&EPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600/1-79-045
December 1973
Research and Development
Effects of
Endogenous
Ammonia on
Neutralization of
Inhaled Sulfuric
Acid Aerosols
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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 unhe'althful 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-600/1-79-045
December 1979
EFFECTS OF ENDOGENOUS AMMONIA
ON NEUTRALIZATION OF INHALED SULFURIC ACID AEROSOLS
by
Susan M. Loscutoff
Biology Department
Bat-tell e Memorial Institute
Pacific Northwest Laboratories
Richland, Washington 99352
Contract No. 68-03-2665
Project Officer
William E. Pepelko
Laboratory Studies Division
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Environmental Research Center,
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 recommen-
dation for use.
n
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FOREWORD
The U. S. Environmental Protection Agency was created in response
to increasing public concern about the dangers of pollution to the
health and welfare of the American people and their environment. The
complexities of environmental problems require an integrated program
of research and development using input from a number of disciplines.
The Health Effects Research Laboratory was established to provide
sound health effects data in support of the regulatory activities of
the EPA. An important segment of this data bank includes information
delineating the acute effects of inhaled sulfuric acid upon lung func-
tion.
This report presents the results of a study designed to determine
the effects of increased exogenous and endogenous ammonia upon neutra-
lization of inhaled sulfuric acid in the lungs of dogs. An understanding
of the neutralization process and its relationship to lung function
is important in determining the potential health effects of sulfuric
acid emitted from automobile exhaust dn7i*other pollutant sources.
R. John Garner
Director
Health Effects Research Laboratory
m
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ABSTRACT
Nine male beagles were exposed by inhalation to 0, 6, and 10.5 mg/m3
sulfuric acid aerosols with normal ammonia, increased blood ammonia, and
increased inhaled ammonia (ammonium sulfate exposures) to determine whether
the addition of ammonia affected the toxicity of sulfuric acid aerosols.
Toxicity was evaluated from measurements of pulmonary function before,
during, and after exposure. Exhaled concentrations of ammonia, sulfuric
acid, and ammonium bisulfate were measured to determine the neutralization
of inhaled aerosol by ammonia in the lung. Exhaled ammonia was measured
continuously by chemiluminescence after conversion to NO. Exhaled
sulfates were monitored with a flame photometer after selective vaporization
and absorption to separate H2S04 and NH4HS04.
Unneutralized sulfuric acid aerosol was measured in exhaled air during
exposure to 10.5 mg/m3 H2S04 with approximately 25% of the total exhaled
sulfate measured as H2S04. Comparable exposures conducted while blood
ammonia was increased tenfold by continuous infusion of ammonium acetate
showed no unneutralized H2S04 in the exhaled air.
The pulmonary function parameters most sensitive to changes caused by
aerosol exposure were closing volume, residual volume, and functional
residual capacity. H2S04 exposures with and without increased blood ammonia
resulted in nearly identical dose related changes in closing volume suggest-
ing no effect of blood ammonia on the pulmonary response to inhaled H2S04
aerosols. Functional residual capacity and residual volume, however, were
significantly lower following the 10.5 mg/m3 H2S04 exposure compared with
the 10.5 mg/m3 exposure with increased blood ammonia. In considering both
of these results, increased blood ammonia may effect the pulmonary irrita-
tion caused by inhaled sulfuric acid aerosols. However, further experiments
are required to substantiate this effect.
Five ppm NH3 and 15 mg/m3 ammonium sulfate produced similar changes in
pulmonary function. Tidal volume during exposure was increased and the
slope of phase IV of the closing volume maneuver was steeper in both groups
compared with controls. These changes were not seen during exposure to
7 mg/m3 ammonium sulfate.
These results indicate that neutralization of inhaled sulfuric acid
aerosols can be increased by increasing blood ammonia concentrations. How-
ever, the increased neutralization does not seem to markedly effect the
toxicity of inhaled acid aerosols.
iv
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This report was submitted in fulfillment of Contract No. 68-03-2665 by
Battelle Memorial Institute under sponsorship of the U.S. Environmental Pro-
tection Agency. This report covers the period from April 18, 1978 to June
18, 1979 and work was completed as of May 18, 1979.
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CONTENTS
Foreword iii
Abstract iv
Figures viii
Tables ix
Acknowledgment x
1. Introduction 1
2. Conclusions 3
3. Recommendations 4
4. Materials and Methods 5
5. Results and Discussion 18
References 36
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FIGURES
Number Page
1 Apparatus for exposing dogs to H2S04 aerosols 6
2 Sulfuric acid aerosol generation system 8
3 Connections of the exposure mask to the aerosol chamber
and exhaled sampling apparatus 10
4 Mask used for dog exposures with exhaled ammonia and
sulfate sampling lines attached 11
5 Position of sample lines for analysis of exhaled ammonia
and sulfate concentrations 12
6 Pulmonary function testing apparatus 15
7 Mask, mouthpiece, and esophageal balloon for measuring
pulmonary resistance and dynamic pulmonary compliance
in awake dogs 16
8 Record of exhaled ammonia from a dog with normal
blood ammonia 23
9 H2S04 concentration vs. closing volume; normal,
increased endogenous, and increased exogenous ammonia 31
vm
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TABLES
Number Page
1 Exposure Groups and Order of Exposures 7
2 Characteristics of the Inhaled Sulfuric Acid Aerosols .... 19
3 Analysis of Exhaled Sulfate 20
4 Blood Ammonia Concentration 22
5 Ammonia Concentration in Exhaled Air 24
6 Pulmonary Function Data: Tidal Volume Before and
During Exposure 26
7 Pulmonary Function Data: Respiration Rate Before and
During Exposure 27
8 Pulmonary Function Data: Minute Volume Before
and During Exposure 28
9 Pulmonary Function Data: Pulmonary Resistance 29
10 Pulmonary Function Data: Pulmonary Compliance 30
11 Pulmonary Function Data: Measurements From
Closing Volume Maneuvers 32
12 Pulmonary Function Data: Lung Volumes 33
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ACKNOWLEDGMENT
The author wishes to acknowledge the excellent technical assistance
of Mr. Bruce W. Kill and, who contributed greatly to the performance of
these studies.
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SECTION 1
INTRODUCTION
Ammonia has been measured as a normal constituent of expired air in
both dogs and humans (Jacquez et al., 1959; Kupprat, et al., 1976;
Larson et al., 1977). Exhaled ammonia concentrations in tracheostomized
dogs ranged from 2.3 to 6.4 nmoles/£ alveolar air, while concentrations
in humans ranged from 1.7 to 30 nmoles/2 when breathing by mouth and
0.8 to 2.7 nmoles/A when breathing by nose. Larson et al. (1977) reported
that the last airway traversed by respired gas before sampling appears to
have the greatest effect on exhaled ammonia concentrations. Higher levels
of ammonia were measured with the mouth traversed last, apparently from
high concentrations of ammonia in the mouth due to bacterial action on
saliva. Concentrations of ammonia measured in respired air from the nose
and tracheobronchial tree appeared to reflect blood ammonia concentrations.
Robin et al. (1959) reported that exhaled ammonia in dogs could be
increased to 22.4 nmoles/2 by increasing blood ammonia levels. This con-
centration represents a tenfold increase over the average concentration
in exhaled air reported by Jacquez et al. (1959).
Ammonia has been shown to neutralize sulfuric acid aerosols both in
the atmosphere and in the respiratory tracts of humans. Submicronic
aerosols measured around Chicago (Cunningham and Johnson, 1976), St. Louis
(Charlson et al., 1974) and in Western Sweden (Brosset et al., 1975) were
primarily mixtures of sulfuric acid and neutralization products of sulfuric
acid and ammonia (ammonium sulfate, (NH4)2S04, and ammonium bisulfate,
NH4HS04). Larson et al., (1977) reported neutralization of 0.6 mg/m3
inhaled sulfuric acid aerosols in humans, with an average ammonium to
sulfate molar ratio in exhaled air greater than or equal to one.
Neutralization of inhaled sulfuric acid aerosols may be important
in terms of acute and chronic toxicity. Amdur (1978) showed that the
toxicity of inhaled sulfate compounds differed, depending on the assoc-
iated cations. Ammonium sulfate aerosols were less toxic to guinea pigs
than sulfuric acid aerosols of similar mass concentrations and particle
size. In addition, Sim and Rattle (1957) found that symptoms of coughing,
lacrimation, and rhinorrhea, caused by exposure of human subjects to a
39 mg/m3 H2S04 aerosol, disappeared when ammonia was added to the aerosol.
Since sulfuric acid may be neutralized by ammonia in the respiratory tract,
it is important to determine the capacity of respiratory ammonia to neutralize
inhaled sulfuric acid aerosols and to determine whether the toxicity of
inhaled sulfuric acid aerosols is affected by neutralization with ammonia in
the respiratory tract.
