».670/1-73-036
January 1973
Environmental Health Effects Research Series
ANNUAL REPORT FOR CALENDAR YEAR 1972
ENVIRONMENTAL TOXICOLOGY RESEARCH LABORATORY
NATIONAL ENVIRONMENTAL RESEARCH CENTER
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
-------
REVIEW NOTICE
The National Environmental Research Center - Cincinnati,
U.S. Environmental Protection Agency has reviewed this
report and approved Its publication. Mention of trade
names or cownerldal products does not constitute en-
dorsement or recommendation for using.
-------
A-670/1-73-036
January 1973
ENVIRONMENTAL TOXICOLOGY RESEARCH LABORATORY
NATIONAL ENVIRONMENTAL RESEARCH CENTER
Cincinnati, Ohio
ANNUAL REPORT
FOR CALENDAR YEAR 1972
Issued January 1973
by
The Staff of the ETRL
J. F. Stara, Director
-------
FOREWORD
Pursuant to environmental legislation (Sec. 103 of
the Clean Air Act as amended In 1970), the Environmental
Protection Agency was charged to evaluate potential toxic
effects of fuels and fuel additive emissions from mobile
and stationary sources and to conduct definitive toxi-
cologlc testing of individual potentially hazardous air
pollutants. Because of this charge, the Agency has di-
rected its National Environmental Research Centers to
address the problems of the potential toxic effects of
these emissions. The Environmental Toxicology Research
Laboratory, NERC - Cincinnati, is concerned with the toxi-
cological evaluation of emissions and potentially hazardous
pollutants in laboratory animal model systems.
There is a critical need to test and clarify the
potential destructive effects of environmental contami-
nation through advanced toxlcologic techniques 1n order
to establish safe population standards. Existing methods
must be used and new methods developed to evaluate and
control the impact of pollution on man's health and well-
being. Toxicologic research must be conducted primarily
in non-human biological test systems since most chemicals
found 1n the environment cannot be safely tested in man.
Furthermore, the need for more experimental animal models
with clearly defined characteristics is now well estab-
lished and the utilization of such models has proven
extremely successful over the years. In fact, many of
the advances in biology and medicine have been derived
from animal studies.
The probability of reproducing the human response
in animals increases with judicious selection of the animal
species. For this reason, an Increasing number of mamma-
lian species are being used in this Laboratory to develop
appropriate models for toxicologlcal evaluation of po-
tentially harmful pollutants. In its planning, the
Laboratory gives a special emphasis to certain factors
which may influence-susceptibility such as age-sensitivity,
relevant routes of exposure, and different chemical forms
of compounds found 1n the environment. In studying the
toxic process of a disease, several "standard" animal
species, e.g., mice, rats, guinea pigs, rabbits, and dogs
are routinely used; others were added whenever indicated,
e.g., Syrian hamsters, non-human primates and cats.
111
-------
The problems concerned with the extrapolation of
animal data to man are minor when compared with the great
advantages of animal Investigation 1n determining the toxic
potential of various agents. The following factors favor
animal experimentation: (a) strict control of exposure
concentrations; (b) strict control of duration of expo-
sure; and (c) opportunity to make a detailed biological
examination of tissues and organs not possible In man.
Possibly, the most cogent arguments for the use of animals
1n toxicologies! studies are: (1) they are necessary for
determination of dose:effect relationships because levels
much greater than those found 1n nature may be used; (2)
they are necessary to assay potential threats of new agents
which may be Introduced Into the environment 1n the future
due to technological advancement.
The research approach for determining the potential
toxic effects of mobile source emissions Is complicated
by the rapidly changing scope in fuels and fuel additives
marketing, and development of new emission systems and
control devices, e.g., catalytic converters. The goals
of ETRL are to provide answers to these problems using a
mult1d1sc1p!1nary toxicologic research approach outlined
In Table I.
The program plans of ETRL call for conducting re-
search in two major areas of environmental pollution:
A. Inhalation and 1ngest1on studies of single
pollutants with particular emphasis on
hazardous substances (trace metals).
B. Inhalation exposure to fuel and fuel additive
emissions from mobile sources, which represents
a complicated bu realistic mixture of pollu-
tants in the environment.
The specific studies are designed to provide data
which will supplement and add to existing Information
on various pollutants. Such Information 1s required for
the compilation of criteria documents, which in turn
serve as background material for enforcement actions and
for establishment of safe, accurate and imagainative
environmental pollution standards.
J. F. Stara
1v
-------
Table I
ETRL MODEL FOR DEFINITIVE TOXICOLOGIC TESTING
(FUEL ADDITIVES, FUELS, POLLUTION CONTROL DEVICES, TRACE METALS)
—
OFFAR
REVIEW COHHITTEE
CHEMIC
CHAR
OF
M_ AND PHYSICAL
ICTERIZATION
EMISSIONS
•— ^.
»
w
DEFINITIVE
TOXICOLOGY
TESTING
1
GENERATION AND
ANIMAL EXPOSURES
OF FUEL AND FUEL ATDITIVE
EMISSIONS
TOXICOLOGY
SCREENING
REPORT
GENERATION AND
ANIMAL EXPOSURES
OF TRACE ELEMENT
AEROSOLS
METABOLISM
TISSUE BURDENS
STUDIES
UPTAKE
DISTRIBUTION
EXCRETION
BlOTRANSFORMATION
ROUTINE
BIOCHEMICAL
AND
PATHOLOGICAL
TESTS
ASSESSMENT
PHYSIOLOGICAL
FUNCTION
CARDIAC,
RESPIRATORY,
RENAL
NEUROLOGIC
REPRODUCTIVE
HEMATOPOIETIC
SPECIAL TESTS:
E.G. BIOCHEMICAL
BEHAVIORAL
EMBRYOTOXIC
DEPENDENT ON
COMPOUNDS UNDER
STUDY
TOXICOLOGY
TEST
REPORT
T
-------
TABLE OF CONTENTS
SINGLE, INTER-MEDIA. POLLUTANT STUDIES
MANGANESE
Hyte.ll, Mooie.,
, Cn.oo.ke.ti
Mop-ne, Hyte.lt,
Malanchak, S tafia.
Cfioc.ke.fi, Stafia
Moo fie., Hyte.lt,
Moo/te, Column
Gage.
Stafia
LEAD
Stafia,
Ne,ihe,
-------
Staia, Mooie,
Lzwkow&k*.
Environmentally Bound Lead:
III. Effects of Source on
Blood and Tissue Levels of
Rats 28
Lead: Placental Transfer,
Central Nervous System
Effects, and Immune Response
Alteration 30
CADMIUM
Moote, Sta.na,
Cn.oo.ke.rL
Moon, Sta.na.,
Malanchuk,
Mo one., Matanchu.k,
., C/iocfeet
, Moo/te,
G.I. Absorption of Different
Compounds of '»5mcd and the
Effect of Different Concen-
trations in the Rat
Effect of Different Routes of
Administration of 115mcdCl2
Upon Whole Body Retention ...
Retention of 115mCdCl2 and
HSmcdO Following Inhalation
Exposure
Effect of Cadmium Ingestion on
Blood Pressure in Monkeys
32
35
36
37
MERCURY
Lee, 8ti>fcte/L,
Pannet, Johnson,
Mc.M4JU.an, Moon.*.,
Sta.n.0.
