).
In the present studies we have examined the comparative
irritant potency of ammonium sulfate, ammonium bisulfate, cupric
sulfate and sodium sulfate. We have also determined whether
or not these sulfate salts potentiate the response to low concen-
trations of sulfur dioxide.
C. Results
The effects of the four sulfate salts on resistance and
compliance are presented in Table 2. In order that the various
salts may be compared in irritant potency, the response is also
expressed as percent change per microgram of sulfate.
Ammonium sulfate caused a statistically significant de-
crease in compliance at all the concentrations and particle
sizes tested. Two of the exposures produced an increase in
resistance and two did not. The resistance increase produced
by the 0.3 ym particles at a concentration of 1 mg/m was in
good agreement with values found on a smaller group of animals
in a previous study (Amdur and Corn, 1963). With the exception
of the fact that there was a minimal response to the 0.2 \im
particles, the response per ug of sulfate was greater as the
particle size decreased.
-15-
-------
Ammonium bisulfate was also a mild respiratory irritant.
All the exposed groups showed a statistically significant in-
crease in resistance and decrease in compliance. The; degree of
response per microgram of sulfate increased as the pcirticle size
decreased. As was the case with ammonium sulfate, the response
was minimal to the 0.8 um particles. An overall consideration
of the data suggests that ammonium bisulfate is less irritant
than ammonium sulfate. At the smallest size (0.13 urn) the
percent change in resistance per ug of sulfate was 0.. 063 for the
sulfate and 0.019 for the bisulfate; the corresponding values
for compliance were -0.074 and -0.019. A similar pattern emerges
when one compares the data for 0.3 um ammonium sulfate with the
data for 0.5 um ammonium bisulfate. For what it's worth, these
comparisons would tend to suggest that the response to ammonium
sulfate is of the order of three times that to ammonium
bisulfate.
The data for copper sulfate permit the direct comparison of
two concentrations at similar particle sizes and of two particle
sizes at similar concentrations. At a particle size of 0.1 um a
concentration of 0.4 mg/m produced a slight but statistically
— 16 —
-------
significant decrease in compliance. The slight decrease in resis-
tance was not statistically significant. At a higher concentration
of 2 mg/m both an increase in resistance and a decrease in com-
pliance were observed. Exposure to 2 mg/m at a particle size of
0.3 urn produced a statistically significant change in resistance
and compliance, but the response was slightly less than that produced
by the smaller particles.
Sodium sulfate at a particle size of 0.1 ym produced no change
in resistance. The slight decrease in compliance was not statisti-
cally significant.
Data for sulfur dioxide alone at a concentration of 0.3 ppm
and for the combination of sulfur dioxide and the aerosols at a
particle size of 0.1 pm at the lowest concentration used are presented
in Table 3. In all exposures there was a statistically significant
increase in resistance and decrease in compliance. Figure 3 com-
pares the responses to the combined exposure with the sum of the
responses to the sulfur dioxide and aerosol given alone. The com-
bination of the copper sulfate and sulfur dioxide was more than
additive. The response to the other combinations could be predicted
on the basis of a simple additive response.
D. Discussion
Of the four sulfate salts examined, ammonium sulfate appeared
to be the most irritant, followed by ammonium bisulfate, copper sul-
fate, and sodium sulfate. There is evidence that isolated guinea
pig lung fragments release histamine when incubated with solutions
of ammonium sulfate but not when incubated with solutions of sodium
-17-
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sulfate (Charles and Menzel, Arch. Environ. Health 31:314, 1975).
Intratracheal injections of ammonium sulfate solutions produced
bronchoconstriction in isolated perfused lungs but sodium sulfate
did not. Ammonium ions also increased the absorption of sulfate
by the rat lung in vivo (Charles and Menzel, Res. Comm. Chem. Path.
Pharm. 12_:389, 1975). Sulfate removal was differentially enhanced
and presumably there was increased flux by various associated cat-
ions across the mast cell where histamine is stored. Among the
most active in this regard were ferric and zinc ions. In earlier
work in our laboratory (Amdur and Corn, 1963; Amdur and Underhill,
1968), both ferric sulfate and zinc sulfate were found to be irri-
tant. The pharmacological findings correlate well with the data
obtained in our studies using mechanics of respiration as a means
of determining relative irritant potency.