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In this study, dogs were exposed to 0, 6, and 10.5 mg/m3 H2S04 aerosols
at normal ammonia levels, increased blood (endogenous) ammonia, and
increased inhaled (exogenous) ammonia. Previous studies conducted in this
laboratory on dogs exposed to 0, 1, 3.5, and 6.3 mg/m3 H2S04 showed signi-
ficant levels of unneutralized H2S04 in exhaled air only at the highest
acid level. Acute pulmonary effects were also greatest following exposure
to the 6.3 mg/m3 aerosol. In this study, ammonia was added to the blood
to determine if neutralization of H2S04 in the lung was influenced by the
blood ammonia concentration and to determine whether increased neutraliza-
tion of acid aerosols in the lung affected acute toxicity. An additional
group of exposures were conducted in which the dogs were exposed to an
aerosol of NH3 neutralized H2S04 ((NH4)2S04) to assess the relative toxi-
city of neutralized sulfate vs. sulfuric acid alone.
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SECTION 2
CONCLUSIONS
This study showed that, in dogs, increasing the ammonia concentration
in blood tenfold (from 20 to 200 umoles/2) increased the neutralization of
inhaled sulfuric acid aerosols to the point that no unneutralized H2S04 was
detected in exhaled air during exposure to 6 and 10.5 mg/m3 H2S04. Without
increased blood ammonia, unneutralized H2S04 comprised approximately 25% of
the total exhaled sulfate during exposure to 10.5 mg/m3 H2S04.
The pulmonary function parameters most sensitive to changes.caused by
aerosol exposure were closing volume, residual volume, and functional
residual capacity. H2S04 exposures with and without increased blood ammonia
resulted in nearly identical dose related changes in closing volume.
Functional residual capacity and residual volume were significantly lower
following the 10.5 mg/m3 H2S04 exposure compared with the comparable exposure
with increased blood ammonia. In considering both of these results,
increased blood ammonia may influence the pulmonary irritation caused by
inhaled sulfuric acid aerosols. However, further experiments are required
to substantiate this effect.
Five ppm NH3 and 15 mg/m3 (NH4)2S04 produced different responses from
those measured during H2S04 exposures. The increased tidal volumes during
NH3 and (NH4)2S04 exposures were probably caused by nasal irritation at
least for the 5 ppm NH3 exposure. The increased slope of Phase IV of the
closing volume maneuver could also be due to reflex effects of nasal
irritation since ammonia, due to its high solubility, probably did not
penetrate beyond the nose. Although inhaled ammonia and ammonium sulfate
were not innocuous, the dogs responses to these compounds were signifi-
cantly different from their responses to H2S04 aerosols indicating a
different site or mechanism of effect.
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SECTION 3
RECOMMENDATIONS
Experiments should be conducted to evaluate the site and time course
of neutralization of inhaled sulfuric acid aerosols by ammonia in the lung.
These studies should be conducted in experimental animals in which the
degree of neutralization from the nose or mouth to the trachea can be
evaluated at different flow rates and blood ammonia concentrations. The
relationship between blood and exhaled ammonia or ammonia within the air-
ways certainly requires further investigation. The retrograde catheter
technique developed by Macklem and Mead (1967), to measure peripheral
resistance in dogs, could also be used to evaluate the neutralization of
acid aerosols in the lung from the trachea to a 2-mm airway.
Further studies on awake dogs inhaling acid aerosols may not be par-
ticularly useful because of the very high acid concentrations required to
produce significant functional effects, and the limited information
available in evaluating mixed exhaled air. These studies showed that the
respiratory system of the dog is very effective in neutralizing inhaled
sulfuric acid aerosols. The question now is to determine where, and how
rapidly, this neutralization occurs.
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SECTION 4
MATERIALS AND METHODS
GENERAL
Nine male beagles approximately 3 years old were exposed to 0, 6, and
12 mg/m3 sulfuric acid aerosols at normal ammonia levels, increased blood
(endogenous) ammonia, and increased inhaled (exogenous) ammonia. Since the
reactions between NH3 and H2S04 are very rapid, exposures to increased
exogenous ammonia will be referred to as exposures to ammonium sulfate
((NH4)2S04) aerosol.s throughout this report. The apparatus for conducting
these exposures is shown in Figure 1. The exposure groups and the order
of exposures are shown in Table 1. All dogs were exposed to each condi-
tion at intervals of approximately one week. Parameters measured during
exposure included respiration rate, tidal volume, minute volume, and
concentrations of exhaled sulfuric acid, ammonium bisulfate, and gaseous
ammonia. Pulmonary function and blood ammonia were evaluated before and
after exposures. Data were analyzed using an unpaired t test making all
possible comparisons between exposure groups. A paired i test was used
to compare values before and after exposure. Statistical significance
was established as p < 0.05.
AEROSOL GENERATION
Sulfuric acid aerosols were generated by vaporization/condensation
of concentrated sulfuric acid using the system shown in Figure 2. High-
purity N2 was blown across a reservoir of sulfuric acid heated in a three-
necked flask. This H2S04-N2 stream was diluted with a second N2 stream
before being combined with the main air supply to the aerosol chamber. The
H2S04-air stream traveled through a tube 30-cm-long, 1.2-cm in diameter,
heated to about 220°C before reaching the exposure chamber. Mass concen-
tration of the aerosol was controlled by adjusting the N2 flow througlvthe
three-necked flask. Particle size could be adjusted by changing the
temperature of the tube leading to the exposure chamber. Air flow to
the chamber was 16 £/min and approximately 50% relative humidity. Before
mixing with the H2S04-N2 stream, chamber air was passed through a Gelman,
47-mm, type AE glass fiber filter pad. The air was not scrubbed further
since no appreciable ammonium sulfate was detected in sulfuric acid aerosols
generated in this manner. During exposures, sulfuric acid concentration
in the aerosol chamber was monitored with a Meloy SA160 flame photometric
sulfur analyzer.
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1. Aerosol Chamber
2. Aerosol Generation System
3. Dog Mask
4. H2S04 Separation Apparatus
5 & 6. Meloy SA160-Sulfur Analyzers
7. Thermo Electron Model 12A NO/NO Analyzer
8. Grass Model 7 Polygraph
9. Temperature Regulators
10. Breath Counter and Tidal Volume Meter
Figure 1. Apparatus for exposing dogs to H2S04 aerosols.
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TABLE 1.
EXPOSURE GROUPS AND ORDER OF EXPOSURES
NH3 LEVEL H2S04 CONCENTRATION ORDER CODE*
(mg/m3)
C 0 2 CC
C 6 4 C6
C 12 6 C12
EN 0 3 ENC
EN 6 1 EN6
EN 12 5 EN12
EX - 5 ppm NH3 0 7 EXC
EX 6 (NH4)2S04 9 EX6
EX 12 (NH4)2S04 8 EX12
C = Normal
EN = t Endogenous
EX = t Exogenous
*
Abbreviations for each exposure group.
Mass concentration and particle size distribution of the aerosol were
determined from filter pad and cascade impactor samples collected on days
before and after dog exposures. Air flow through the sampling apparatus
was controlled with critical orifices positioned between the apparatus
and house vacuum. Flows were checked with a bubble flow meter before
and after collecting samples. If flows differed by more than 5%,
the samples were discarded. Gelman, type AE glass fiber filters were
used in early sample collection. Because of high background sulfate
levels and interference with our method for sulfate analysis caused by
particles released from the filter pad, 0.45 urn HAWP filters (Mi111pore;
Bedford, MA) and 0.25 urn FGLP filters (Millipore; Bedford, MA) were used
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Figure 2. Sulfuric acid aerosol generation system.
for most sulfate analysis. Impactor samples were collected on a seven-
stage Mercer impactor. Untreated, 22-mm glass cover slips were used to
collect H2S04 samples. Cover slips sprayed with Dow Corning Antifoam A
to prevent particle bounce were used to collect (NH4)2S04 samples.
Sulfuric acid concentrations of impactor and filter pad samples were
determined fluorometrically, based on the decreased fluorescence of a
thorium-morin complex due to the precipitation of Th(S04)2. Filter pads
and glass cover slips were placed in vials containing 10 ml of distilled
water and allowed to stand for at least 24 hours. Two ml of the sulfate
sample, 0.5 ml of 10 ug/ml Th 4, 5 ml of 95% ethanol, and 2.5 ml of 0.002%
morin were then added consecutively to a beaker. The pH was adjusted to
2.00 + 0.05 with 1.0 N HC1. After 20 minutes, fluorescence was read on a
spectrofluorometer, Mark I, Farrand Optical Co., at 420 nm excitation and
8
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505 nm emission wavelengths. The working range of this procedure was from
0.5 to 100 |jg H2S04/ml, with a detection limit of 0.1 ug H2S04/ml. Percent
reproducibility ranged from 3 to 10%, depending on the sulfate concentra-
tion.
