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observed by scanning electron
microscopy. The data were correlated to
determine which parameters were the
most sensitive indicators of toxic effects
of the sulfates.
Materials and Methods
Three- to 4-week old male and female
CDi mice and 4-week old male Syrian
golden hamsters were used. During
exposures to the "pollutants and the
infectious challenge, the animals were
held in special stainless steel wire-
mesh cages which held up to 24
animals in individual compartments.
Exposures to the air pollutants were
carried out in 432 liter (1 20x60x60 cm)
Plexiglas chambers supplied with
filtered room air. Deotized cage boards
were placed on the floor of the
chambers to prevent ammonia buildup
during the exposure periods
The pollutant aerosols were
generated by means of a nebulizer
containing a 1 percent (w/v) aqueous
solutions of cupric sulfate, 102.3
percent purity; aluminum sulfate, 99.8
percent purity; aluminum ammonium
sulfate, 99.9 percent purity. The sulfate
solutions were replenished at 30 min
intervals during the 3-hr exposure. The
sulfate mass concentration in the
aerosol was determined from two
parallel samples collected over the 3-hr
exposure period on 0.2 fjm membrane
filters. The sulfates collected on the
filters were assayed by a barium sulfate
turbidimetric method and expressed in
mg/m3 of the sulfates.
Particle size was determined with an
8-stage cascade impactor only for the
aluminum ammonium sulfate aerosol
The amount of sulfate on each impactor
stage was assayed by the barium sulfate
turbidimetric method and used to
determine the mass median aerodyana-
mic diameter (MMAD) and geometric
standard deviation (erg) of the aerosol.
Particle size measurements were not
made during other acute exposures
since the aerosol generation and
delivery systems were identical in all
experiments. During one multiple
exposure to cupric sulfate, aerosol
particle size was determined with the
quartz crystal micro-balance-based
cascade impactor.
The animals were exposed to the
experimental atmospheres for a single
3-hr period or in the multiple exposure
experiments for 3 hr/day, 5 days/week
for 1 or 2 weeks. Since, due to the size of
the chambers, it was not possible to
expose a sufficient number of animals
to measure all experimental parameters
on a single day, replicate exposures
were carried out on 2 or more days
To determine changes in resistance to
respiratory infection, the mice were
challenged with an aerosol of
Streptococcus pyogenes (Group C) as
described by Ehrlich et al (Environ. Res.
14, 233, 1977).
Pulmonary defense mechanisms
were studied using the methodology
described by Aranyi (EPA-600/51-81-
003, 1981) and Aranyi et al. (In, Short-
Term Bioassays in the Analysis of
Complex Environmental Mixtures,
Plenum Press, New York, N.Y., 1981)
Briefly, the intrapulmonary bacterial
mactivation was determined in
individual mice after exposure to viable
35S-labeled K. pneumoniae. The ratio of
viable bacterial count to radioactive
count in each animal's lung defined the
rate at which bacteria were destroyed at
a given time after infection. The
pulmonary free cell population was
studied using mice killed within 1 or 24
hr after the final pollutant exposure. The
pulmonary cells were isolated from the
lavage fluids and total cell counts were
made using a hemocytometer. Percent
distribution of alveolar macrophages,
polymorphonuclear leukocytes and
lymphocytes were determined by
differential counts. Viability was
determined by dye exclusion using 1%
trypan blue Cellular adenosine
triphosphate (ATP) concentrations in
the lavaged cells were determined with
a Luminescence Biometer.
Changes in tracheal ciliary beating
frequency and morphology were
determined using techniques described
by Schiff et al (Environ. Res. 19, 339,
1979) Following exposure to a
pollutant, tracheas were removed from
mice and hamsters and organ cultures
established. Cilia beating frequency
was measured at each quadrant of the
lumen. The percentage of normal
epithelium, i e., a smooth lummal
surface with beating cilia, was
determined for each tracheal ring In
addition, histopathologic examination
was made of the tracheal ring explants.
Scanning electron microscopic
examination of the respiratory tract was
made using methods described by
Ketels et al. (Scanning Electron
Microscopy/1977 Vol. II. Proc.
Workshop on Biomedical Applications,
519, 1977).
The Chi-square (X2) test with 2x2
contingency table was used to test
hypotheses regarding mortality rates.
Student's t test was used to determine
the statistical significance of treatment
differences in mean survival time,
pulmonary bactericidal activity, and
pulmonary free cell parameters.
Hypotheses involving cilia beating
frequency were tested with two-way
analysis of variance. Duncan's New
Multiple-Range Test was used to detect
treatment differences. Treatment
differences in percent normal
epithelium were determined from the
Chi-square distribution Statistical
analysis of the mortality rate data was
conducted also with log-linear modeling
procedures which provide maximum
likelihood estimates, standard errors,
and tests of significance for the mam
effects and interactions of discrete or
qualitative data.