Early Biochemical Effect of
Methylmercury Chloride in Rats
40
MISC. POLLUTANTS
Lee, Vanne,*.,
Button., Menzel,
Stana
Early Biochemical Effects of
03 and N02 and Influence of
Vitamin E on These Effects .,
44
-------
Lee, Pannes,
, S waltz,
Stan.a,
Campbe.tt,
Ca.mpbe.ll, Halt
fUmme-C, Mooie,
Hy6e.lt, Stafia
Alveolar Protein Accumulation:
A Sensitive Indicator of Low
Level Oxidant Toxicity
Chronic Exposure Effects of
Ozone in Beagles
49
51
Influence of Exposure Pattern
on Toxic Response to Nitrogen
Dioxide
Hexachlorophene Teratogenicity
53
56
TOXICOLOGIC ASSESSMENT OF MOBILE EMISSIONS (TAME) STUDIES
Sta)ia Toxicological Assessment of
Mobile Emissions (TAME):
Overall Study Approach and
Objectives 63
fUnne/u, Buikant, Design and System Performance
Ittl* for Mobile Emissions Bioeffect
Studies at ETRL 66
Uatanchmk, Contne.fi, Atmospheric Characterization
State.*, in Auto Exhaust Emissions 74
Ua.tanc.hu.kf Cohen Particulate Sampling Procedures
for Auto Exhaust Emissions 78
Ca.mpbe.tt, Ma.tcLnc.kuk, Differences in Fuel Emission
State,*., Contnti, Components in TAME "E" and "F":
lltl&, Zu.nka.nt, Tentative Inferences as to
Hi.nne.ru, Vana, Staia Possible Influence of MMT 81
Hy4e.tt, Mooie. Changes in Pathology of Rats
and Hamsters Following Inhalation
Exposure to Mobile Emissions 86
, Moo*e, Effects of Mobile Emissions on
Body Weight and Tissue Levels
of Mn i n Rats 90
1x
-------
Lee, Pannei,
McMillan,
Staia
Halt, Wat king ton,
Ball, Adam*,
Ca.mpbe.tl
Halt, Malanckak,
Coften, Adam.6,
Campbe.ll, Staia
Halt, Washington,
Ball, Adam*,
Campbe.ll
Gage, Vang,
Co ken, Stana
Gage
Gage, Scftne
-------
METHODS DEVELOPMENT STUDIES
Hy&ttt., Moo-te., The Use of Cornea! Mitotlc
Rate As A Measure of Ocular
Irritation 133
, lttJi& Blood Pressure of Monkeys 135
Correlation of Evoked
Potential and Spinal Cord
Responses as a Method for
the Evaluation of the Bio-
logical Effects of Environ-
mental Pollutants 142
yang Estimation of Relative
Toxicity: A Proposed
Treatment of Bioeffect
Data 143
lttJi& The Approach to Data
Analysis in ETRL 147
PUBLICATIONS 149
ETRL ROSTER 155
ACKNOWLEDGMENT 157
xi
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SINGLE, INTER-MEDIA, POLLUTANT STUDIES
-------
ORAL TOXICITY OF MMT IN RATS
D. Hysell, W. Moore, R. Miller, and W. Crocker
The paucity of information on the toxicity of MMT
(per National Academy of Science request) prompted a
series of studies to provide necessary data on its
metabolic behavior and biological effects.
The organometallic compound 2-methylcyclopenta-
dienyl manganese tricarbonyl (MMT) is of current In-
terest because of its possible use as an anti-knock
compound in unleaded gasoline. The compound is also
marketed as a combustion improver for fuel oils under
the trade name "Ethyl" Combustion Improver 2 (CI 2) and
as a smoke suppressant for diesel engines and stationary
jet fuel power sources. The compound is an orange
colored liquid having low volatility, a distinctive
herbaceous odor, and thermal stability, and is subject
to rapid photochemical decomposition. The calculated
manganese content is 25.2%. The proposed concentration
of manganese per gallon of gasoline is approximately
0.125 g.
Eighty COBS rats, weighing 200-250 g, were divided
into eight groups of 10 animals each and given a single
oral dose of Mn tricarbonyl for determination of oral
toxicity. The Mn tricarbonyl was diluted with Wesson
011 and given by intragastric tube. The concentrations
ranged from 15 to 150 mg/kg (based on Mn content of MMT),
and one group of animals serving as controls.
In animals given the high concentrations (80-150
mg/kg), deaths occurred within 24-48 hours after dosing.
The progressive effects consisted of huddling, roughened
hair coats, tremors, progressive weakness, labored res-
piration, seroganuineous nasal discharge and terminal
coma. The incidence of clinical symptoms in each group
essentially paralleled the mortality rate except the
lowest dosage groups showed the roughened hair coat and
huddling for the first 24 hours.
All deaths occurred within 6 days after exposure,
and by 14 days, the survivors appeared normal. The
study was terminated at this point and tissues taken for
histopathologlcal examination and Mn analysis. The
mortality data are presented in Table 1.
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TABLE 1. MORTALITY IN RATS FOLLOWING ORAL
ADMINISTRATION OF Mn TRICARBONYL
Dose
mg/kg
15
30
45
60
80
100
150
Control
MortalUy
*— ~ Dosed
0/10
0/10
5/10
6/10
6/10
8/10
10/10
0/10
Length of survival
of animals dying
2-6 days
2-3 days
<24 hr. - 3 days
<24 hr. - 3 days
<24 hr. - 2 days
<24 hr. - 2 days
4
-------
Other investigators had found that the oral 1059
varied in the rat depending on age, sex, and the medium
used for dilution of the Mn tricarbonyl. The range for
the male rat varied from 17-176 mg/kg, and from 9-96
mg/kg for the female rat.
Necropsies were performed on a representative number
of animals dying during the study as well as selected
animals euthanatized at the end of 14 days. In animals
dead within 24 hr, the gross necropsy findings consisted
of large sacular atonic stomachs, severely congested
livers, and severely congested lungs that, on sectioning,
exuded a sero-sanguineous fluid from the cut surfaces.
In animals dead between 24-72 hr, the pulmonary and
hepatic changes were the same. The small intestine was
distended with clear watery contents and the walls appeared
thin and friable. By 14 days, the organs appeared grossly
normal except for the livers from animals receiving the
higher dosage levels, which were a tannish yellow in
appearance. At necropsy, specimens of heart, lung, liver,
kidney, duodenum, and brain were collected for chemical
assessment of tissue manganese. The mean Mn concentration
in selected tissues for those animals that died following
exposure and those that were sacrificed 14 days post ex-
posure are given in Table 2.
Specimens of heart, lung, liver and kidney were
collected for histologic preparation.
TABLE 2. CONCENTRATION OF Mn IN DIFFERENT TISSUES FOLLOWING
IN6ESTION OF 2-METHYLCYCLOPENTADIENYL MANGANESE
TRICARBONYL
ANIMALS DYING FOLLOWING EXPOSURE
TISSUE
Duodenum
Kidney
Liver
Lung
Heart
Brain
ug/g Dry Weight
CONTROL
ANIMALS
TISSUE
Duodenum
Kidney
Liver
Lung
Heart
Brain
45
34.7
18.3
22.8
12.0
4.00
7.18
SACRIFICED
60
38.
25.
22.
10.
4.
8.