None of the sulfate salts tested in the present studies or in
our earlier work are as irritant as sulfuric acid itself. The per-
cent increase in resistance per ug of sulfate as sulfuric acid
is 0.432 for 0.1 um particles and 0.410 for 0.3 vim particles (Amdur,
Proc. 4th Symp. Statistics and the Environment, pg. 48, 1976). Data
for all the sulfates tested in these and earlier studies are avail-
able at the 0.3 ym size except sodium sulfate, which was only studied
as 0.1 urn particles. If one assigns a value of 100 to the 0.410
obtained with sulfuric acid and relates the values for the sulfate
salts to it, it is possible to rank these compounds for irritant
potency as shown in Table 4. The ten-fold less irritant potency
of ammonium sulfate as compared to sulfuric acid would fit the
-18-
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observation made twenty years ago that neutralization with ammonia
eliminated the lethality of sulfuric acid to guinea pigs (Pattle,
Burgess and Cullumbine, J. Path. Bact. 72:219, 1956).
The irritant potency of the sulfate species varies so widely
that the term "suspended sulfate" is toxicologically meaningless.
The practical implication of this fact is that a better analytical
measurement than "suspended sulfate" will be needed in research
epidemiology before definitive data relating to health effects of
particulate sulfur species are likely to emerge.
The range of particle size of the aerosols used in the present
study (0.1 to 0.8 ym) was too narrow to demonstrate a rational
relationship between particle size and degree of reponse. Such
relationships were reported earlier for zinc ammonium sulfate over
a size range of 0.3 to 1.4 ym (Amdur and Corn, 1963) and for sul-
furic acid over a size range of 0.1 to 2.5 ym (Amdur, 1976). The
data for any given compound in the present study, however, showed in
general a greater degree of response at a smaller particle size.
Overall, data from various studies in our laboratory indicate that
measurement of mass median diameter would be much more meaningful
than "respirable size" in attempts to assess the health effects of
atmospheric aerosols .
Ammonium sulfate, ammonium bisulfate, and sodium sulfate in
these studies did not potentiate the response to sulfur dioxide.
These exposures were all performed at an exposure chamber relative
humidity of 50%. There is evidence that increasing the relative
humidity to 80% markedly increases the potentiating effect of sodium
chloride (McJilton, Frank and Charlson, Science 182:503, 1973).
-19-
-------
Whether or not increased relative humidity would effect these sul-
fate salts in a similar manner is not known.
It was previously reported that salts of manganese, vanadium,
and ferrous iron were strong potentiators of the response to sul-
fur dioxide (Amdur and Underhill, 1968). At concentrations of 1.4
_ o
to 1.8 x 10 millimoles of metal per cubic meter the metal salts
increased the response to 3 to 4 times that observed in exposures
to sulfur dioxide alone. These metals were known to promote the
conversion of sulfur dioxide to sulfuric acid. Under our exposure
conditions about 10% conversion was found at sulfur dioxide concen-
trations of 0.2 ppm. When this amount of sulfuric acid was combined
with 0.2 ppm sulfur dioxide, the response observed from sulfur di-
oxide and the metal aerosols was reproduced (Amdur, Amer. Indust.
Hyg. Assoc. J. 35^:589, 1974).
It had been reported that copper sulfate also promoted the
conversion of sulfur dioxide to sulfuric acid (Cheng et al., Atmos.
Environ. 5^:987, 1971). On this basis, our observation that aero-
sols of copper sulfate potentiated the response to sulfur dioxide
would have been predicted. It is interesting, however, to note
that copper appears to be a more powerful potentiator of sulfur
dioxide than the other metals. The concentration of copper used
was 1.3 x 10 millimoles/m , or about one tenth of the concen-
tration of the other metals. The response increased four-fold. A
possible explanation for the greater effectiveness of copper may
be provided by the observation made at the Center for Thermochemi-
cal studies at Brigham Young University that aerosols containing
-20-
-------
copper can complex with sulfur dioxide in such a manner that the
sulfur is relatively stable as S and is to some extent protected
from further oxidation (Hansen et al., Proc. Int. Conf. Environ.
Sensing and Assessment, 1975). Their samples were from within a
smelter or from the atmosphere when wind patterns brought material
from known point sources. It has also been reported that sodium
bisulfite is a much more powerful irritant than sulfur dioxide
(Alarie, Wakisaka and Oka, Environ. Physiol. Biochem. 3_:182, 1973).
These data raise the interesting speculation that perhaps the
copper potentiation is mediated by the formation of a sulfite or
bisulfite complex rather than by the formation of sulfuric acid.