DOG EXPOSURES
Preceding exposures, dogs were treated with 0.75 mg/kg Acepromazine
Maleate (Ayerst) injected intravenously as a tranquilizing agent. Each dog
was then connected to the exposure apparatus for 30 minutes. He inhaled
room air for 10 minutes and chamber air for the remaining 20 minutes through
the system shown in Figure 3. The volume of air between the aerosol chamber
and the dog mask was approximately 65 ml. Air flow was measured as the
pressure drop across the venturi, and the flow signal was integrated and
summed to determine total volume. Respiration rate was counted from
pressure swings as air passed through the venturi. Mean minute volume
(inhaled volume per minute) was calculated by dividing total volume by time.
Mean tidal volume (inhaled volume per breath) was calculated by dividing
total volume by total breaths. Respiration rate, tidal volume, and minute
volume were recorded for 5-minute intervals throughout the exposures.
The mask used for dog exposures is shown in Figure 4. This mask was
designed to minimize dead space and resistance to air flow by incorporating
low-resistance, molded spiral valves (Hans Rudolph) for separating inspired
from expired air into the body of the mask. The positions of exhaled
sulfate and ammonia sample lines are also shown in Figure 4. During
exposures, the end of the dog mask and the exhaled air line were heated
to about 45°C to prevent condensation of water vapor.
EXHALED SULFATE ANALYSIS
Sulfuric acid and ammonium bisulfate concentrations in exhaled air
were measured using the apparatus shown in Figure 3. This technique,
developed by J. J. Huntzicker (1976), is based on the selective vaporization
and absorption of sulfuric acid and ammonium bisulfate at different temper-
atures. Our apparatus was designed to first remove any gaseous ammonia by
drawing the sample through a diffusion tube consisting of a 21-cm-long by
1.5-cm-diameter piece of flexible stainless steel tubing containing a
cylinder of Gelman, type AE glass fiber filter material soaked in dilute
phosphorous acid. The air sample was then divided into two streams and
drawn through two parallel heated diffusion tubes consisting of 12-cm-long
by 1.2-cm-diameter pieces of pyrex tubing containing cylinders of Gelman,
type AE glass fiber filter material soaked in dilute sodium hydroxide
solution. One tube was heated to 30°C for transmission of all exhaled
sulfate particles. The other tube was heated to 120°C for vaporization
and absorption of H2S04 and transmission of ammonium sulfate (NH4)2S04
or ammonium bisulfate (NH4HS04) particles.
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1. Aerosol Chamber
2. Directional Valve to Room Air or Aerosol Chamber
3. Venturi
4. Exposure Mask
5. Exhaled Air-Reservoir
6. Ammonia Sampling Apparatus
7. Sulfate Sampling Apparatus
Figure 3. Connections of the exposure mask to the aerosol
chamber and exhaled sampling apparatus.
10
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Figure 4. Mask used for dog exposures with exhaled ammonia and
sulfate sampling lines attached.
Uniform heating was assured by surrounding the diffusion tubes with
a 25-cm-length of 1.6-cm-diameter pyrex tubing wrapped with heater tape.
Teflon tape wrapped at both ends of the 1.2-cm-diameter diffusion tubes
assured a continuous air space between the 1.2 and 1.6-cm tubing and
secured a Chromel-Alumel (type k) thermocouple in the air space between
the tubes. Outputs of the thermocouples were displayed on a voltmeter
and temperature was controlled by adjusting the voltage supplied to the
heater tape. A third diffusion tube, heated to 220°C, was used initially
to absorb H2S04 and NH4HS04 and transmit other less volatile sulfates (e.g.,
(Na)2S04). Since no material passed through this tube, we discontinued
using the 220°C tube and concentrated on comparing total sulfate (30°C tube)
with total sulfate minus H2S04 (120°C tube).
During exposures, sulfate concentrations from the two diffusion tubes
were measured alternately for 5 minute intervals. The tube not connected
to the analyzer was exhausted at about 200 ml/min to prevent build-up of
material. Total flow through the apparatus was about 400 ml/min.
The sulfur analyzer used to measure sulfur concentrations in exhaled
air was calibrated following dog exposures. The aerosol generation system
was adjusted so that the concentration of H2S04 in the chamber, when
measured through the 30°C tube, was similar to aerosol concentration
normally measured in exhaled air. Sulfate concentrations in the aerosol
chamber were then determined from samples collected on 0.45 urn HAWP
millipore filter pads and analyzed fluorometrically.
11
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EXHALED NH3
The concentration of ammonia in exhaled air was measured with a
Thermoelectron Model 12A NO-NO analyzer containing a heated stainless
steel catalyst to convert NH3 £o NO. We modified the instrument by replac-
ing the sampling line with a 32-cm-length of P.E. 360 tubing, and by
turning the temperature controller on the NH3 converter from 750°C to
600°C. At 600°C the response time of the instrument was faster and
output higher than at 750°C. The apparatus for withdrawing ammonia samples
is shown in Figure 5. Because of the solubility of NH3 in water, great
care must be taken to prevent condensation of water vapor from exhaled
NH3 Sample
to Analyzer
NH3 Sampling
Apparatus
Figure 5. Position of sample lines for analysis of exhaled
ammonia and sulfate concentrations.
breath. To prevent water condensation, the dog mask and exhaled air lines
were heated to about 45°C. The ammonia sampling tip consisted of a 2.5-cm
by 0.5-mm I.D. glass capillary wrapped with nichrome wire, surrounded by
heat shrink tubing and heated to about 60°C. This sample tip was attached
to a 25-mm filter holder. A stream of dry, ammonia free air was introduced
into the filter holder through a hole next to the sample tip. This dry,
12
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ammonia-free air diluted the exhaled gas sample by about 2.5:1 (clean air :
exhaled gas). The combination of reduced pressure caused by the capillary
restriction and dilution of the sample with clean air was sufficient to
yield stable readings of exhaled ammonia concentrations. Clean dry air for
dilution was obtained by passing compressed air through silica gel,
chromium-trioxide-soaked glass fiber filter material to convert NO •* N02,
activated charcoal to remove N02, and a phosphorous-acid-soaked 47-mm filter
pad to remove NH3. The experimental design called for measuring exhaled
ammonia before and during exposure to H2S04. Initial experiments with
Gel man, type AE 25-mm glass fiber filter pads soaked in dilute NaOH and
placed in the filter holder showed good transmission of ammonia and removal
of acid. However, bits of filter material released from the filter pad
plugged the capillary tubes in the NH3 analyzer, interfering with the
response. In addition, although specifications on the instrument state
that sulfur compounds do not interfere with analysis, particulate sulfates
produced substantial positive readings. Because of these complicating
factors, we measured exhaled ammonia concentrations only during the
10-minute period preceding acid exposure. In analyzing ammonia concentra-
tions in exhaled breath, we found considerable background levels of NH3
and NO in the room. Exhaled ammonia concentrations reported here reflect
the difference between exhaled ammonia and background NH3 plus NO levels
in the room.
The ammonia analyzer was calibrated by diluting a standard gas
containing 5 ppm NH3 in nitrogen (Precision Gas Products, Santa Clara, CA)
with clean, dry air. This gas mixture was sampled simultaneously by the
Model 12A ammonia analyzer and by three glass bubblers (Ace Glass #752907
and 752915) filled with 10 ml of 0.01M H2S04, and arranged in parallel.
Flow rates through the bubblers were approximately 350 ml/min. Preliminary
experiments showed that all the ammonia was removed with a single bubbler.
Samples were collected for one hour after which the ammonia concentrations
in the bubblers were measured with a specific ion electrode (Orion Model
95-10).
BLOOD AMMONIA
Ammonia concentration in blood was measured using a "Blood Ammonia
Test" kit (Hyland, Costa Mesa, CA). Jugular venous blood samples were
collected before and immediately after exposure in heparinized syringes.
During exposures with increased endogenous ammonia, samples were collected
before beginning ammonia infusion, after 3 minutes of constant ammonia
infusion, and immediately following exposure before discontinuing infusion.
Samples were placed in a refrigerated centrifuge within two minutes of
collection and spun at 2000 rpm for 20 minutes. The separated plasma was
transferred to another sample tube, frozen in a dry-ice/water bath and
stored at -70°C until analyzed. Initial samples were analyzed 24 hours
after collection. However, repeated analysis of the same sample 24 hours
and 4 days after collection showed no significant difference in ammonia
concentrations. Therefore, some samples were stored for as long as 4 days
before analysis.