14
4
3
9
2
83
68
80
65
17
31
15
4
9
DAYS POST
pg/g
CONTROL
3.32
5.81
7.05
1.86
5.89
15
4.89 3
4. 64 5
7.14 9
4.40 4
1.83 3
7.18 8
30
.81
.62
.11
.87
,77
.68
.8
.4
.6
.4
.67
.66
100
142.
48.
32.
28.
7.
7.
5
0
0
1
84
53
150
177.6
40.0
36.5
28.6
6.06
8.16
INGESTION .
Dry Height
45
5.03
3.16
10.52
3.67
3,00
9.68
60
6.49
2.62
9.55
5.56
9.66
80
6.48
3
11
3
7
.59
.33
.13
.53
100
7.50
4.99
10.82
6.39
8.16
-------
Microscopically, the lungs of animals dead in 24 hours
showed severe congestion, perivascular and alveolar edema and
alveolar hemorrhage (Fig. 2).
Figure 2. Microscopic appearance of pulmonary tissue from
animals dead within 24 hr.
-------
From 24-72 hr there was,
f ibrinopurul ent pneumonia with
filtrate. (Fig. 3)
tn addition, a severe
prominent macrophage
Figure 3. Pulmonary changes in animals dead 48-72 hr
after exposure to MMT.
In animals surviving 14 days, the lungs showed extensive
areas of consolidation, thickened alveolar septa and focal
areas of alveolar macrophage activity (Fig. 4).
-------
Figure 4
Chronic pro!1ferat1ve pneumonia at 14 days
surviving MMT exposure.
The hepatic lesions progressed from acute centrolobular
passive congestion at 24 hr, to hepatic parenchymal necrosis
and leukocytlc Infiltration at 48-72 hours, and extensive
cytoplasmic vacuolar change (probably Hpidic) by 14 days.
(Figs. 5, 6, & 7)
8
-------
Figure 5. Hepattc changes at 24 hr.
Figure 6. Hepatic changes at 48-72 hr.
Figure 7. Hepattc changes at 14 days.
9
-------
The early renal changes were hyaline droplet change
and cytoplasnrtc vacuolatlon of proximal convoluted tubules
plus dlstentlon of the glomerular space and tubule lumens
with a material that was finely granular and stained
lightly basophlllc. (F1g. 8)
Figure 8. Renal changes at 24 hr.
By 48 hr there was severe tubular degeneration as Indicated
by nuclear pyknosls and cell lysis. (F1g. 9)
10
-------
Figure 9. Renal tubular degeneration and necrosis
at 48-72 hr.
In those animals surviving 14 days, no renal changes were
noted.
11
-------
INHALATION TOXICITY OF MMT VAPOR IN RATS
W. Moore, D. Hysel1, M. Malanchuk, and J. Stara
A group of rats was exposed to MMT vapor at a con-
centration of 2 mg/nr for 4 hr (F1g. 1). Animals, 1n
groups of six, were sacrificed Immediately following ex-
posure and at 1, 2, 4, 8 and 16 days and necropsled.
No gross abnormalities were noted. Tissue specimens of
heart, lung, liver, and kidney were collected for histo-
loglc preparation and microscopic examination. One eye
was extirpated from each animal, prepared, and examined
for determination of corneal mltotic rate. Tissue
specimens of brain, heart, lungs, liver, and kidneys
were collected for chemical analysis of tissue Mn.
Figure 1 . Aerosol
exposure chamber.
Microscopically, the hearts and kidneys showed no
treatment-related lesions. Pulmonary tissues showed changes
12
-------
consistent with mild chronic respiratory disease of rats.
No accentuation of the disease process due to treatment
was apparent. Livers from control and immediate-sacrifice
groups were normal. At 24 and 48 hr., there appeared to
be cloudy swelling of the hepatic parenchyma. By 96 hr,
there was a prominent cytoplasmic vacuolar change of the
hepatocytes in several animals (Fig. 2).
Figure 2.
Hepatic changes at
exposed animals.
96 hr in the MMT vapor
Morphologically, the changes were identical to the
hepatic changes seen in the oral toxicity survivors
euthanatized at 14 days and compatible in appearance with
a lipidic degeneration of the liver. All animals sacri-
ficed at 8 days showed the vacuolar hepatic change; by
16 days, however, the process was apparent in only two
animals.
The results of the corneal mitotic rate determina-
tion are shown in Figure 3. It is not apparent that the
changes in rate represent a direct ocular effect. It has
13
-------
-»v»v»
33
.2
u.
j300
M
i
|TOO
a
= TOO
i
• Mean
• Observed Values
a «
m
0
_ • 0
• 8
1 *
A •
5 *
\ « ' .:
a «
' > 1 1 1 1 1 1 1 1 | 1 1 1 1 t 1 I
&ntnl 2*4 4*8 % #2 I&M 888
Hours, After Exposure
Figure 3. Cornea! mltottc rates.
long been known, for Instance, that partial hepatectomy
results In a stimulation of cornea! mltotlc rate (Exoerl-
entla, 2£:569-70, June 15, 1968) perhaps mediated by
adrenal gland function. Since the mltotlc rate changes
paralleled the hepatic damage, a similar physiologic
response may be functioning 1n this case.
In the tissue analysis for Mn, there was a slight
elevation 1n Mn levels In the lungs, liver, and kidney
Immediately after exposure and at 24 hr. After longer
Intervals of time, the Mn levels approached normal levels.
14
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UPTAKE, DISTRIBUTION AND EXCRETION OF 54Mn TRICARBONYL
W. Moore, D. Hysell, W. Crocker, and J. Stara
Through the cooperation of the Ethyl Corp., a
small quantity of Mn tricarbonyl was prepared for use
in tracer studies. 54Mn has a half-life of 303 days
and an 0.83 MeV gamma, which makes it ideal for this
type of approach. Although the values are reported as
5*Mn,..th1s should not be interpreted as to indicate that
the Mn was split off the compound. The fate of the
Mn tricarbonyl molecule following absorption is not
known. A further step must be the determination of the
metabolic products and whether or not Mn is split off
the molecule.
A group of fasted COBS rats, weighing approximately
200 gm, was given by intragastric intubation 2.5 mg (0.625
mg 54Mn) 54Mn tricarbonyl diluted in Wesson Oil. Whole
body counts were made immediately after dosing and periodi-
cally thereafter to determine the retention of Mn.
Twenty-four-hour urine and feces samples were collected
on the days the animals were counted. Another group of
animals was given the same dose of Mn tricarbonyl and
then sacrificed at different intervals of time to determine
the 54Mn distribution in the tissues.
The percent of Mn retained with time following
dosing is shown in Figure 1. Mn was rapidly eliminated
from the body with approximately 27% remaining after 24
hr. It was evident that the retention curve is composed
of several components with one component having a con-
siderably longer half-life (T^/2 = 24 days). Data Indi-
cated that the feces contained more 54Mn than the urine
(Figure 2). The urine/feces ratio varied from approxi-
mately 0.68 to 0.25; this ratio is in contrast to the
normal elimination of Mn, which is primarily fecal with
very little appearing in the urine. Whether the 54Mn in
the urine exists as a metabolite of 5^Mn tricarbonyl or
as the Mn salt has not been determined; however, these
findings plus other evidence would suggest that a Mn
metabolite of Mn tricarbonyl is present In the urine. In
other studies (Kettering report) where rabbits were either
Injected or painted with tritium-labeled Mn tricarbonyl,
nearly all of the tritium label was excreted in the urine
as an acid metabolite. The presence of Mn in the acid
metabolite was not determined.
