United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-81-038 June 1981
Project Summary
Effect of Conventional and
Advanced Coal Conversion
By-Products on the
Pulmonary System
Catherine Aranyi and Jeannie Bradof
To evaluate the environmental impact
of different energy technologies, fly
ash samples collected from a coal-
fired and from an oil-fired electric
power plant were used in aerosol
inhalation exposures of mice. The
effects of multiple 3-hr exposures to
the fly ash particles at 2 and 1 mg/m3
aerosol mass concentration and <0.5
fan mass median aerodynamic diam-
eter were evaluated in male and female
mice by examining the changes in
their pulmonary free cells, in their
susceptibility to streptococcus infec-
tion, and in the bactericidal activity in
their lungs to inhaled Klebsiella
pneumonias. Generally no consistent
differences could be discovered in the
effects of the exposures between the
two sexes. However, in a combined
evaluation of both sexes, more and
greater significant changes relative to
controls were observed in the experi-
mental parameters after inhalation of
the oil power plant fly ash than after
exposure to the coal fly ash. Thus, the
overall results of the study indicate
that the pulmonary defense system of
mice was more adversely affected by
the oil-fired power plant fly ash, a true
stack emission effluent, than by the
coal fly ash collected by electrostatic
precipitator, an in-plant control device.
This Project Summary was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park,
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction and Summary
A study was conducted under carefully
controlled conditions to examine the
effects of exposure to aerosols of respi-
rable-size particulate by-products of
coal-fired and oil-fired electric power
plant on pulmonary defense mecha-
nisms. The study objectives were to
determine if inhalation of the aerosols
in short-term and intermediate multiple
3-hr exposures (a) produces change in
number and function of alveolar macro-
phages or (b) potentiates an experimen-
tally induced infection. Mice were ex-
posed to aerosols of test compounds,
and the effect of inhalation of these
substances on susceptibility to respira-
tory infection was examined by deter-
mining mortality rates and survival
times after challenge with airborne
pathogenic bacteria. The effect on
bacterial clearance after aerosol expo-
sure was evaluated by measuring change
in bactericidal activity to inhaled,
radiolabeled bacteria in animal lungs. In
addition, total number, cellular distribu-
tion, viability, andadenosinetriphosphate
(ATP) levels were examined in cells
obtained by lung lavage after aerosol
exposure.
-------
Methods
Two fly ash samples were used, one
collected from a coal-fired and one from
an oil-fired power plant. The coal-fired
power plant fly ash originated from
high-sulfur-containing Eastern coal and
was collected as an electrostatic precip-
itator hopper ash from a conventional
power plant. Although this fly ash
cannot be considered a true effluent
sample, previous study of this particulate
demonstrated that the <3 urn aerody-
namic size fraction used in this study
can act as a qualitative surrogate for the
constituents adsorbed on the stack ash.
The oil-fired power plant fly ash was
collected from stack emission at 400°F
in a fabric filter collection jar as a true
effluent sample. Exploratory in vitro
testing of the oil-fired ash with the
rabbit alveolar macrophage (RAM) assay
indicated a greater relative cytotoxicity
than that documented for the coal fly
ash.
Male and female CDi mice quarantined
8 to 14 days were used for the inhalation
exposure studies. The mice were housed
in individual cages during exposure to
the test aerosols and infectious chal-
lenge. The animal exposure chambers
and the aerosol generation and dilution
systems were housed in second cham-
ber enclosures for exposures to infec-
tious and radioactive particulates. The
actual inhalation chambers were main-
tained at negative pressure relative to
the outside chamber systems, and the
latter were negative relative to the
pressure in the room.
For animal exposure to aerosols of
test fly ash particles, caged mice were
confined within a 453-liter compartment
of a Plexiglas exposure chamber. The
various components of the aerosol
generation, dilution, and monitoring
systems were located on a shelf sus-
pended over the exposure chamber.
Aerosol was introduced into the expo-
sure chamber near the bottom at one
end and exhausted along the top at the
opposite end.
For animal exposures to bacterial
aerosols, a 380-liter chamber containing
the caged mice was installed in a
second housing unit. For exposures to
Streptococcus pyogenes, the chamber
was installed within a microbiological
safety cabinet. For exposures to S-
labeled Klebsiella pneumonias, the
chamber was installed in a glove box.