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INCREASED ENDOGENEOUS AMMONIA
Blood ammonia concentrations were increased by infusing a solution
of 0.35 M ammonium acetate in sterile saline. The ph of the solution was
adjusted from 7.0 to 7.4 by the addition of sodium bicarbonate. The
solution was infused through a 20-g, 1.5-inch catheter needle (Becton-
Dickinson) positioned in the radial vein using a Sage Instrument Model
01341 syringe pump. The initial infusion rate was set at approximately
5 ml Ann for one minute to produce a rapid increase in blood ammonia.
The infusion rate was then decreased to 0.6 ml/min during exposure.
The dog was allowed to stablize for about 5 minutes at the slower rate
before beginning exposure.
INCREASED EXOGENOUS AMMONIA
The concentration of exogenous ammonia selected as being most
comparable to the increased endogenous ammonia exposures was the concen-
tration of ammonia which produced the same H2S04/NH4HS04 ratio in the
aerosol chamber as measured in the expired air during exposure to
10.5 mg/m3 H2S04, plus increased endogenous ammonia. Since no unneutralized
H2S04 was measured in exhaled breath during this exposure, exogenous
ammonia (4950 ppm NH3 in nitrogen, Matheson) was added to the main exposure
air stream until no unneutralized H2S04 was measured in the aerosol chamber
at an initial acid concentration of 10.5 mg/m3. The ammonia concentration
required to produce this neutralization was approximately 5 ppm.
PULMONARY FUNCTION
Pulmonary function measurements included pulmonary resistance and
dynamic compliance on awake dogs, before and immediately (within 5 minutes)
after exposure, and closing volume, lung volumes, quasi-static compliance,
pulmonary resistance, and dynamic pulmonary compliance on penthothal-
anesthetized dogs after exposure. Apparatus used for pulmonary function
testing is shown in Figure 6. The mask, mouthpiece, and balloon for resis-
tance and compliance measurements are shown in Figure 7. The esophageal
balloon was positioned in the thoracic esophagus by advancing the balloon,
stiffened by inserting a 0.6-mm cardiac catheter guide wire (Picker,
Spokane, WA), through the mouth, down the esophagus, to the cardiac
sphincter. The guide wire was then removed and the balloon withdrawn to
a predetermined position which produced optimal respiratory oscillations
with minimal cardiac artifact. Pulmonary resistance and dynamic compliance
were determined by electronically manipulating signals of flow, volume, and
transpulmonary pressure to achieve a horizontal line on a scope displaying
transpulmonary pressure versus flow. This technique was developed by Miller
and Simmons (1960) as a modification of the subtracter method of Mead and
Whittenberger (1953). Because three of the nine dogs became very nervous
during these measurements, and the time required to calm these dogs delayed
additional function testing, measurements of awake resistance and compliance
were made on the six calmer dogs.
14
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Figure 6. Pulmonary function testing apparatus.
Anesthetized tests were conducted with the dog lying prone in a sling.
Quasi-static pressure vs. volume maneuvers, and closing volume maneuvers,
were performed by inflating the lungs with either compressed air or oxygen
and deflating with a vacuum. Flow was controlled at 75 ml/sec during both
inflation and deflation by needle valves in the respiratory lines. Flow
and volume were measured with a pneumotachometer. Nitrogen concentrations
were measured with a fast-responding nitrogen analyzer (Hewlett-Packard
Model 47302A). Transpulmonary pressure was measured as the difference
between esophageal pressure and pressure at the end of the endotracheal
tube, and transpulmonary pressure was used to control the level of inflation
and deflation. Maximum inflation (total lung capacity), was defined as
30 cm H20 transpulmonary pressure. Maximum deflation (residual volume),
was defined as minus 10 cm H20 transpulmonary pressure. At these pressures,
relays were triggered which automatically stopped inflation or deflation.
15
-------
Figure 7. Mask, mouthpiece, and esophageal balloon for measuring
pulmonary resistance and dynamic pulmonary compliance
in awake dogs.
Vital capacity maneuvers were carried out by inflating the lungs from
residual volume to total lung capacity and deflating to residual volume.
During all tests, three vital capacity maneuvers were performed consecu-
tively: during the first vital capacity, lungs were inflated with air to
establish a constant volume history; during the second, lungs were again
inflated with air, and transpulmonary pressure vs. volume curves were
recorded for measurements of quasi-static compliance and vital capacity;
during the third vital capacity, lungs were inflated with oxygen and closing
volume curves were recorded. Following the third vital capacity, lungs were
again inflated with air and the dogs were allowed to exhale passively through
the pneumotachometer to functional residual capacity (FRC). Pulmonary
resistance and dynamic compliance were measured with the dogs ventilated at
25 breaths per minute, 200 ml per breath, with a Harvard Model 613 ventilator.
The FRC was measured using the method of DuBois, et al. (1956). Dogs were
sealed in the plethysmograph, and plethysmograph pressure vs. mouth pressure
was recorded as the dog attempted to inhale against a closed airway. The
airway was closed at end exhalation by covering the airway opening. At
least three FRC maneuvers were recorded on each dog, allowing three or four
breaths between maneuvers. To facilitate analysis of pulmonary function
16
-------
data on anesthetized animals, key points on each chart were digitized, and
calculations were performed with a Hewlett-Packard 9825 calculator and
peripheral digitizer.
17
-------
SECTION 5
RESULTS AND DISCUSSIONS
Characteristics of the inhaled sulfuric acid aerosols are shown in
Table 2. Mass concentrations for exposures specified at 6 mg/m3 were
approximately 6.5 mg/m3 for the normal and increased endogenous ammonia
exposures and 7.8 mg/m3 for the ammonium sulfate exposure. The particle
size of the 6 mg/m3 aerosols was between 0.15 and 0.3 [jm with a geometric
standard deviation of 1.5. Mass concentrations for exposures specified at
12 mg/m3 were approximately 10.5 mg/m3 for the normal and increased endo-
genous ammonia exposures, and 15 mg/m3 for the ammonium sulfate exposure.
The higher levels for the 6 and 12 mg/m3 ammonium sulfate exposures resulted
from high readings for postexposure samples. The reasons for these higher
values are not known, since generation conditions were the same before,
during, and after exposure. The 7 and 15 mg/m3 ammonium sulfate aerosols
were slightly larger than H2S04 aerosols; however, all aerosols were less
than 0.5-(jm mass median aerodynamic diameter (MMAD).
EXHALED SULFATE
The sulfate concentrations measured in exhaled air are shown in
Table 3. Total exhaled sulfate concentration (H2S04 + (NH4)2S04) is
measured through the 30°C sample tube and the ammonium bisulfate (NH4HS04)
concentration is measured through the 120°C sample tube. Preliminary
experiments showed that all the exhaled sulfate was either H2S04 or an
ammonium salt of H2S04. Throughout the exposures, no significant back-
ground was measured when the dogs breathed room air. Ammonium sulfate
disassociates to NH4HS04 plus NH3 in the 120°C tube with the free NH3
combining with previously unneutralized H2S04 (Huntzicker, 1976).
Therefore, a greater sulfate concentration measured through the 30°C tube,
compared with the 120°C tube, indicates a molar ratio of S04 to NH3 in
exhaled air of greater than one.
The retention of inhaled aerosols measured in these studies was greater
than 95% and is probably unrealistically high. Stauffer (1974) calculated a
maximum deposition of 80% for 0.035 (jm particles and results of the Task
Group on Lung Dynamics (1966) predict 45% deposition for inert, non-hygro-
scopic aerosols of 0.2-|jm MMAD, with a geometric standard deviation of 2.
Because of the hygroscopic nature of H2S04 and its tendency to conglomerate,
retentions of greater than 45% are expected. Amdur, et al. (1952), measured
a 77% average retention in 22 human subjects inhaling l-pm H2S04 aerosols.
In addition, Swift et al. (1977) measured greater depositions of inhaled
aerosols in dogs compared with humans under comparable conditions. Based
18
-------
TABLE 2.
CHARACTERISTICS OF THE INHALED SULFURIC ACID AEROSOLS
Mass Cone.
Exposure Filter
Group* Type**
C6
C12
EN6
EN12
EX6
EX12
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
Pre
Post
GF
MP
FP
GF
GF
FP
MP
MP
MP
MP
MP
(mg/m3)
X SD
6.54
9.5
11.9
6.52
6.54
10.4
9.5
6.78
8.9
11.3
18.9
0.35
0.8
2.4
0.33
0.35
0.8
0.8
1.2
1.8
2.9
1.3
Particle Size
N
3
3
2
2
3
3
3
4
6
6
6
MMAD*** (pm) GSD****
0.19
0.22
0.21
0.22
0.16
0.19
0.19
0.21
0.29
0.41
0.39
1.4
1.3
1.7
1.7
1.8
1.4
1.7
1.7
1.5
2.0
1.5
*First Symbol - NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol - Inhaled H2S04 Concentration
6=6 mg/m3
12 = 12 mg/m3
**GF Gelman Type AE Glass Fiber
FP Millipore 0.2umiFGLP (Fluoropore)
MP Millipore 0.45um HAWP (Millipore)
***MMAD - Mass Median Aerodynamic Diameter
****GSD - Geometric Standard Deviation
19
-------
TABLE 3.