Overall, these data emphasize the importance of analysis of
specific trace metal content of atmospheric aerosols in studies
attempting to unravel the complexities of the health effects of the
sulfur oxides-particulate pollution complex.
-21-
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Table 2
Respiratory Response to guifato salts
Sol fate Salt ft«4>J«>4
020 0.30 0.81 0.13 0.52 0.77 0.11 0.13 0.33 0.1)
HUD -|i n " . n 214 + 023 102 + 011 9.54 + 0.94 0.93+_0.09 2.60 + 0.29 10.98 +_ 1.64 0.43+0.17 2.05j_0.?n 7.41 «_ 0.31 0.90^0j.l]
Salt - nxy™3 ifi-l 1553" " '«&" 6926~ 775 2168 9157 2" 12K 1™R fit)8 i
lig S04/m3 Jo 10 10 10 19 10 1O 23 30 35 10
No. of Animals
Resistance cm II 0/ml/sec
control
exposure
airterence
S x 3
r < h
% change
\ change/ w SO4
0 43
+ 0 . 10
O.041
0.05
+ 23
0.063
0.53
0.51
-0.02
0.043
N.S.
-4
0.65
0.83
+ 0.18
0.052
0 01
+ 29
0 039
0.60
0.60
0
-
0
o
0.60
0.69
+ 0.09
0.035
O.O2
+ 15
0.019
0.87
1.11
+ 0.24
0.059
0.01
+27.5
0.013
0.59
0.73
+0.14
0.043
O.O2
+23
0.002
0.44
0.40
+0.04
0.021
NS
<9
0.44
0.55
HO. 11
O.O32
0.01
125
0.07.0
0.49
0.5C.
+0.07
O.OPl
0.01
H4
0.009
O.G6
0.67
+0.01
~
MS
1-2
O.OO.l
CompJ i ance ml/cm
control
exposure
di f fer once
S v a
X
„ x b
p <
% chanqe
0.29
0.21
-0.08
0.019
0.01
-0 . 074
0.23
0 .20
-0.03
0.013
0.05
-13
-0.008
0.30
0 23
-0.07
0.012
0.001
-23
-0.032
0.26
0.23
-0.03
0.013
0.05
-12
-0.002
0.26
0.22
-0.04
0.011
0.01
-15
-0.019
0.23
0.16
-0.07
O.O13
0.001
-30
-0.014
0.26
0.21
-0.05
0.011
O.01
-19
-0.002
0.27
0.24
-0.03
0.017
0.02
-11
-0.043
0.77
0.23
-0.04
O.OOS
0.001
-15
0.012
0.77
0.74
-0.01
O.O06
0.001
-11
-0.000
0 . 26
0.24
-0.02 .
0.01'f
NS
-7
-0.01(1
a stiindnrrt F.rror of Difference
b Students paired tost
-------
Table 3
Response to 0.1 ym Sulfate Salts and SO2
SO2 - ppm 0.32
Sulfate Salt
Cone. mg/m3
Number of Animals 10
0.30
(NH4)2S04
0.5
10
0.32
NH4HS04
0.9
10
0.36
CuSO4
0.4
10
0.31
Na2SO4
0.9
10
Resistance -
CmH20/ml/sec
control
exposure
difference
Sx
P <
% change
0.62
0.69
+0.07
0.023
0.02
+12
0.50
0.66
+0.16
0.051
0.02
+31
0.61
0.74
+0.13
0.047
0.05
+ 21 '
0.49
0.78
+0.29
0.067
0.01
+59
0.53
0.59
+0.06
0.024
0.05
+11
Compliance -
control
exposure
difference
Sx
P <
% change
0.22
0.20
0.02
0.012
NS
-10
0.23
0.18
•0.05
0.020
0.05
-22
0.28
0.21
-0.07
0.019
0.01
-25
0.30
0.19
-0.11
0.021
0.001
-37
0.31
0.27
-0.04
0.017
0.05
-13
-23-
-------
Table 4
Relative Irritant Potency of Sulfates
Sulfuric Acid 100
Zinc Ammonium Sulfate3 33
Ferric Sulfateb 26
Zinc Sulfate3 19
Ammonium Sulfate 10
Ammonium bisulfate 3
Cupric Sulfate 2
Ferrous Sulfate 0.7
Sodium Sulfate0 0.7
Manganous Sulfate^ -0.9d
Data of Amdur and Corn, 1963
Data of Amdur and Underbill, 1968
Particle size O.lym
d
Resistance decreased; change N.S.