Conclusions and
Recommendations
The results of the studies,
summarized on Tables 1 and 2,
indicated that changes in mortality rates
and mean survival time after respiratory
challenge with airborne Streptococcus
pyogenes were the most consistent and
sensitive indicators of damage due to
inhalation of sulfate aerosols. The other
health effect parameters produced
evidence of damage but generally only
at the higher pollutant concentrations.
Cupric sulfate was the most toxic of the
three sulfates but relative toxicity of
aluminum sulfate and aluminum
ammonium sulfate could not be clearly
established. A number of apparent
between-sex differences were found,
but the differences were inconsistent
and probably not related to the sex of the
mice
Thus, it is apparent that the change m
mortality of mice infected with S.
pyogenes is the most sensitive indicator
of the effects of exposure to metallic
sulfates. Future studies should focus on
further definition of this assay in
laboratory animals other than mice,
thus enabli ng the ranking of toxicity of a
given compound in several experimental
hosts. Future studies also should
include the evaluation of the effects of
inhaled metallic sulfates on the immune
response. This includes both the cell-
mediated as well as humoral immunity.
Results of this study showed that the
tracheal epithelium of the CDi mouse
was less suitable for evaluation of
sulfates than was that of the hamster.
As a result, hamsters rather than mice,
should be used for this parameter test.
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Table 1. Summary of the Health Effects of Single 3-Hr Exposures to Various Sulfates
Mortality
Rate
SO43
Pollutant mg/m3
Cupric
Sulfate 2 S3
293
0.43
Aluminum
Sulfate 2 71
231
7 84
Aluminum
Ammonium 3 79
Sulfate 2 W
1 47
CD,
M F M&F M
+ +
0 +
0 0
0 0
0 +
0 0
-
0
0
0
0
0
MSJ*
CD,
Total
Cells*
CD,
F M&F M F
0
0
0
- 0 +
0 0
ND
0
ND
0 0
0 0
0 0
ND
Total
Cel/sc
CD,
M F
ATPb
CD,
M F
00 +
0 ND 0 0
ND ND
- 0 0
ND
ND
ND
0 0
ND
ND
0 0
+ +
0 0
ND
ATPC PBA"
CD, CD,
M F M F
0 ND 0 0
ND ND
0 0 + +
ND ND
ND 0 0
ND ND
0 + +
ND ND
Cilia Beat/
Mm
CD,
0
0
ND
0
ND
ND
ND
0
ND
Hamster
0
ND
0
ND
0
0
ND
Normal
Epithelium, %
CD,
0
0
ND
e
ND
ND
ND
0
ND
Hamster
0
ND
ND
-
-
0
ND
Mean Survival Time
bMice killed within 1 hr after exposure
cMice killed 24 hr after exposure
^Pulmonary Bactericidal Activity
"Unable to make evaluation due to technical difficulty
^Significantly greater than control fp<0.05)
"Significantly lower than control (p<0.05)
°No significant changes
ND/V0 data
Table 2. Summary of the Health Effects of Multiple 3-Hr Exposures to Cupric Sulfates
No. of
Exposures
5
10
SO*
mg/m3
Mean SO
0.09
0.09
0.10
0.01
0.01
0.02
Mortality
Rate
CD,"
M F M&F M
0000
MST"
CD,
F M&F
0 0
Total
Cells"1
CD,
M F
0 0
0 0
ATPh
CD,
M F
0 0
0 0
PBAC
CD,
M F
0 -
Cilia Beat/Min
CD,
M F
e e
e e
Hamster
0
0
Normal
Epithelium %
CD,
M F
- 0
0
Hamster
0
0
aMean survival time
t'Mice killed within 1 hr after exposure
0Pulmonary Bactericidal Activity
d CD, strain of mice
e Unable to make evaluation due to technical difficulty
+Significantly greater than control (p<0.05)
"Significantly lower than control (p<0.05)
0 No significant changes
John G. Drummond, James D. Fenters, Richard Ehrlich, Catherine Aranyi. and
Leonard Schiff are with IIT Research Institute, Chicago, IL 60616.
Judith Graham is the EPA Project Officer (see below).
The complete report, entitled "Short- Term Screening Test for Quick Responses
to Pollutants Where Health Effects Data are Lacking," (Order No. PB 82-234
303; Cost: $7.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
US GOVERNMENT PRINTING OFFICE 1982-559-017/0755
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Environmental Protection Information Environmental
Agency Cincinnati OH 45268 Protection
Agency
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Official Business
Penalty for Private Use $300
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