After daily 3-hr animal exposures to
aerosols of the test particulates for 5,
10, or 20 consecutive days, effects were
evaluated by observing changes in the
animals' susceptibility to respiratory
bacterial infection and in pulmonary
bactericidal activity to inhaled radio-
labeled K. pneumoniae, and by exami-
nation of the pulmonary free cells
lavaged from their lungs. The effects
were assayed within 1 hr after the last
exposure to aerosol of the test particu-
lates.
For the isolation and characterization
of cells, at least 200 cells selected at
random were counted microscopically
to determine differential counts and
viability. ATP concentrations in the
lavaged cells were determined using a
DuPont 760 luminescence biometer.
Results
Male and female mice inhaled aerosols
of the coal-fired power plant fly ash or
the oil-fired power plant fly ash at 1 and
2 mg/m3 mass concentration in multi-
ple daily (5 days/week) 3-hr exposures.
The aerosol mass concentrations were
determined by taking the averages of
three daily filter readings and calculat-
ing the means and standard deviations
(SD) over the total number of exposure
days. The means and SD for the coal fly
ash were 2028 ± 322 ,ug/m3 and 1030
± 187/ug/m3; and for the oil power plant
fly ash 1993 ± 222 fjg/m3 and 1053 ±
208 fjg/m3.
The effects were determined after 5,
10, and 20 exposures by examining
changes in the free cells lavaged from
the lungs, susceptibility to respiratory
streptococcal infection, and pulmonary
bactericidal activity to inhaled radio-
labeled K. pneumoniae. Results are
summarized in Table 1 and are expressed
for the pulmonary free cell data as
percentage of the control responses,
and for mortality rates (%), mean survival
times (days) and pulmonary bactericidal
activity (%) as the change between ex-
posed and control observations. Changes
in the total number and ATP content
(representing phagocytic competence)
of free cells lavaged from the lungs were
clearly affected by inhalation of the two
fly ash samples.
In susceptibility to inhaled strepto-
coccal infection, a greater number of
significant changes were observed
(Table 1) following exposure to the oil-
fired power plant fly ash than after
inhalation of the coal fly ash aerosols.
Mean survival times in general showed
significant decreases, paralleling the
significant increases in mortality rates
for both of the fly ashes. On the other
hand, significant increases in mean
survival time occurred, correlating with
the significant mortality decreases in
both sexes after 20 exposures to aerosols
of the oil power plant fly ash at 1053
fjg/m3. In general, the changes in
bactericidal activity following inhalation
of the coal fly ash were increases com-
pared to the control values, whereas
changes after exposure to aerosols of
the oil power plant fly ash were de-
creases.
For a better review and comparison of
the effects of the two fly ash samples,
the overall results in terms of significant
increase, decrease, or no change are
presented in a summary table (Table 2).
The effects of each individual fly ash
sample on the two sexes appeared to be
generally similar, with the exception of
susceptibility to respiratory infection,
i.e., the coal fly ash produced more
significant changes in female and the oil
power plant fly ash in male mice. Thus,
the results of this essentially qualitative
evaluation indicate no more than random
variations between the responses in the
two sexes; however, no in-depth statis-
tical analysis was applied to this aspect
of the studies.
In terms of an overall comparison of
all experimental parameters and across
both sexes, it is immediately apparent
from Table 2 that the oil-fired power
plant fly ash produced more significant
changes than coal fly ash (a total of 25
vs. 20). Generally the same trend could
be observed for each individual experi-
mental parameter with the exception of
pulmonary free cellular ATP levels.
In the case of the pulmonary cellular
lavage studies, it is evident that there
are more numerous and greater signifi-
cant increases in cell counts after ex-
posure to the oil power plant fly ash (8
increases) than to the coal fly ash (5
increases). In contrast, for ATP levels,
the coal fly ash produced more signifi-
cant and consistent changes (7 de-
creases) than the oil fly ash (3 increases
and 1 decrease).
There is a greater incidence as well as
magnitude of effect in the significant
increases in mortality due to strepto-
coccal infection after inhalation of the
oil power plantflyash(6increases)than
after inhalation of the coal fly ash (4
increases). In addition, 2 significant
decreases in mortality were caused by
exposure to the oil fly ash. Significant
changes in pulmonary bactericida
activity to K. pneumoniae were generally
depressions (4 out of 5) for the oil power
plant fly ash, whereas only significant
increases (4) were found after exposure
to the coal fly ash.