-24-
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SULFATE
COMBINATION
I
NJ
U1
I
ncrease %
Resistance
ance c&crease
E
o
o
A\\\
A\\\
A\\\
A\\V
--
\\\v •:>•••:•:•:•"•
NH4 HS04 Cu S04
Na2 S04
\\\V
\\\\
FIGURE 3 - COMPARISON OF RESPONSE TO COMBINATION OF SO, AND
SULFATES WITH SUM OF THE RESPONSES TO EACH GIVEN
ALONE.
-------
V. Ozone and Sulfur Dioxide
A. Methods of Generation and Measurement
The ozone was generated by passing oxygen through a high voltage
electric field. To prevent interference from sulfur dioxide, a
chemiluminescent ozone detector was used to measure ozone concentra-
tions. The method of generation and measurement of sulfur dioxide
was as described in Section IV. The ozone and sulfur dioxide were
mixed with the main air stream prior to contact so that there was
no chance for chemical interaction of the two gases at high concen-
trations. During one run at levels of 0.8 ppm of both gases, a
sample was collected on a Millipore filter and analysed for par-
ticluate sulfate. No detectable amount of sulfate was present.
B. Background for Present Studies
Hazucha and Bates (Nature 3_3_:50, 1975) reported that human
subjects exposed for two hours with intermittent exercise to a
combination of 0.37 ppm ozone and 0.37 ppm sulfur dioxide showed
greater effects on pulmonary function than could be accounted for
on the basis of simple addition. It was suggested that these
effects might have occurred in response to sulfuric acid formed
in the respiratory tract by chemical interaction of ozone and sul-
fur dioxide.
The guinea pig preparation we use is very sensitive to sulfuric
acid. Thus it appeared worthwhile to expose animals to the com-
bination of ozone and sulfur dioxide. The backlog of dose-response
data on small-size sulfuric acid would permit a possible estimation
-26-
-------
of the amount formed. Our initial protocol was to expose groups
of animals for two hours to 0.2, 0.4, or 0.8 ppm of each gas or to
combinations of the two at equal concentrations. On the basis of
the results of the exposures to the two higher levels, the planned
experiments on the combination of the lowest concentration were
not carried out.
C. Results
The response to ozone alone is shown in Table 5. As had been
previously observed, ozone concentrations of this order of magnitude
do not alter pulmonary flow resistance. The two higher concentra-
tions produced a decrease in compliance of 22-24% below control val-
ues after one hour of exposure and 28-29% below control values by
the end of the two-hour exposure. The post-exposure period of
30 minutes was not long enough for complete reversal. Earlier
work showed that control values would be reached within two hours
after exposure. The decrease in compliance resulted in a decrease
in the time constant of the lungs and was accompanied by an increase
in respiratory frequency. At the lowest concentration the increase
in frequency was the only statistically significant change produced
by the two-hour exposure.
The response to the exposure to sulfur dioxide alone is shown
in Table 6. In these particular groups of animals the response
was minimal and no statistically significant alterations in respira-
tion were produced. There was no progressive increase in flow-
resistance produced by the extension of the exposure time to two
hours.
-27-
-------
The response to the combined exposures to 0.4 and 0.8 ppm
of each gas are shown in Table 7. The responses observed are
essentially the same as those produced by the exposure to these
concentrations of ozone alone. This response pattern is not
typical of the response pattern produced by sulfuric acid.
D. Discussion
The results of the exposure to the combination of ozone and
sulfur dioxide do not indicate a synergism between the two gases
under the exposure conditions prevailing in these experiments.
The response typical of ozone exposure, i.e. an increase in fre-
quency, a decrease in compliance, minimal change in resistance
and a decrease in the time constant, was observed in response to
the combination of ozone and sulfur dioxide. This pattern of
response is not similar to the changes produced in the quinea pig
by exposure to sulfuric acid.
Since this work was done, Bill and Hackney at Rancho Los Amigos
in Los Angeles have done further exposures of human subjects to
combinations of ozone and sulfur dioxide. Their overall finding
was that the effect was much less dramatic than that originally
observed by Hazucha and Bates in Montreal. Together the two groups
explored the various possible reasons for the observed difference.