ANALYSIS OF EXHALED SULFATE
(nmoles S04/£ Exhaled Air)
Exposure Group*
(Inhaled 30°C Tube 120°C Tube
Concentration)
C6
(66 nmoles/£)
C12
(109 nmoles/2)
EN6
(66 nmoles/£)
EN12
(101 nmoles/A)
EX6
(80 nmoles/A)
EX12
(154 nmoles/A)
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
10
2.8
0.3
6
2.9
0.2
9
2.4
0.2
8
2.8
0.3
9
2.3
0.4
9
3.9
0.4
9
Min
6
6t
g
D
g
\J
6tt
d-5)tt
20
2.
0.
6
3.
0.
9
2.
0.
5
2.
0.
9
2.
0.
9
4.
0.
9
8
2
0
2
4
2
9
2
3
4
3
5
Min
6
6t
g
\J
g
LI
6tt
(l-5)tt
15
2.
0.
6
2.
0.
9
2.
0.
8
2.
0.
9
2.
0.
9
5.
0.
9
.
6
2
2
2
4
2
7
2
6
4
2
6
Min
6
6t
g
U
g
\j
6tt
(l-5)tf
25
2.5
0.2
6
2.3
0.2
9
2.5
0.2
5
2.8
0.2
9
2.9
0.6
9
5.5
0.6
9
Min
6
6t
g
\J
g
\J
6tt
(l-5)tt
*First Symbol - NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol - Inhaled H2S04 Concentration
6=6 mg/m3
12 = 12 mg/m3
tOutput of 30°C tube > 120°C tube (p < 0.05; paired r test)
ttOutput of 30°C tube < 120°C tube (p < 0.05; paired T test)
Superscripts indicate groups which are significantly different (p < 0.05;
unpaired t test).
1 = C6 4 = EN12
2 = C12 5 = EX6
3 = EN6 6 = EX12
20
-------
on these factors, deposition of between 80 and 90% of the inhaled aerosol
in the respiratory tract is possible.
During exposure to 15 mg/m3 ammonium sulfate, the total exhaled
sulfate concentration was higher (p < 0.05, unpaired t test) than in all
other groups. The lack of any significant difference between exhaled
sulfate concentrations during exposures to 6 and 10.5 mg/m3 H2S04 may be
caused by the very high retention of inhaled aerosol masking small differ-
ences in the exhaled concentration.
The presence of unneutralized sulfuric acid in the exhaled air was
determined by comparing the sulfate concentrations measured through the 30°
and 120°C sample tubes, with a higher output from the 30°C tube indicating
unneutralized H2S04. Significant (p < 0.05, paired i test) levels of unneu-
tralized H2S04 were measured only during exposure to 10.5 mg/m3 H2S04. The
unneutralized acid comprised about 25% of the total exhaled sulfate concen-
tration. During ammonium acetate infusions, no unneutralized H2S04 was
measured in exhaled air.
In our previous study on the neutralization of inhaled sulfuric acid,
we found significant unneutralized acid in the exhaled air during exposure
to 6.3 mg/m3 H2S04. Although in the study reported here the outputs of the
30°C tube tended to be higher than the 120°C tube during exposure to
6 mg/m3 H2S04, the difference between the two sample tubes was not signifi-
cant, based on a paired t test. Two factors may contribute to the
difference between these studies. In the first study, apparatus dead
space was greater, contributing a greater concentration of uninhaled aerosol
to exhaled air. In addition, dogs were not tranquilized in the first study;
their more rapid, shallow breathing (caused by general excitement and by the
irritating effects of the acid) could decrease the time for neutralization
in the respiratory tract. Although the reaction between NH3 and H2S04 is
practically instantaneous, the ammonia concentration in the lung is so low
that ammonia must continually diffuse from the respiratory surface into the
airways to produce any substantial neutralization. With the increased
respiration rates, less time would be available for diffusion.
Table 3 shows that during ammonium sulfate exposures, the sulfate
concentration measured through the 120°C tube was greater than through the
30°C tube (p < 0.05, paired t test). The reason for this difference is not
known. When ammonium sulfate aerosols were sampled from the aerosol
chamber, the output of both tubes was the same.
BLOOD AMMONIA
Blood ammonia concentrations measured before and after exposure are
shown in Table 4 and averaged 21 umoles/A pre-exposure. Minor differences
were measured among several of the pre-exposure groups reflecting day-to-day
variations. However, these differences were small compared with the tenfold
increase in bipod ammonia during ammonium acetate infusion. The infusion
schedule used in this study produced a reproducible increase in blood
ammonia which did not change significantly during the 30 minute exposure.
21
-------
TABLE 4.
BLOOD AMMONIA CONCENTRATION (u MOLES/A BLOOD)
Significant**
Exposure
Group*
CC
C6
C12
ENC
EN6
EN12
EXC
EX6
EX12
t
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
Pre-Infusion
—
--
--
—
—
—
--
--
--
25
1.6
9
23
1.5
9
19
2.1
9
—
--
—
—
—
—
—
—
--
Pre-Exposure
23
1.8
9
21
1.6
9
19
1.7
9
208
26
8
189
39
9
214
24
9
21
1.4
9
16
1.0
9
16
0.5
9
Postexposure
27
2.2
9
27
1.6
5
24
2.3
6
240
22
8
198
30
9
204
16
9
26
2.1
9
22
2.6
9
20
1.3
9
Difference
Pre-Post
ND
p < 0.05
ND
ND
ND
ND
p < 0.05
p < 0.05
ND
^First Symbol - NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol - Inhaled H2S04 Concentration
C - 0 mg/m3
6=6 mg/m3
12 = 12 mg/m3
**Paired T test comparing pre- and postexposure values for each
ND = Not significantly different
dog.
22
-------
During exposure to 6 ntg/m3 H2S04) 5 ppm NH3, and 6 mg/m3 (NH4)2S04
blood ammonia was significantly higher (p < 0.05, paired t test) post-
exposure than pre-exposure. The increased ammonia postexposure in dogs
exposed to 5 ppm NH3 and 6 mg/m3 (NH4)2S04 may be the result of inhalation
of ammonia. However, factors responsible for the increased ammonia
following exposure to 6 mg/m3 H2S04 are not known. This increase could
be related to the exposure since ammonia also tended to increase following
exposure to air only and 10.5 mg/m3 H2S04.
EXHALED AMMONIA
Ammonia concentrations in exhaled air were measured during the
10 minute period before aerosol exposure and mean values for the nine expo-
sures are shown in Table 5. Figure 8 is a record of ammonia exhaled from a
dog preceding exposure to 6 mg/m3 H2S04. This record shows that during the
10 minute sampling time, the measured concentration of exhaled ammonia
1.7 n MOLES
EXHALED AIR
ROOM AIR
Figure 8. Record of exhaled ammonia from a dog
with normal blood ammonia.
increased to a relatively constant level which was about twice the back-
ground concentration measured with the sample tip drawing in room air.
The background NH3 + NO level in the room was equivalent to about
1.2 nmoles NH3/£. Ammonia concentrations after subtracting background
and at normal levels of blood ammonia were approximately 2.0 nmoles/£.
These values agree well with values of approximately 1.5 nmoles/£
measured by Larson et al. (1977), in humans exhaling through the nose
and 2.5 nmoles/£ measured by Larson et al. (1979) in trachea! air of
dogs. During ammonia infusions, exhaled ammonia was generally higher
than values with normal blood ammonia although the increase was not as
great as might be expected from the tenfold increase in blood ammonia.
23
-------
TABLE 5.
AMMONIA CONCENTRATION IN EXHALED AIR (nmoles/£)
Exposure
Group* _X SE
CC 2.01 0.31 8
C6 1.25 0.08 4
C12 -
ENC 1.81** 0.18 7
ENS 2.11** 0.37 7
EN12 2.90** 0.30 9
EXC 2.57 0.17 6
EX6 1.43 0.13 8
EX12 1.96 0.30 8
*First Symbol - NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol - Inhaled H2S04 Concentration
C = 0 mg/m3
6=6 mg/m3
12 = 12 mg/m3
**Exhaled ammonia did not increase significantly during ammonia infusions
which increased blood ammonia tenfold.