-------
Table 1. Significant Change" in CD, Mice Following Exposures to Test Aerosols
Effects of Inhaled Ash Plus Infective Agent
Effects of Ash Inhalation
Aerosol Mass
Cone fag/m3)
Sample
Coal-fired
power plant
fly ash
Oil-fired
power plant
fly ash
Sex
M
M
F
F
M
M
F
F
Mean
2028
1030
2028
1030
1993
1053
1993
1053
SD
322
187
322
187
222
208
222
208
No of
Daily 3-hr
Exposures
5
10
20
5
10
20
5
10
20
5
10
20
5
10
20
5
10
20
5
10
20
5
10
20
n"
84
18
49
33
32
32
67
23
19
32
51
74
31
32
32
36
34
32
32
35
33
34
37
34
Free Cells
Lavaged
from Lung*
1% of Control)
113**
205**
117
124
150**
94
96
140*
147*
109
111
105
107
224***
165***
95
134**
140**
104
178***
178***
115
153***
177***
n
76
16
49
32
32
32
56
16
15
30
47
68
31
32
32
33
32
31
31
31
31
33
30
33
Pulmonary
Cell ATP
Content"
(% of Control)
94
91
97
85**
78**
73**
89**
60***
113
85**
82**
99
97
106
94
116*
102
128**
85**
96
118**
112
89
96
Susceptibility
to Streptococcus
n
382
395
237
318
267
296
475
385
137
295
375
334
278
229
306
237
145
273
330
310
146
297
239
212
Mortality
1% of Control) MSr
0
-0.1
8.4
3.7
-2.1
4.9*
5.9**
5.3
17.9**
1.1
0.1
4.5**
18.2***
18.8***
10. 7**
-0.7
9.3**
-5.6**
25.2***
5.1*
2.6
1.9
-0.1
-12.3**
0
0.1
-0.9
-0.4
0.4
-0.4*
-0.2
-0.6
-1.2**
-0.1
0.1
-0.6*
-1.6***
-1.5***
-0.9***
-0.1
-0.8**
0.4**
-2.5***
-0.5*
-0.4
-0.2
0
0.7**
Bactericidal Activity
to 355-Af. pneumonias
n
47
45
23
45
40
36
58
44
24
43
86
61
43
38
40
44
45
44
43
44
44
46
43
45
K, pneumonias
killed
!% of control)
6.6**
-2.0
2.1
2.7*
0.4
-3.1
3.6*
1.7
1.4
-2.3
-0.9
6.7***
2.7*
-4.0**
3.8
1.1
-2.4
0.8
-5.4**
-2.1
1.7
-2.5*
1.5
-3.5*
"Significant change from corresponding control mice determined by two-way analysis of variance (total cell counts, ATP/cells);
Chi-square (% mortality) and Student's t-test (Mean Survival Time; % bactericidal activity): *p<0.1, **p<0.05, ***p<0.001.
^Number of mice.
c Number of cells expressed as count x 10s.
"ATP content expressed as 106 fg/10s cells.
"MST = mean survival time in days, expressed as difference in MST days for exposed mice compared to that for controls.
Table 2. Summary of Significant Changes in Experimental Parameters After Exposure to Aerosols of a Coal-Fired Power Plant
Fly Ash or an Oil-Fired Power Plant Fly Ash"
Aerosol Mass ^Q Qf
Cone, ffjg/m3) nailu ,.hr
Sample Mean
Coal-fired 2028
power plant
fly ash
1030
Oil-fired 1993
power plant
fly ash
1053
SD Exposures
322 5
10
20
187 5
10
20
222 5
10
20
208 5
10
20
Total Cells
M
-t-
+
0
O
+
0
0
+
+
0
+
+
F
0
+
+
0
0
0
0
+
+
0
+
+
Cellular ATP
M
0
0
0
.
-
-
0
0
0
+
0
+
F
.
.
0
_
-
0
_
0
+
0
0
0
Mortality
M
0
0
0
0
0
+
+
+
+
0
+
-
F
+
0
+
0
0
+
+
+
0
0
0
-
Bactericidal
Activity
M
+
0
0
+
0
0
+
_
0
0
0
0
F
+
0
0
0
0
+
0
0
.