The most likely explanation appears to be the fact that the condi-
tions in the Montreal exposure chamber led to the production in the
exposure atmosphere of perhaps up to 200 yg/m of acid sulfate, most
likely sulfuric acid (Bill e_t al. , Am. Indust. Hyg. Assoc. J., in
press). The negative results obtained in our studies, in which no
-28-
-------
sulfate was present in the chamber, suggest that there was no
interaction of the ozone and sulfur dioxide after inhalation,
as was originally postulated, at least in the guinea pig lung.
-29-
-------
Table 5
Response to Ozone
PPM
Number of Animals
Resistance
cm H-O/ml/sec
Compliance
ml/cm H20
R x C
sec
Frequency
breaths/min
Tidal Volume
ml
Minute Volume
ml
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
0.2
10
0.74
0.62
0.56
0.56
0.25
0.23
0.22
0.22
0.185
0.143
0.123
0.123
80
89
94*
93*
2.3
2.3
2.3
2.1
184
205
216
195
0.4
10
0.67
0.65
0.67
0.50
0.25
0.19*
0.18*
0.20*
0.167
0.123
0.120
0.100
84
93
99*
96*
2.4
2.2
2.0
2.3
201
205
198
221
0.8
10
0.60
0.50
0.44
0.47
0.27
0.21*
0.19*
0.21*
0.162
0.105*
0.084*
0.099*
86
97*
115*
113*
2.6
2.2
1.8
2.0
223
213
207
226
*Statistically significant: p < 0.05 or better.
-30-
-------
Table 6
Response to Sulfur Dioxide
PPM
Number of Animals
Resistance
cm H20/ml/sec
Compliance
ml/cm H_0
R x C
sec
Frequency
breaths/min
Tidal Volume
ml
Minute Volume
ml
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
0.2
10
0.62
0.64
0.59
0.58
0.24
0.21
0.21
0.20
0.149
0.134
0.124
0.116
87
93
94
89
2.3
2.2
2.3
2.1
200
204
216
187
0.4
10
0.59
0.63
0.57
0.56
0.20
0.20
0.19
0.18
0.118
0.126
0.108
0.101
89
95
92
94
2.0
2.1
2.0
2.1
178
199
184
197
0.8
10
0.62
0.66
0.64
0.60
0.23
0.23
0.24
0.22
0.143
0.152
0.154
0.132
78
80
76
74
2.1
2.3
2.2
2.1
169
184
167
155
-31-
-------
Table 7
Response to Combination of Ozone
and Sulfur Dioxide
PPM
Number of Animals
Resistance
cm H20/ml/sec
Compliance
R x C
sec
Frequency
breaths/min
Tidal Volume
ml
Minute Volume
ml
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
Control
1 hr
2 hr
Post Exp.
0.4
10
0.42
0.40
0.33
0.31
0.27
0.23
0.22*
0.28
0.113
0.092
0.072*
0.087*
91
104*
108*
111*
2.6
2.2
2.1
1.9
237
229
227
211
0.8
10
0.55
0.56
0.42
0.37
0.28
0.23
0.21*
0.24
0.154
0.129
0.088*
0.089*
71
89*
88*
99*
2.6
2.4
2.1
2.1
184
214
185
208
*Statistically significant: p < 0.05 or better.
-32-
-------
VI. Oil Mists and Sulfur Dioxide
This work is described in detail by Daniel L. Costa in a
thesis: The Physical and Physiological Effects of Oil Mists and
Sulfur Dioxide (Harvard School of Public Health, May, 1977). Two
manuscripts for publication are currently being prepared from this
material.
When simultaneous exposures were made to 1 or 10 ppm sulfur
dioxide and 10 mg/m motor oil, the irritant effects of sulfur
dioxide (resistance increase) were antagonized. Mineral oil
(medicinal grade napthene oil) did not protect against sulfur di-
oxide. When either the detergent or the dispersant component of
the "additive package" used in the motor oil were added to mineral
oil, partial protection was obtained. The addition of both of these
components to mineral oil essentially reproduced the protection re-
sulting from the motor oil. We were unable to elicit true coopera-
tion from the manufacturers due to the proprietary nature of their
product. Our attempts to negotiate with them bore more resemblance,
to a game of twenty questions than to a scientific inquiry.
Neither motor oil nor medicinal mineral oil protected animals
from the irritant effects of formaldehyde, suggesting that the
protection observed with the motor oil was specific for sulfur di-
oxide and not a general protection against irritant action per se.