The reason for the insignificant change in exhaled ammonia with a
tenfold increase in blood ammonia is not known. Robin et al. (1959)
measured exhaled ammonia concentrations of 22 nmoles/£ (approximately ten
times the values reported here) in dogs during ammonium acetate infusions
which increased blood ammonia to 500 umoles/A. This work suggests that
the increase in exhaled ammonia is relatively less than the increase in
blood ammonia. However, the relationship between blood and exhaled ammonia
requires further investigation. Because of the complex chemistry of blood
and the extremely low concentrations of ammonia present in blood, there may
not be a one-to-one relationship between blood and exhaled ammonia.
24
-------
Pulmonary Function
Breathing patterns (tidal volumes, respiration rates, and minute
volumes) were measured before and during exposures and the results are
shown in Tables 6-8. After breathing through the exposure apparatus for
5 minutes, respiration was fairly well stabilized and breathing patterns
during the last 5 minutes pre-exposure are reported here. Values during
exposure were averaged for the first 10 minutes and last 10 minutes of
exposure. Most of the significant differences among exposure groups were
measured as differences in tidal volume, Table 6.
Breathing patterns pre-exposure were not significantly different
among any of the exposure groups. Tidal volumes during exposure to
6 mg/m3 H2S04 plus increased endogenous ammonia (EN6) were significantly
lower (p < 0.05, unpaired t test) than during several other exposures.
EN6 was the first exposure and although the dogs were tranquilized and had
previously been conditioned to breath through the exposure apparatus, they
seemed more apprehensive than during later exposures. This anxiety was
probably responsible for the lower tidal volumes.
Tidal volumes during exposure to 5 ppm NH3 (20-30 min) were signifi-
cantly higher than during the control exposure (CC). Five ppm ammonia is
at the odor threshold of ammonia for humans (Mangold, 1971). With the dogs
more sensitive olfactory system, this concentration of ammonia was probably
readily detected causing increased tidal volumes. Ammonium sulfate is
generally considered to be much less irritating to the respiratory tract
than sulfuric acid (Amdur, 1970; Sim and Rattle, 1957). However, tidal
volumes showed similar changes during exposure to 10.5 mg/m3 H2S04 and
15 mg/m3 (NH4)2S04. During both exposures, tidal volumes tended to increase
while tidal volumes during the control exposure decreased significantly
compared with pre-exposure values (p < 0.05; paired t test). The signifi-
cant increase during exposure to 15 mg/m3 (NH4)2S04 compared with control
but not during exposure to 10.5 mg/m3 H2S04 compared with control is
probably due to the higher concentration of the ammonium sulfate aerosols.
These results indicate that ammonium sulfate and sulfuric acid aerosols
have similar effects on breathing pattern under these exposure conditions.
The mechanical properties of the lungs, shown in Tables 9 and 10, were
not affected by acid exposure. A decrease in dynamic compliance following
exposure to 10.5 mg/m3 H2S04 plius increased endogenous ammonia compared with
increased endogenous ammonia alone was the only significant change asso-
ciated with aerosol exposure. Significant day-to-day variations were
detected in measurements of awake resistance and compliance. However,
these changes were not related to exposure and to avoid confusion they
were not marked on the tables.
25
-------
TABLE 6.
PULMONARY FUNCTION DATA:
PRE-EXPOSURE
TIDAL VOLUME BEFORE AND DURING EXPOSURE
DURING EXPOSURE
Exposure
Group*
CC X
SE
N
C6 X
SE
N
C12 X
SE
N
ENC X
SE
N
EN6 X
SE
N
EN12 X
SE
N
EXC X
SE
N
EX6 X
SE
N
EX12 X
SE
N
*First Symbol
5-10 Min
Tidal
Volume
(ml)
t
165T
7.6
8
160
13.1
6
159
10.4
9
174
7.9
8
153
7.3
5
162
11.5
8
174
13.8
9
160
13.0
9
174
15.7
8
- NH3
10-20 Min
Tidal
Volume
(ml)
1539
7.5
8
152
18.5
6
167
13.1
9
1665
6.2
8
1384,6,7,9
4.5
5
1695
9.5
9
175
12.3
9
159
14.8
9
1921'5
14.4
9
20-30 Min
Tidal
Volume
(ml)
1467'9'*
7.9
9
139
21.8
5
1725
12.5
9
1685
7.4
8
1323,4,6,7,9
6.8
4
1615
8.7
9
1871'5
14.1
9
163
16.7
9
1851'5
15.7
9
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol Inhaled H2S04 Concentration
C = 0 mg/m3
6=6 mg/m3
12 = 12 mg/m3
tSignificant Difference Pre vs. During (p < 0.05; paired
Statistical Significance: Superscripts indicate groups
significantly different (p < 0.05; unpaired i test)
T test)
which are
1 = CC
2 = C6
3 = C12
4 = ENC
5 = EN6
6 = EN12
7 = EXC
8 = EX6
9 = EX12
26
-------
TABLE 7.
PULMONARY FUNCTION DATA: RESPIRATION RATE BEFORE AND DURING EXPOSURE
Exposure
Group*
CC X
SE
N
C6 X
SE
N
C12 X
SE
N
ENC X
SE
N
EN6 X
SE
N
EN12 X
SE
N
EXC X
SE
N
EX6 X
SE
N
EX12 X
SE
N
Pre-Exposure
5-10 Min
Respiration
Rate
(BPM)**
17.1
1.9
9
17.4
1.7
6
15.8
1.6
9
16.6
1.8
8
16.3
2.0
5
15.7
1.5
8
15.8
2.7
9
17.1
3.5
9
17.9,
2.2
8
DURING
10-20 Min
Respiration
Rate
(BPM)**
16.1
1.3
9
17.1
1.8
6
15.5
1.2
9
17.8
1.8
8
18.4
1.9
5
15.9
1.2
9
14.8
2.3
9
15.7
2.5
9
15.1
1.8
9
EXPOSURE
20-30 Min
Respiration
Rate
(BPM)**
17.6
1.4
9
15.5
1.7
5
14.4
1.3
9
18.0
2.0
8
16.0
1.2
4
15.3
0.9
9
14.4
1.9
9
14.9
2.8
8
15.6
1.7
9
*First Symbol - NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol - Inhaled H2S04 Concentration
C = 0 mg/m3
6=6 mg/m3
12 = 12 mg/m2
**BPM = Breaths/minute
27
-------
TABLE 8.
PULMONARY FUNCTION DATA: MINUTE VOLUME BEFORE AND DURING EXPOSURE
Pre-Exposure
5-10 Min
DURING EXPOSURE
Exposure
Group*
CC X
SE
N
C6 X
SE
N
C12 X
SE
N
ENC X
SE
N
EN6 X
SE
N
EN12 X
SE
N
EXC X
SE
N
EX6 X
SE
N
EX12 X
SE
N
Minute
Volume
(ml/min)
2860
250
8
2843
446
6
2392
124
9
2867
292
8
2142
595
5
2421
125
8
2526
227
9
2487
275
9
2920
229
8
10-20 Min
Minute
Volume
(ml/min)
2519
171
8
2673
485
6
2464
121
9
2951
312
8
2515
250
5
2611
155
9
2393
167
9
2264
177
9
2801
186
9
20-30 Min
Minute
Volume
(ml/min)
2531
220
9
2185
493
5
2391
179
9
29695'8
281
8
20994'9
91
4
2444
155
9
2491
159
9
21 604
193
8
27655
218
9
*First Symbol -
NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Statistical Significance: Superscripts indicate groups which are
significantly different (p < 0.05; unpaired T test)
1 = CC 3 = C12 5 = EN6 7 = EXC 9 = EX12
2 = C6 4 = ENC 6 = EN12 8 = EX6
Second Symbol -
Inhaled H2S04 Concentration
C = 0 mg/m3
6=6 mg/m3
12 = 12 mg/m3
28
-------
TABLE 9.
PULMONARY FUNCTION DATA: PULMONARY RESISTANCE
Exposure
Group
CC
C6
C12
ENC
EN6
EN12
EXC
EX6
EX12
*
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
Pre-Exposure
31.0
3.5
8
23.4
4.5
4
22.4
2.5
5
28.5
3.3
7
—
—
—
19.4
3.7
5
22.4
1.9
6
27.3
4.6
6
29.3
4.7
6
Pulmonary Resistance
(cm H20/£/sec)
Postexposure
28.7
3.6
8
34.4
10.4
3
29.8
5.0
5
28.0
2.1
6
22.7
2.3
6
30.0
4.1
5
24.7
2.9
4
30.4
2.4
6
Anesthetized
2.5
0.1
9
2.6
0.1
6
2.6
0.2
9
2.3
0.2
8
2.6
0.2
7
2.5
0.2
9
2.5
0.1
9
2.5
0.1
9
2.5
0.1
9
First Symbol -
NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Second Symbol -
Inhaled H2S04 Concentration
C = 0 mg/m3
6=0 mg/m3
12 = 12 mg/m3
29
-------
TABLE 10.