0
-
'+: Significantly greater than control (p<0.10); -: Significantly lower than control (p<0.10); 0: No significant change from control.
3
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Previous studies have consistently
demonstrated that aerosols of paniculate
samples with higher relative in vitro
cytotoxicity and partial solubility (see
discussion of oil fly ash in Methods
section) produced significant depression
in pulmonary bactericidal activity to
inhaled K. pneumoniae and increased
mortality from experimentally induced
streptococcal pneumonia, whereas
inhalation of more inert particles (such
as the coal fly ashes) could be correlated
with bactericidal activity increases and
with no change in mortality.
In terms of the present study, this
further confirms the evaluation of the oil
power plant fly ash as the relatively
greater inhalation hazard than the coal
fly ash. To keep this observation in
proper perspective it should be remem-
bered, however, that the coal fly ash
was collected from an electrostatic
precipitator, and samples obtained from
such in-plant pollution control devices
are generally removed from the effluent
stream at relatively high temperatures
prior to cooling and dilution. The more
volatile compounds, which may also be
more toxic, are not collected and there-
fore are not present on these particles.
The oil-fired power plant fly ash was an
actual fugitive emission sample collected
from the stack gases, and thus the
particles probably contained more haz-
ardous trace metals as well as aliphatic
and/or aromatic hydrocarbon compounds
condensed on their surfaces.
Conclusions and
Recommendations
Inhalation exposure to the oil fly ash
produced more adverse health effects
than exposure to the coal fly ash. The
results demonstrated no differences in
the effects due to sex. However, when
comparing all experimental parameters
and all conditions tested across both
sexes, the oil-fired power plant fly ash
produced more statistically significant
changes than the coal fly ash. This trend
also was found generally for each
individual experimental parameter with
the .exception of the ATP levels in
pulmonary free cells. Thus, there were
more significant changes in the total
number of cells lavaged from the lungs,
in the susceptibility to respiratory in-
fection, and in pulmonary bactericidal
activity after inhalation of the oil fly ash
than the coal fly ash.
As in the case of a previously examined
copper smelter dust, water-soluble
compounds were released from the oil
fly ash particles that were cytotoxic to
alveolar macrophages/>? vitro. However,
this was not the case for any of the coal
fly ash samples investigated. Since
inhalation of both the copper smelter
dust and the oil fly ash also significantly
depressed pulmonary bactericidal activ-
ity and increased susceptibility to respi-
ratory infection (whereas the coal fly
ashes did not), there may be a correlation
between the relative inhalation hazard
of an aerosol and the partial solubility of
its constituent particles in an aqueous
milieu; however, more research must
be performed in this area to prove the
hypothesis.
We recommend the examination of
the effects on health of other energy
technologies capable of generating by-
products that can become potentially
hazardous environmental air contami-
nants. The original plans of this project
called for comparison of effluent by-
products of conventional and advanced
coal conversion technologies. Because
of the limited number of advanced
process sites available for sampling, the
emission sample from an oil-fired power
plant was substituted for examination.
A fly ash collected from an in-plant
control device was used to represent the
by-product of a conventional coal con-
version process. The results of our
investigations provided important in-
formation demonstrating that this oil
power plant fly ash represented more of
an inhalation hazard than the coal fly
ash examined. However, the ultimate
objective, in view of the abundant coal
resources of the country, is comparison
and evaluation of the various coal-
based energy technologies. If oil-based
processes are becoming of interest, the
by-products of extracting oil from oil
shale should be considered for their
impact on health. In addition, future
plans should concentrate on collecting
true stack emission samples in order to
formulate a consistent and meaningful
evaluation of the potential inhalation
hazard contributed to the environment.
Based on our observations in this and
other studies on the effects of the
solubility characteristics and cytotoxicity
of the test particles on their potential
inhalation hazard as aerosols, we
recommend preliminary testing for
solubility in aqueous media or in vitro
prescreening of the particles with the
RAM assay.
Catherine AranyiandJeannie Bradof are with IITResearch Institute, Chicago, IL
60616.
Judith A. Graham is the EPA Project Officer (see below).
The complete report, entitled "Effect of Conventional and Advanced Coal Con-
version By-Products on the Pulmonary System," (Order No. PB 81-190 506;
Cost: $6.50, 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
4
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