15
-------
54
The distribution of Mn among the organs of the
rat followed a pattern similar to that reported for the
normal distribution of this element 1n animal tissues.
At one day post-exposure, the highest concentrations of
54Mn were found 1n the liver, lung, kidney, and pancreas,
liver, and kidney. Smaller amounts of 54Mn were found
In bone, brain, testicles, lungs, and blood.
1000.000-.-
KXJ.OOG -
Figure 1. Whole body
retention of 5*Mn
following 1ntra-
gastrlc administration
of Mn trlcarbonyl.
1QOOD -
f:
4
3
Urine/Fecal fatio
1 .557
3 .689
6 .309
10 .471
14 .250
It .381
Urine
6 8 10 12 M 16 18
Dnys, offer dosing
Figure 2. Excretion of
54
Mn following 1ntra-
gastrlc administration
of Mn trlcarbonyl.
30-
26-
22-
II
1
8 14
1*0 20 30 4'0 SO
Diys, after exposure
70 80
16
-------
UPTAKE AND DISTRIBUTION OF 54Mn IN PREGNANT RATS AND
FETUSES FOLLOWING ORAL ADMINISTRATION OF
54Mn TRICARBONYL
W. Moore, D. Hysell, and T. Wessendarp
An Initial study op the uptake, distribution, and
fetal concentration of b4Mn trlcarbonyl 1s reported.
Additional studies on the level of
fetal toxtdty are In process. In
rats were bred and they were given
of 54Mn trlcarbonyl (2.5 mg MMT).
were sacrificed on the 20th day of
fetuses examined for abnormalities
changes. No gross abnormalities were seen. The fetuses
and tissues from the dams were counted.for determination
of 54Mn. The mean concentrations of 54Mn for selected
tissues, measured as mean count l/m1n/g, are:
MMT required for
this study, female
a single oral dose
The pregnant females
gestation and the
and histologlcal
Maternal blood — 470
Fetus 88
Maternal Placenta 173
Bone 91
Liver 916
Lung 455
Kidney 470
11 Pancreas 658
17
-------
EFFECTS OF MMT ON LUNG CELLS GROWN IN TISSUE CULTURE
W. Moore, M. Colvin, and J. Everts
Arrangements were made with the Eastern Environ-
mental Radiation Laboratory to study the effects of MMT
on cells grown tn tissue culture. Lung cells were
chosen for the study as Inhalation 1s probably the most
important route of exposure. In an effort to delineate
the toxlclty of MMT from Mn toxldty, the study was
designed to compare MMT and MnSO, or MnCl« on primary
lung cells or on passaged lung cells that were Initially
grown out 1n bottles. In this way, the effect of these
compounds could be ascertained on cells taken directly
from the animals as well as on cells that had gone
through more than one division 1n tissue culture. For
chromosomal analysis, only primary cultures were used.
Approximately 1-mo-old Chinese hamsters were sacri-
ficed and the lungs removed for tissue culture. All
the lungs were pooled, trypslnzed, and seeded either
Into roller tubes at 7 x 105cells/tube or Into bottles
at 7 x 10b cells/ml. The medium consisted of Eagles
plus fetal serum and antibiotics. Since no significant
differences were noted 1n the effects of MMT on primary
cells and passaged cells, only the data pertaining to
the primary cells will be reported. The growth and
appearance in six tubes were analyzed for each dilution
using the standard procedure of scoring where 1 indi-
cates approximately 25% of the cells affected, 2 Indicates
50%, 3 Indicates 75%, and 4 indicates all the cells are
dead. A summary of three replicates of this study are
presented in Table 1.
There were no marked differences between the effects
of MMT and MnSO* although subjectivly there appeared to
be additional changes in the cells exposed to MMT that
did not occur 1n MnSO^ or MnCl2 exposures.
In the study for chromosomal effects, Lelghton
tubes containing cover slips were seeded along with the
roller tubes used 1n the toxiclty study. After 3 days,
Col chimed was added to the Lelghton tubes and the cover
slips were harvested 4 hr later using the routine pro-
cedure for preparing cells for chromosomal analysis. The
results of two replicates are presented 1n Table 2. Most
of the aberrations consisted of chromatld type lesions.
18
-------
TABLE 1. ANALYSIS OF GROWTH OF LUNG CELLS FOLLOWING
EXPOSURE TO MMT OR MnSO.
Compound
HMT
MnS04
Dilution
of Mn
9.8 x TO"7
9.8 x TO"8
9.8 x 10"9
9.8 x TO"10
9.8 x 10~7
9.8 x 10'8
9.8 x TO"9
9.8 x 10'10
Growth, davs after inoculation
1
+
-
-
-
+
-
.
-
Z
3
2
1
-
2
+
.
-
3
3
2
1
-
3
2
-
-
Contents
Sparse cell population
many granular cells,
very few growing
Approx. half of cells
growing
Some cells granular in
appearance and rounded
Growth normal
Many dead, other cells
granular, some growth
Some dead cells
Growth normal
Growth normal
1 Slight changes in appearance
1 Approximately 25% of the cell sheet affected
2 Approximately 50% of the cell sheet affected
3 Approximately 75% of the cell sheet affected
4 All of the cells are rounded up or detached
TABLE 2. PERCENT OF ABERRANT LUNG CELLS FOLLOWING EXPOSURE
TO Mn TRICARBONYL AND MnSO,
Compound
MKT
MnS04
Control
Exposure dilution
of Mn
9.8 x 10"7
9.8 x 10"8
9.8 x 10"9
9.8 x 10"10
9.8 x 10"11
9.8 x 10"12
9.8 x 10*13
9.8 x 10"7
9.8 x 10"8
9.8 x 10'9
9.8 x 10"10
9.8 x 10"11
9.8 x 10"12
9.8 x 10"13
Cells
scored
150
171
120
326
509
473
461
Cells
Cells
270
272
409
380
368
386
Number of
aberrants
5
6
4
14
23
12
9
died
died
6 •
9
12
2
4
6
,
3.33
3.50
3.33
4.29
4.51
2.53
1.95
2.22
3.30
2.93
0.52
1.08
1.55
19
-------
MANGANESE EFFECTS ON FIXED-INTERVAL PERFORMANCE
OF MONKEYS
M. I. Gage
Symptoms of manganese (Mn) toxicity are similar to
those observed 1n patients who have Parkinson's Disease;
notably tremor, difficulty 1n executing movements, and
other extrapyramldal motor system symptoms. Relief from
the symptoms of Mn poisoning has been obtained by the
administration of the drug 1-DOPA. The motor difficulties
associated with Mn toxicity have been produced In the
Macaque monkey and the chimpanzee by subcutaneous In-
jection of manganese dioxide (Mn02). A decrease 1n do-
pa mine In the caudate nucleus of Squirrel monkeys Injected
with Mn02 has also been reported. Based on these findings,
a pilot study was begun to determine 1f an objective, re-
peatable behavioral measure of motor responses can be
used to estimate the toxic threshold dose level of Mn.
Four young rhesus monkeys (Macaca mulatta), one male
and three female, were trained to push a round, plastic
button to obtain banana flavored food pellets on a fixed-
Interval, 1-mln. schedule of reinforcement (FI l-m1n.).