PULMONARY FUNCTION DATA: PULMONARY COMPLIANCE
Exposure
Group*
CC
C6
C12
ENC
EN6
EN12
EXC
EX6
EX12
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
Dynamic Pulmonary
Compliance (ml/cm H20)
Pre-Exposure
49.8
5.8
8
44.2
10.5
4
59.7
6.6
5
37.6
3.6
7
—
—
—
62.1
3.3
5
69.1
9.5
6
42.9
8.9
6
46.4
9.2
6
Postexposure Anesthetized
45.0
3.7
8
56.8
20.6
3
47.6
3.2
5
46.8
8.4
6
—
—
—
52.6
7.0
6
60.1
10.5
6
64.1
18.8
4
43.8
8.7
6
* First Symbol -
NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
87.9
6.1
9
85.3
8.3
6
83.7
4.4
9
98. 56
7.3
8
116
18.3
7
78. 84
5.8
9
88.5
7.8
9
89.6
7.3
9
96.3
7.8
9
Second Symbol -
Inhaled H2S04 Ci
C = 0 mg/m3
6 = 6 mg/m3
12 = 12 mg/m3
Quasi-Static
Pulmonary
Compliance
(ml/cm HgO)
107
6.8
9
98
5.6
6
109
8.3
9
107
7.8
8
120
12.0
8
98
7.1
9
101
6.8
9
98
6.3
9
107
7.5
9
Concentration
Statistical Significance: Superscripts indicate groups which are
significantly different (p < 0.05; unpaired i test)
1 = CC 3 = C12 5 = EN6 7 = EXC 9 = EX12
2 = C6 4 = ENC 6 = EN12 8 = EX6
30
-------
Differences were also measured between values for awake and anesthe-
tized resistance and compliance. These differences were expected due to
the effects of anesthesia and endotracheal intubation. They do not indicate
any effects of aerosol exposure.
Measurements of closing volume and lung volumes (Tables 11 and 12)
appeared to be most useful in comparing the toxicity of different exposures.
H2S04 exposures with and without increased blood ammonia resulted in nearly
identical dose related changes in closing volume (Figure 9). Closing
volumes measured following the 10.5 mg/m3 exposures with and without
increased blood ammonia were both significantly higher than following
control and 5 ppm NH3 exposures. If changes in closing volume are
related to an acute toxic effect of aerosol exposure then the similar
dose response relationships between normal and increased blood ammonia
suggest little effect of blood ammonia on the response of the lung to
inhaled sulfuric acid aerosols.
100
75
o
o
CO
Q
50
25
NORMAL NH,
*VS'
4 EN
TEX
NH,
NH,
VALUES SIGNIFICANTLY
DIFFERENTP<0.05
(UNPAIRED t TEST)
X±SE
12
18
INHALED
(MG/M'
Figure 9. H2S04 concentration vs. closing volume; normal, increased
endogenous, and increased exogenous ammonia.
31
-------
CO
IM
TABLE 11.
PULMONARY FUNCTION DATA: MEASUREMENTS FROM CLOSING VOLUME MANEUVERS
Exposure Anatomic Dead M-3 M-4
Group* Space (ml) (X N,/«) (X N,/£)
CC
C6
C12
ENC
EN6
EN12
EXC
EX6
EX12
* First
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
X
SE
N
Symbol
125
7
9
141
8
6
124
7
9
128
8
8
137
9
8
129
8
9
125
7
9
121
6
9
127
6
9
- NH3
0.62
0.05
9
0.579
0.05
6
0.67
0.04
9
0.64
0.04
8
0.70
0.04
8
0.64
0.03
9
0.70
0.09
9
0.79
0.08
9
•)
o.?r
0.03
9
7 q
4.71/>9
0.34
9
4.97
1.28
5
4.107'9
0.24
9
5.12
0.63
7
4.247'9
0.38
8
4.047'9
0.31
9
6.731'3'5'6
0.41
8
5.07
0.51
9
1 0 C C
6 48l,J,b,b
0.66
9
Closing
Volume
(ml)
3 f,
60. 7J'6
4.7
9
61.8
16.7
6
84. O1'7
8.0
9
64.0
12.6
8
70.3
8.6
8
85.11'7
8.8
9
53.63'6
8.4
9
74.6
5.7
9
67.4
7.4
9
Closing
Closing Volume/ Capacity
Vital Capacity (%) (ml)
C
5.6b
0.5
9
5.2
1.4
6
7.37
0.7
9
5.8
1.1
8
6.0
0.6
8
7.51'
0.6
9
4.83>
0.7
9
7.07
0.6
9
6.1
0.7
9
Second Symbol
443
58
8
448
41
6
3586
26
9
445
34
8
407
42
6
7 4613'7
31
9
6,8 3746
24
9
419
22
9
389
22
9
- Inhaled H2S04
Closing
Capacity/
Total Lung
Capacity (%)
30
2.7
8
29
1.6
6
256,8
1.4
9
30
2.2
8
27
1.8
6
313
2.0
9
268
0.8
9
293,7
1.1
9
27
0.9
9
Concentration
C = 0 mg/m3
6=6 mg/m3
12 = 12 mg/m3
C = Normal
EN = t Endogenous
EX = t Exogenous
Statistical Significance: Superscripts indicate groups which are significantly different (p < 0.05;
unpaired T test)
1 = CC 2 = C6 3 = C12 4 = ENC 5 = EN6 6 = EN12
7 = EXC 8 = EX6 9 = EX12
-------
TABLE 12.
PULMONARY FUNCTION DATA: LUNG VOLUMES
Vital
Exposure Residual Functional Residual Capacity
Group* Volume (ml)
CC X 383
SE 58
N 8
C6 X 3863
SE 33
N 6
C12 X 2752'4'6'8
SE 22
N 9
ENC X 3813
SE 31
N 8
EN6 X 334
SE 38
N 6
EN12 X 3763
SE 27
N 9
EXC X 320
SE 25
N 9
EX6 X 3443
SE 22
N 9
EX12 X 321
SE 19
N 9
* First Symbol -
NH3
C = Normal
EN = t Endogenous
EX = t Exogenous
Statistical Significance:
significantly different (p
1 = CC 3 = C12
2 = C6 4 = ENC
Capacity (ml)
589
66
8
600
48
6
5186
31
9
604
32
8
546
48
6
6303
40
9
559
41
9
575
33
9
547
36
9
(ml)
1100
43
9
1167
41
6
1140
46
9
1105
52
8
1143
53
8
1137
54
9
1117
45
9
1088
46
9
1114
46
9
Total Lung
Capacity (ml)
1455
81
8
1553
64
6
1415
58
9
1486
50
8
1476
84
6
1513
53
9
1438
65
9
1433
61
9
1435
61
9
Second Symbol -
Superscripts
< 0.05; unpai
5 = EN6
6 = EN12
Inhaled H2S04
C = 0 mg/m3
6=6 mg/m3
12 = 12 mg/m3
indicate groups
red t test)
7 = EXC 9
8 = EX6
Concentration
which are
= EX12
33
-------
Although closing volumes were increased by the same amount following
the 10.5 mg/m3 H2S04 exposures with normal and increased blood ammonia,
closing capacity, functional residual capacity, and residual volume were
significantly lower following exposures with normal ammonia (C12) compared
with increased blood ammonia (EN12). The lower closing capacity indicates
that the airways in the lung began to close at lower lung volumes during
the C12 than during the EN12 exposures. Since closing volumes in the two
groups were the same, the lung was actually drawn to a lower residual
volume following the C12 compared with the EN12 exposure (Table 12).
Lower values of functional residual capacity following the C12 exposure
suggest that the elastic recoil of the lung was increased compared with
the EN12 exposure.
Based on these measurements of lung volume, we can predict that inhala-
tion of 10.5 mg/m3 H2S04 at approximately 2-um MMAD causes a greater
narrowing of small airways than comparable aerosol exposures with blood
ammonia increased tenfold. This narrowing is not great enough to produce
significant changes in dynamic or quasi-static compliance but.it is suffi-
cient to increase the elastic recoil of the lung resulting in decreased
values of residual volume and functional residual capacity. The increased
closing volumes measured in both 10.5 mg/m3 H2S04 exposures (C12 and EN12)
although of a similar magnitude, do not necessarily indicate similar effects
of exposure since the airways actually began closing at lower lung volumes
during the C12 exposure.