One curious finding, which was not included in the thesis
write-up, was the fact that the addition of a-tocopherol to the
medicinal mineral oil would protect against the irritant action of
sulfur dioxide. We currently have no rational explanation to offer
-33-
-------
for this. It is probably worth further work to determine whether
it was a local effect in the lung or would also be observed if
vitamin E were administered by more usual routes.
VII. Preliminary work on Sulfites
The finding by the group at Brigham Young University that
in the presence of some trace metals, sulfur remains as SIV
rather than being oxidized to S points up the need for toxicolog-
ical work on sulfites and bisulfites.
Alarie e_t al. (Environ. Physiol. Biochem. 3^:182, 1973) reported
that sodium bisulfite was more irritating than sulfur dioxide and
that sodium sulfite was not irritant. They were using reduction
of respiratory frequency in mice as the criterion of irritant
response. The concentrations are all reported as S09 equivalents.
If one aerosolizes a solution of sodium bisulfite, one ends
up by generating sulfur dioxide, with no sulfur in a particle
mode. Alarie used a glycol to stabilize the aerosol phase, but
i
the paper does not define how much sulfur was in the aerosol phase
and how much was present as sulfur dioxide gas. A phone call
indicated that as they had not used a filter in their sampling
system, no effort had been made to determine this factor. We
tried their system, using the glycol. With the bisulfite, some
was indeed present on the filter, but some was also present as sul-
fur dioxide gas (unless of course it came off the filter rather
than being present in the chamber atmosphere).
-34-
-------
The system had other disadvantages, as there is evidence
that some liquid aerosols capable of dissolving sulfur dioxide will
cause potentiation of response to the gas. In order to properly
cope with this possibility, we would have had to run a separate
series with the glycol plus sulfur dioxide gas. Overall, the
system seemed too inoptimum to spend further time on at this
moment. We also had a problem with an infection in our supply
colony of guinea pigs. These factors, combined with problems of
moving myself and my lab from Harvard to M.I.T., led me to throw
in the sponge on this project for the time being. I plan to
return to the problem of S -aerosol complexes, but not via this
particular generation system.
-35-
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TECHNICAL REPORT DATA
/Please read Instructions on the reverse before completing)
1 REPORT NO.
EPA-600/1-78-021
2
4. TITLE AND SUBTITLE
PHYSIOLOGICAL RESPONSE TO ATMOSPHERIC POLLUTANTS
7. AUTHOR(S)
Mary 0. Amdur
9. PERFORMING ORGANIZATION NAME Al>
Department of Physiology
Harvard University School c
665 Huntington Avenue
Boston, Massachusetts 021 IE
•ID ADDRESS
f Public Health
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory RTP,NC
Office of Research and Development
U.S. Environmental Protection Agency
Rpsearr.h Triangle Park, NT ?7711
15. SUPPLEMENTARY NOTES
3 RECIPIENT'S ACCESSION NO.
5. REPORT DATE
March 1978
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
1AA601
11. CONTRACT/GRANT NO.
R-802030
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA 600/11
16. ABSTRACT
During the period of this grant several materials were examined as air pollutants of
interest for their irritant effects. These included sulfuric acid, a series of
inorganic sul fates, and a combination of ozone and sulfur dioxide. Some attention
was also given to the effect of various oil mists on the irritant response to sulfur
dioxide. The method used for measuring irritant response was by simultaneous
tracings of intrapleural pressure, tidal volume, and rate of flow of gas in and out
of the respiratory system. By relating the intrapleural pressure change to the
change in flow rate at points of equal lung volume, it was possible to calculate
the flow resistance; by relating pressure change to volume at the beginning and
end of inspiration, it was possible to calculate compliance. The concentrations
used in these studies are well within the range of human exposure. These studies
indicate that the irritant response previously observed at higher concentrations
of sulfuric acid is also observed at concentrations below 1 mg/m^. The failure
of alterations in resistance to return promptly to control values following
termination of exposure has been a consistent finding in the work with various
irritant aerosols. The lowest concentrations used in these studies (100 yg/nr)
are in the range of concentrations which have been reported as short-term maxima in
urban atmospheres.
17.
a. DESCRIPTORS
KEY WORDS AND DOCUMENT ANALYSIS
b. IDENTIFIERS/OPEN ENDED TERMS
air pollution
sulfuric acid
sul fates
lung
toxicity
13 DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
20. SECURITY CLASS (This page )
UNCLASSIFIED
c. COSATI Held/Group
06 F, P, T
21. NO OF PAGES
40
22. PRICE
EPA Form 2220-1 (9-73)
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