A pellet was dispensed for the first press made after
61.44 sec had elapsed since the last pellet was dispensed.
The monkeys were given half-hour sessions dally, 5 days/wk.
After performance on this schedule stabilized and the
pattern of presses during the Interval assumed the typi-
cal scallop shape (the frequency of responses Increased
as the Interval approached termination), two of the mon-
keys were given a single subcutaneous Injection of 400
mg/kg MnO~ suspended In olive oil in the mid torso area
and the other two monkeys were Injected with a similar
volume of olive oil. Performance continued to be ob-
served on a dally basis after the injections. Approxi-
mately 3 mo after the manganese was administered, blood
and 24-hr urine samples were analyzed for Mn and urine
was analyzed for delta am1no1eve1lin1c add concentration.
The experiment 1s still in progress so only pre-
liminary analysis of the findings can be presented.
Three monkeys (the two injected with MnO« and the male
control) showed a slight decrease In response rates on
20
-------
the FI 1-min. schedule beginning about 3 mo after MnO?
administration. The male's decrease, however, startea after
the decrease began in the treated monkeys. This rate de-
crease seemed to be progressive, but it is too soon to
fully evaluate this trend. Blood and urinary Mn levels
taken soon after the onset of the behavioral change were
low and in the normal range in all monkeys; however, Mn
levels in the treated animals were slightly higher than
the levels in the controls.
In other reported experiments, the onset of observable
motor symptoms of manganism began about 3 mo after Mn02 in-
injections in chimpanzees but began 9 mo after Mn02 was
administered to rhesus monkeys. The cause of this vari-
ability is unknown. Monkeys in the present pilot study
are not displaying any obvious motor difficulties during
the fourth month since MnOp injections. Possibly enough
time has not yet elapsed for the observable symptoms of
manganism to begin.
The behavioral measure of response rate on a learned
operant task may be quite sensitive for evaluating perform-
ance changes due to small increases of Mn in the body.
The decreases seen in the injected monkeys may represent
early signs of manganism. The small sample size in the
present study, however, makes it difficult to determine if
the test is a good general purpose screening tool. The
experiment needs to be continued until the injected mon-
keys display obvious abnormalities of motor behavior and
needs to be repeated on a larger sample of monkeys.
21
-------
EFFECT OF MANGANESE ON THE RAT VISUAL EVOKED POTENTIAL
J.P. Lewkowski, W. Moore, and J.F. Stara
The application of various computer averaging tech-
niques has enabled investigators to observe changes in
the visual evoked potential. Consequently, a wealth of
information has been accumulated on the topographical
distribution of various waveforms and the effects of drugs
on these evoked potentials.
Furthermore, although some work has been done on the
effects of pollutants on the visual evoked potential, few
investigations have been performed on the effects of
various toxicological agents such as the heavy metals. The
effect of manganese is currently being tested since this
cation 1s presently used as a fuel additive.
Preliminary experiments have indicated that the intra-
venous administration of low levels of manganese elicits a
transient but highly reproducible change in the rat visual
evoked potential. Figure 1 shows the results of one such
experiment. A is the control evoked potential before the
manganese injection. Immediately after the control was
recorded, 1.4 mg/kg of manganese was administered. The
effect 5 min. after the injection can be observed in B.
However, the control waveform is elicited 10 min. after the
manganese administration (C).
The fact that the averaged evoked response is reprodu-
cible and remains somewhat constant can be seen by comparing
C and D which were recorded 10 and 30 min. after the man-
ganese administration. C and D are also comparable to A,
which was recorded before the manganese injection.
An additional 1.4 mg/kg of manganese was administered
immediately after D. Again, a change in the evoked po-
tential is elicited approximately 90 msec, after the light
flash. Control evoked potential waveforms are again ob-
served 10 min. after the manganese injection.. Immediately
after record G, a comparable volume of saline was administered.
Little, if any, change is evident in H which was recorded
5 min. after the saline injection.
Thus, the administration of low levels of manganese
has been shown to elicit a reproducible change in the rat
visual evoked potential. Further work is currently under-
way to determine the mechanism responsible for the observed
change as well as to determine the effects of various oher
cations.
22
-------
A. THE CONTROL AVERAGED EVOKED POTENTIAL
PRIOR TO THE ADMINISTRATION OF MN
E. FIVE MINUTES AFTER ANOTHER
ADMINISTRATION OF 1.1 MG/KG
FIVE MINUTES AFTER THE INTRAVENOUS
ADMINISTRATION OF 1.4 MG/KG
F. TEN MINUTES AFTER THE
ADMINISTRATION
C. TEN MINUTES AFTER THE
ADMINISTRATION
G. FIFTEEN MINUTES AFTER THE
ADMINISTRATION
D. THIRTY MINUTES AFTER THE Mw
H. FIVE MINUTES AFTER A SALINE
ADMINISTRATION OF COMPARABLE
VOLUME
Figure 1. Effect of low levels of Mn, administered
intravenously, on rat visual evoked
potential.
23
-------
ENVIRONMENTALLY BOUND LEAD: I. BLOOD LEVELS IN RATS
FOLLOWING A SINGLE ORAL DOSING
O.F. Stara
M.K. Richards, S.
W. Moore and K.
Neiheisel
Bridbord
Y.Y. Yang
For the process of setting meaningful environmental
standards, it 1s necessary to obtain biological data for
exposure to lead as 1t actually occurs 1n the environ-
ment. As a source of environmentally bound lead, dust
samples were collected 1n New York's Queens Tunnel, the
Los Angeles Freeway, and the Immediate vicinity of the
El Paso smelter. The dust samples were separated by
sieving and analyzed for lead content. The fine parti-
cles were used for animal exposures since they contained
higher amounts of lead (0.6-2.4%) and also because of
their greater potential biological activity. Lead
analyses of the dust samples were performed at four
different laboratories (Table 1).
TABLE 1. LEAD CONTENT (%) IN THREE DUST SAMPLES
Site of collection
Queens Tunnel, N.Y.
Los Angeles Freeway
Vicinity of
El Paso Smelter
Laboratory Analyses
A*
5.78
3.20
2.81
B
1.7
1.03
0.77
C
2.53
1.06
0.64
D
2.43
1.04
0.61
*Values from Lab A were discarded because of analytical
error.
Gelatin capsules filled
of sufficient quantity to de
were orally administered to
samples (0.2 ml) were taken
36 days following Pb Ingesti
for lead content by the New
Health Laboratory. The mean
in Figure 1. Twenty-four ho
blood level in the experimen
with dust from Queens Tunnel
liver a dose of 10 mg of Pb
a group of rats. Daily blood
from the orbital sinus up to
on. All samples were analyzed
York State Department of
blood levels are presented
urs after dosing, the lead
tal group rose to an average
24
-------
of 45 g/100 ml and thereafter decreased sharply so
that by day 15, it did not differ significantly from
controls. The mean blood levels of the control animals
were approximately 10-15 g/100 ml; these agree with
values reported in the literature. Similarly, tissue
analyses showed increased concentration of lead in the
bone and G.I. tract of the experimental animals.