Because of the different response patterns seen between measurements
of closing volume and measurements of other lung volumes no conclusions can
be made on the effect of increased blood ammonia on the pulmonary response
to inhaled H2S04 aerosols.
Exposure to 5 ppm NH3 and 15 mg/m3 (NH4)2S04 produced similar effects
on pulmonary function while exposure to 6 mg/m3 (NH4)2S04 had little effect.
During 5 ppm NH3 and 15 mg/m3(NH4)2S04 exposures, the slope of Phase IV of
the closing volume maneuver (Table 11, measured as the slope of the volume
vs. N2 curve from closing volume to residual volume) was significantly
steeper than following control exposures. This increased slope may reflect
an increased gradient in N2 concentration in the upper (independent) lung
regions after airways in the lower (more dependent) regions have closed.
Tidal volumes during .exposure (Table 6) were also increased in both of
these exposure groups compared with control indicating two parameters
affected in a similar manner by exposure to 5 ppm NH3 and 15 mg/m3
(NH4)2S04.
Similar response to inhaled NH3 and (NH4)2S04 would not be predicted
from the expected deposition patterns of these two compounds. Gaseous
ammonia is highly soluble and would be expected to be completely absorbed
in the nose similar to the absorption of S02 measured by Frank et al.
(1969). Ammonium sulfate aerosols of 0.4 urn MMAD would be expected to
34
-------
deposit in the deep lung with perhaps only 10 percent deposition in the
nose (Task Group on Lung Dynamics, 1966). However, results of these
exposures suggest that the fractional deposition of (NH4)2S04 in the
nose produces irritation similar to that caused by inhalation of
5 ppm NH3.
Although responses to inhaled sulfuric acid and ammonium sulfate
aerosols were not the same, dogs were affected by 15 mg/m3 (NH4)2S04.
Changes caused by ammonium sulfate exposure suggested nasal irritation
similar to effects seen during inhalation of 5 ppm NH3. Changes caused
by H2S04 exposures (e.g., closing volume and lung volume changes) suggested
effects on small airways.
35
-------
REFERENCES
Amdur, M. 0., L. Silverman, and P. Drinker. 1952. Inhalation of Sulfuric
Acid by Human Subjects. A.M.A. Arch. Ind. Hyg. Occup. Med. 6:305-313.
Amdur, M. 0., J. Bayles, V. Ugro and D. W. Underhill. 1978. Comparative
Irritant Potency of Sulfate Salts. Environ. Res. 16:1-8.
Brosset, C., K. Andreasson, and M. Perm. 1975. The Nature and Possible
Origin of Acid Particles Observed at the Swedish West Coast. Atmos.
Environ. 9:631-642.
Charlson, R. J., A. H. Vanderpol, D^ S. Covert, A. P. Waggoner, and N. C.
Ahlquist. 1974. H2S04/(NH4)2S04 Background Aerosol: Optical Detection
in St. Louis Region. Atmos. Environ. 8:1257-1267.
Cunningham, P. T. and S. A. Johnson. 1976. Spectroscopic Observation of
Acid Sulfate in Amospheric Particulate Samples. Science. 191:77-79.
DuBois, A. B., S. Y. Botelho, G. N. Bedell, R. Marshall and J. H. Comroe,
Jr. 1956. A Rapid Plethysmographic Method for Measuring Thoracic Gas
Volume: A Comparison with a Nitrogen Washout Method for Measuring
Functional Residual Capacity in Normal Subjects. J. Clin. Invest.
35:322-326.
Frank, R., R. E. Yoder, J. D. Brain and E. Yokoyama. 1969. S02 (35S
Labelled) Absorption by the Nose and Mouth Under Conditions of Varying
Concentration and Flow. Arch. Environ. Health. 18:315-322.
Huntzicker, J. J., L. M. Isabelle and J. G. Watson. 1976. The Continuous
Measurement of Particulate Sulfur Compounds by Flame Photometry.
pp. 76-31.3, In the Abstracts of the 69th Annual Meeting of the Air
Pollution Control Assoc. Portland, OR.
Jacquez, J. A., J. W. Poppell, and R. Jeltsch. 1959. Partial Pressure of
Ammonia in Alveolar Air. Science. 129:269-270.
Kupprat, L, R. E. Johnson, and B. A. Hertig. 1976. Ammonia: A Normal
Constituent of Expired Air During Rest and Exercise. Fed. Proc. 35:478.
(abstract).
Larson, T. V., D. S. Covert, R. Frank, and R. J. Charlson. 1977. Ammonia
in Human Airways: Neutralization of Inspired Acid Sulfate Aerosols.
Science. 197:161-163.
Larson, T. V , R. Frank, D. S. Covert, D. Holub and M. Morgan. 1979.
Measurements of Respiratory Ammonia and the Chemical Neutralization of
Inhaled Sulfuric Acid Aerosols in Anesthetized Dogs. Am. Rev. Resp.
Dis. 119:226. (abstract).
-------
Macklem, P. T. and J. Mead. 1967. Resistance of Central and Peripheral
Airways Measured by a Retrograde Catheter. J. Appl. Physiol. 22:395-401.
Mangold, C. A. Investigation of Occupational Exposure to Ammonia. Record
of Industrial Hygiene Division Investigation, Puget Sound Naval Shipyard,
November 29, 1971.
Mead, J. and J. L. Whittenberger. 1953. Physical Properties of Human
Lungs Measured During Spontaneous Respiration. J. Appl. Physiol.
5:779-796.
Miller, J. H. and D. H. Simmons. 1960. Rapid Determination of Dynamic
Pulmonary Compliance and Resistance. J. Appl. Physiol. 15:967-974.
Robin, E. D., D. M. Travis, P. A. Bromberg, C. E. Forkner, and J. M.
Tyler. 1958. Ammonia Excretion by Mammalian Lung. Science.
129:270-271.
Sim, V. and R. Rattle. 1957. Effect of Possible Smog Irritants on
Human Subjects. J. Amer. Med. Assoc. 165:1908-1913.
Stauffer, D. 1974. On the Theory of Lung Deposition of Very Small H20-
H2S04 Aerosols. Health Phys. 26:365-366.
Swift, D. L., J. A. C. Cobb and J. C. Smith. 1977. Aerosol Deposition in
the Dog Respiratory Tract. Inhaled Particles, IV, Part 1. W. H. Walton,
editor. Pergamon Press, 237-245.
Task Group on Lung Dynamics. 1966. Deposition and Retention Models for
Internal Dosimetry of the Human Respiratory Tract. Health Phys. 12:173-207.
37
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-79-045
2.
4, TITLE AND SUBTITLE
Effects of Endogenous Ammonia on Neutralization
of Inhaled Sulfuric Acid Aerosols
7. AUTHOR(S)
Susan M. Loscutoff
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle Memorial Institute
Pacific Northwest Laboratories
Richland, Washington 99352
12. SPONSORING AGENCY NAME AND ADDRESS
Heal til Effects Research Lab. - Cinn, OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
December 1979 issuing date
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
10. PROGRAM ELEMENT NO.
IAA601
11. CONTRACT/GRANT NO.
EPA 68-03-2665
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Nine male beagle dogs were exposed by inhalation to 0, 6 and 10.5 mg/m3
sulfuric acid aerosols with normal ammonia, increased blood ammonia, and
increased inhaled ammonia to determine whether the addition of ammonia
affected the toxicity of sulfuric acid aerosols. Exhaled concentrations of
ammonia, sulfuric acid and ammonium bisulfate were measured to determine the
neutralization of inhaled aerosol by ammonia in the lung. Unneutralized
sulfuric acid aerosol was detected in exhaled air only during exposure to
10.5 mg/m3 H2S04 with approximately 25 percent of the total exhaled sulfate
measured as H2SO.. No unneutralized H2S04 was measured after a tenfold
increase in blood ammonia by ammonium acetate infusion. Increased blood
ammonia resulted in a slightly decreased irritant effect of 10.5 mg/m3
H2SC>4 in the lung using residual volume and functional residual capacity as
endpoints. The results indicate that neutralization of inhaled sulfuric acid
aerosols can be increased by increasing blood ammonia concentrations. However,
the increased neutralization does not seem to markedly affect the toxicity
of the inhaled and aerosols.
17.
a. DESCRIPTORS
KEY WORDS AND DOCUMENT ANALYSIS
b.lDENTIFIERS/OPEN ENDED TERMS
Sulfates, lexicological, Pulmonary Func- Health Effects
tion, Mobile Sources and Dogs.
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
20. SECURITY CLASS (This page)
Unclassified
c. COS ATI Field/Group
68G
21. NO. OF PAGES
48
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
PREVIOUS EDITION IS OBSOLETE,,,
JO
OUSGPO: 1980 — 657-146/5530
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