SOir
40
o
o
30
1C
_Exgo$e
-------
ENVIRONMENTALLY BOUND LEAD
II. EFFECT OF DOSE ON BLOOD AND TISSUE LEVELS OF RATS
J. Stara, W. Moore, M. Richards,
S. Neiheisel, R. Miller and K. Bridbord
The effect different dose levels have on lead ab-
sorption in rats was tested by feeding Queen's Tunnel
dust at dose levels of 0.5, 1.0, and 5.5 mg lead/day
in a specially-prepared diet. Figure 1 shows the fitted
curves of the mean blood levels; the dose dependency is
clearly indicated. An expoential equation was used in
fitting a curve to the data. The lead level rose to a
peak of 55 yg/100 ml on day 9 in the high dose group
(5.5 mg). Peak blood levels reached 37 yg/100 ml in
the medium dose group (1.0 mg) and 33 yg/100 ml in the
low dose group (0.5 mg). After the blood levels reached
their highest plateau, a slow descreasing trend was ob-
served. This decreasing level may be due to such factors
as growth, aging with reduced G.I. absorptive capability
for lead, biological saturation of the system, or other
unknowns.
Blood Levels of Rots Fed Different Daily Doses in Their Diet
K m
50
40
-e
§
| 30
S
.a
a-
S20
10
*
/ 1.0 mg f(t)=5IJ3We-*ra7t-4764M«"32'5r~~~
• ' ^^
f / ,' ^^^ """""*-^
/ /it)=49.5755e-w25-l4I3H2e-"7'rf ^""^ ^
^* / * *N^.
' /
//
1 / Control Kf>»44l006e- 03341i434204e-J)6138f
'/-^ ""-- .
Illlllllfll
4 12 20 28 36 44
Days
Figure 1.
Fitted mean blood levels of rats fed different
daily doses of lead in their diet; vehiclerdust
collected in Queen's Tunnel, N.Y.
26
-------
A large accumulation of lead was seen in the skeleton,
and the kidney (Table 2). Other tissues such as blood,
brain, liver, and lung also showed significant increases
in the treated group of animals.
TABLE 2. TISSUE LEVELS OF LEAD IN RATS FED
DIFFERENT DAILY DOSES
Tissue
Femur
Kidney
Liver
Brain
Dose (yg Pb/g Tissue)
5 . 5mg
66.1
9.4
1.3
0.28
1 .Omg
33.6
3.2
0.52
0.055
0. 5mg
25.7
2.5
0.56
0.030
Control
0.75
-
0.13
0.032
Organ
Skeleton*
Kidney
Liver
Brain
Dose (pg Pb/organ)
5.5mg
1487.0
30.8
13.5
1.0
1 .Omg
551.0
7.7
3.8
.15
0. 5mg
412.0
5.9
3.9
0.08
Control
11.9
1.3
.98
.08
*Total bone (skeleton) estimated as 7.41%
of body weight (unpublished data).
27
-------
ENVIRONMENTALLY BOUND LEAD:
III. EFFECTS OF SOURCE ON BLOOD AND TISSUE LEVELS
OF RATS
J. Stara, W. Moore, M. Richards, N. Barkley,
S. Neiheisel and K. Brldbord
To determine if the biological availability of
environmentally bound lead, as measured by G.I. absorp-
tion and resulting blood uptake, may vary with the
source, dust samples collected from Queen's Tunnel, N.Y.,
Los Angeles Freeway, and the vicinity of El Paso Smelter
were used in this experiment. Three groups of rats were
fed a special low-lead diet for 55 days. Each group
received one type of dust that was mixed in the special
diet at the rate of 1 mg Pb/day. The results (Figure 1)
show that the highest absorption, as measured by the
blood level (45 yg/100 ml), were observed in animals fed
the Los Angeles dust samples. The rats fed the New York
dust had intermediate values, and the lowest values
were obtained in the El Paso group of animals. A curve
was fitted to the data by use of an expoential equation.
Analysis of all blood and tissue data is not complete at
this time.
50-
40
30
£20?
10
Blood Levels in Rots fed Dust Containing Img
Pb in Doily Diet
/,
/
1 /
/
./
Control
LA. f(t
N.Y. f(t
I. r. ?tl/=3«p. • «w«rv •^•^wnrrv .,
C f(t)-44J806e-°3Mai3.42Me-fl6138f
_J^_^^^_^^L^^^^^^^^^^^^^^^^^^^^^^^^MB^^M^^^K
412 20 28 36 44
Days
Figure 1
Fitted mean blood levels 1n rats fed 1 mg
lead containing dust in daily diet; collection
sites: Queen's Tunnel, Los Angeles Freeway and
El Paso Smelter.
28
-------
The uptake in tissues and resulting concentrations,
however, indicated that the lead in New York dust was
most readily absorbed. The tissue levels for the New
York dust were, on the whole, higher than in the other
two samples (Table 1). Of the organs examined, the
highest concentrations were observed in the bones, kid-
neys, and liver, in that order.
TABLE 1. TISSUE LEVELS OF LEAD IN RATS FED DAILY
1 mg OF Pb IN DUST COLLECTED FROM THREE
SOURCES
Animal
Source
N.
Y.*
LA
• fi
.+
E. P
*
Control
Organ, yg Pb
Ti
Skeleton
Kidney
Liver
Brain
ssue, vg Pb/am
Femur
Kidney
Liver
Brain
551
7
3
33
3
0
0
.0
.7
.8
.15
.6
.2
.52
.055
532.
6.
2.
•
32.
2.
0.
0.
0
4
3
25
5
7
32
094
375.
5.
2.
•
23.
2.
0.
0.
0
9
5
10
6
5
36
035
11 .9
1.3
.98
.08
0.75
-
0.13
0.032
* Queen's Tunnel, New York City
+ Los Angeles Freeway
* El Paso Smelter
It is extremely difficult, using stable elements
and standard techniques, to determine precisely the
absorption of a metal like lead where the amount absorbed
is very small (less than 5%). Using radioactive tracers,
which permit the detection of very low concentrations in
various tissues, is the most accurate method.
29
-------
LEAD: PLACENTAL TRANSFER, CENTRAL NERVOUS SYSTEM EFFECTS,
AND IMMUNE RESPONSE ALTERATION
Dr. W. B. Buck et al., under a contract with EPA
is investigating the placenta! transfer of lead and
characterizing the neurophysiological and behavioral
effects of lead exposure in lambs. In addition, they
are exploring the effect of lead on the immune response
mechanism.
To investigate the placenta! transfer of lead,
three groups of yearling ewes were fed powdered metallic
lead beginning 3 weeks before breeding, continuing
throughout gestation, discontinuing at parturition. All
animals were then trained and tested on a series of
seven visual discrimination problems (Figure 1). The
results are observed in Table 1. The authors conclude
that lambs from ewes fed subclinical levels of lead
during gestation had statistically significant decreased
performance on visual discrimination tasks 1n comparison
with lambs from nonexposed ewes.
Figure 1.
Analysis of lead
transfer through
the placenta to
the rat fetus are
in progress. These
data will be com-
pared with data
received in the
lamb study. A
determination of
the effect of lead
on learning and
problem-solvi ng
ability is current-
ly being investi-
gated through the
use of a modified Hebb-Wi11iams maze. In addition, experi-
ments to ascertain the effect of lead exposure on serum
immune protein of young sheep and their immune response
to bacterial antigens are also in progress. A comprehensive
treatise on the effects of lead in the three areas studied
will be available upon the completion of these studies.
30
-------
TABLE 1. VISUAL DISCRIMINATION PERFORMANCE AND BLOOD LEAD IN LAMBS
PROM EWES FED SUBCLINICAL LEAD DURING GESTATION
Prenatal Lead
Exposure Group
Control13
(X of 4 animals)
"Low" Leadc
(X of 8 animals)
"High" Lead3
(X of 6 animals)
Group Moan Dayo to Criterion for Visual Discrimination Problems
Problem Number
12 3 " 5 6 - - ? nTf?Lo?rmn Blood ^ in P™
Problem Age (weeks)
VDa OA =1111 VA ®t% CD 0 O 2-4 10-12
3.5 6.7 4.3 3.3 4.8 4.3 12.6 41.8 0.06 0.04
3.1 6.4 4.4 5.5 4.4 -9.8 13.0 46.5 0.17 0.09
3.5 8.3 4.0 6.3 5.3 16.7 29.5* 73.7* 0.27 0.14
Light versus dark.
The lambs in this group are from ewes which received no supplemental lead during gestation. The
mean blood lead of these ewes was 0.06 ppm (6 gg/100 ml) during gestation.
cThe lambs in this group are from ewes which received 2.3 mg lead/kg body weight daily throughout
gestation. Mean blood lead of these ewes was 0.16 ppm (16 ug/100 ml) during gestation.
^The lambs in this group are from ewes which received 4.5 mg lead/kg body weight daily throughout
gestation. Mean blood lead of these ewes was 0.30 ppm (30 ug/100 ml) during gestation.
•Significant at P<0.05.
31
-------
GASTROINTESTINAL ABSORPTION OF DIFFERENT COMPOUNDS
OF 115mCd AND THE EFFECT OF DIFFERENT
CONCENTRATIONS IN THE RAT
W. Moore, J. F. Stara and W. Crocker
Several studies were undertaken to determine kinetic
and metabolic fate of different compounds of cadmium to
provide information not currently available in the
literature.
An investigation was designed to determine whether
or not different chemical forms of this element influenced
the absorption and metabolism of Cd following oral admin-
istration. The retention rates llbm
lom
MDmcadmium
single dose
The retention rates for mcadmium chloride,
sulfate, and ' lomcadmium acetate following a
given via stomach tube are shown in Figure 1.
Figure 1
Retention rates of
three cadmium com-
pounds after rats
received single
doses.
TO •
60
SO
4O-
30-
.2 Z"
H
-I,,
X C <
'CdSGU _
•CcKCI+jCO)2
-j-» **-l*M| **
>CdCI2
8 12 16 20 24
Days, After Dosing
28 32
The results Indicated that the type of cadmium com-
pound did not significantly influence the G.I. absorption
and retention.
32
-------
of 115mCd although the amount of 115mCd retained follow-
ing administration of 115mca(jm-j urn acetate was somewhat
higher. There was an initial rapid clearance during the
first 4 days, which represented the passage of nonabsorbed
cadmium through the intestinal tract. Fasting the animals
for 24 hr did not significantly influence absorption or
the time required for transit of the 115mcd compounds
CdCl2 through the G.I. tract. In the group of rats that
was not fasted before dosing, 58.0% of the M5mca(jmium
chloride was retained at the end of 24 hr and 3.01% at
the end of 4 days, whereas in the fasted group, the values
were 55.6% and 3.1%, respectively. Thus, after 6 days,
between 2.7% and 3.5% of the 115mcd was absorbed when
given as cadmium chloride, cadmium sulfate, or cadmium
acetate.
Radioactive counts of the feces and urine (Figure 2)
showed that the most of llSmcd was eliminated via the
feces and only an extremely small amount was found
Figure 2
Percent of original
dose of 115mcadmium
excreted in feces
and urine following
oral administration,
60
40
3O
1O
6
4
-O
•
O 1.0
!i .6
.3
.08
M
.04
.03
.01
Urin* •—•
F«c«t •—•
10
20
30
Day*
33
-------
in the urine. Twenty-four hours after administration
of the 115mcadmium acetate, 52.1% of the initial dose
was present in the 24-hr feces sample and 0.025% of
the initial dose was in the 24-hr urine sample. Six
days after dosing, 0.22% and 0.013% was found in 24-hr
samples of feces and urine, respectively. After the
first few days, there was a very low continuous rate
of excretion of H5mcd in the feces during the entire
period of study. At 32 days post exposure, 0.026% of
the initial dose was present in a 24-hr feces sample.
After a single oral dose, the only tissues containing
significantly the concentration or distribution of
llbmcd in the tissues.
Three different levels of 115mCdCl2 were given
orally to rats to investigate the effect of dose upon
Cd absorption and body retention. The amount of Cd
given influenced the amount absorbed by the G.I. tract
and resulting tissue concentrations. The mean amount
of Cd in the livers and kidneys (yg/g wet sample) for
the three concentrations is shown in Table 1. The
animals receiving the highest concentration had the
greatest amount of Cd in the liver and kidney although
the increase was not proportional to the increase in
concentration.
TABLE 1. TISSUE CONCENTRATIONS OF CADMIUM
Amount Cd Administered,
mg/animal
0.060
0.75
7.5
yg Cd/gm of Tissue,
wet weight
Kidney
0.073
0.253
0.772
Liver
0.102
0.772
1.564
34
-------
EFFECT OF DIFFERENT ROUTES OF ADMINISTRATION OF
llBmcADMIUM CHLORIDE UPON WHOLE BODY RETENTION IN RATS
W. Moore, J. Stara, M. Malanchuk and R. Iltis
The influence of the route of administration (intra-
venous, intraperitoneal, intragastric, and inhalation) on
Cd retention was determined and biological half-life
calculated (Figure 1). The retention curve for each of the
routes of administration was divided into two components.
The first component reflected the initial rapid clearance
of 115mcd primarily by the G.I. tract and the second com-
ponent indicated the absorption and turnover of 115mcd.
Extrapolation of the second component to the intercept
gave initial retention values of 93%, 91%, 41% and 2.3%
for intraperitoneal , intravenous, inhalation, and oral
routes, respectively. The biological half-life of the
major retention component was greater than 175 days for
all routes.
2 \
S:\
JO »
.1
if
- \
\
•—*..^...,
.. f...^..,
1 MiolotiM
Oral
»7/6o
Days, After Dosing
Figure 1.
Retention curves indication percent
whole body retention of IIDmCd in
rats following inhalation exposure
to H5mcdCl and H5mcdO
35
-------
RETENTION OF 115mCADMIUM CHLORIDE AND
OXIDE FOLLOWING INHALATION EXPOSURE
W. Moore, M. Malanchuk, R. Miller and W. Crocker
5mCdC12 and 115mCdO
hown in Figure 1.
isiderably more '15mCdCl2
[values for the liver
5n»CdCl2, which indicates
r Cd by the lungs. The
rr., 54£ of the initial lung
mCdd2 was present in the lungs 64 days post-
The whole body retention of
following inhalation exposure is s
Following inhalation exposure, con
than 115racdO was retained. Tissue
and kidney were also higher for
greater absorption of this form of
data indicated that approximately
burden of ''5
exposure.
100
90
80
70
6C
SC
30
20
10
—I
•t-t
2 4
W 12 M 16 W 20 22 24 26Z8303Z
D«y$, Afttr
Figure 1. Retention curves indicating percent whole
body retention of HSmcd in rats following
inhalation exposure to «'5mCdCl2 and M5mc
36
-------
EFFECT OF CADMIUM INGESTION ON
BLOOD PRESSURE IN MONKEYS
M.J. Wiester, W. Moore and J.F. Stara
A number of investigators have theorized on the
probability that environmental intake of cadmium may
be intimately involved in various types of pathological
processes throughout the life span of man. Cardio-
vascular, as well as renal, disease has been associated
with cadmium exposure both in animals and man.
Presently, a pilot study is in progress that will
indicate dietary levels of cadmium that will produce a
pressor effect in rhesus monkeys. Parameters other than
blood pressure that are observed are weight, urinalysis,
and terminally, tissue cadmium levels, particularly that
of the kidney cortex. In this pilot study, two rhesus
monkeys are fed 100 mg/day CdCl2 along with their regular
diets. With this regime, we anticipate a pressor effect
after approximately 2 mo. Conclusions drawn from this
brief study will be incorporated in a more extensive
attack on the problem using monkeys as the primary animal.
The animals are monitored once a week throughout the
experiment. Control levels of blood pressure were estab-
lished over a period of 8 wks. before cadmium feeding. To
measure blood pressure, monkeys are tranquilized with
sernylan (2 mg I.M.) and chaired; pressures are read 2-3
hr. later when animals are alert. Measurements are made
by use of noninvasive tail cuff method, which records
systolic and diastolic pressure. This method has been
verified by a series of experiments in which cuff pressures
were compared with directly measured abdominal aortic
pressures. The results agreed within 5-8 mm Hg (personal
obsdrvations). Catherized urine specimens are taken while
the animals are under the influence of the drug. In this
study, no precautions have been taken to keep the animals
endemically cadmium-free or to control zinc intake. A
diet identical to the one used for the last 3 yr. is con-
tinued with the addition of 100 mg/day of CdCl2 during
the experimental period.
Since there is normal variation in blood pressure from
monkey to monkey and small numbers of animals are involved,
each animal will serve as its own control. The control period
pressures for the monkeys are summarized in Table 1. On
12/5/72, the two monkeys were started on the cadmium con-
taminated diet. Analysis of the data will depend on the
magnitude of pressure increase as well as the variations
encountered as the hypertension progresses. Variations in
37
-------
TABLE 1. BLOOD PRESSURE READINGS OF
MONKEYS DURING CONTROL PERIOD
Animal
Monkey
18
Monkey
#29
Systolic Diastolic Mean
pressure, pressure, pressure,
mmHg mmHg mmHg
10-5-72
10-13-72
10-18-72
10-26-72
11-9-72
11-21-72
11-29-72
12-5-72
Mean
for
period
10-5-72
10-13-72
10-18-72
10-26-72
11-9-72
11-21-72
11-29-72
12-5-72
Mean
for
period
114.0
115.5
97.0
108.5
113.3
106.5
98.8
99.5
x 106.6
S.D.+ 7.4
125.0
138.3
134.3
121.0
136.6
113.5
116.6
138.7
x 128.0
S.D.+.10.2
80.0
65.0
84.5
66.5
66.0
70.0
66.2
66.0
x 70.5
S.D.+ 7.5
85.0
91.3
89.3
90.3
94.2
71.2
74.3
99.7
x 86.9
S.D.+ 9.7
91.3
81.3
88.6
80.5
81.7
82.1
77.0
77.2
x 82.5
S.D.+ 5.1
98.3
106.9
104.3
100.5
108.3
85.3
88.4
112.7
X 100.6
S.D.^ 9.6
38
-------
140-
120-
£ 100-
SO-
60-
MONKEY 029
CONTROL PERIOD
9 SYSTOUC
• MEAN
X DIASTOLIC
10-5-72 10-15
10-25
11-4
12-4
DATE
Figure 1. Variations in Tail-Cuff Blood Pressure
From An Unanesthetized Monkey over 8-
Week Control Period
weekly blood pressure measureraents for the control period
are shown in Figure 1. Experimental pressure readings
will be analyzed in a classical way by observing the dis-
tribution trend of pressures versus time, which yields a
definite increase in slope. If variations in weekly
readings are great 4nd the pressor effect extends into
a long period of time, however, mean values and changes
in standard deviation for successive segments of time will
be considered.
An increase in blood pressure, as well as an esti-
mate of renal damage (proteinuria), is anticipated as
the cadmium ingestion period progresses. These changes
will be correlated with time-cadmium ingestion and kidney
tissue cadmium content.
39
-------
EARLY BIOCHEMICAL EFFECT OF
METHYLMERCURY CHLORIDE IN RATS
S. Lee, K. Butler, R. Danner, B. Johnson,
L. McMillan, W. Moore and J. Stara
Several investigators have reported on biological
effects of various pollutants. There still is a
paucity of information concerning the effects of
pollutants on biochemical interactions, however,
especially of those at low, relevant concentrations
of environmental pollutants where in vivo systems in
nonterminal experiments have been use
-------
change in the physiological or biochemical state of the
animal. Gary vibrating reed electrometers were employed
in conjunction with ionization chambers to measure
radioactivity in the expired air. The animal exposure
system and associated instrumentation are depicted briefly
in Figure 1.
ROWMETEftS
ONIZATION 3-ww SOLENOID WUVES
METABOLISM CAGES CHAMBERS fi ~ ' "
1 DRYRITE i Jj
WET TEST METERS
MANIFOLD
\|
4 VIBRATING REED
ELECTROMETERS
MULTIPOINT
RECORDER
COo
RECORDER
Figure 1. Diagram of Radiorespirometry Flow System
Intragastric administration of
tressed '^CO? output following
Following
supp
l-'*C-glucose.
and 0.10 mg/kg body weight,
respectively, in respired '
same treatment was repeated
metabolic suppression of 17
30.8% for the 0.10 mg group
show that methylmercury chloride at low
0.10 mg/kg body weight caused metabolic
methylmercury chloride
intravenous injection of
the initial treatment with 0.05
decreases of 6.6% and 12.1%,
C02 were observed. When the
for each group of animals,
9% for the 0.05 mg group and
were observed. These results
levels of 0.05 and
alteration as
measured by the radiorespirometric technique. This approach
demonstrated changes as early as 24 hr. after ingestion.
The observed effect apparently is cumulative, since a second
dose, given 1 wk. later caused an almost three-fold decrease
in expired 14CO£ when compared with the level recorded
following the initial dose (Table 1).
41
-------
TABLE 1. EFFECT OF REPEATED DOSES OF METHYLMERCURY
CHLORIDE ON CUMULATIVE 14C02 OUTPUT AFTER
14C-1-GLUCOSE INJECTION
Experimental First Dose, Second Dose,
Group % Alteration % Alteration
Control
0.05 mg/kg B.W.
0.10 mg/kg B.W.
0
- 6.6
-12.1
0
-17.9
•
-30.8
In the concentration range used, there is no apparent
dose response to a single-dose administration of CH3HgCl
on blood ?glutathione (Figure 2) . A greater decrease in
blood glutathione concentration was observed with respect
to time, however, after CHsHgCl was administered. The
implication of this finding is a very broad one because
numerous enzymes in our body require presence of optimum
amount of glutathione for their normal functions. In vitro
evidence in the literature supports this contention; however,
our finding suggests the necessity of in vivo studies of
specific enzymes in relation to the time of C^HgCl admin-
istration. With additional experimentation, including the
more time-consuming "constant infusion technique" and use
of '^C-glucose labeled in other positions, it may be
possible to demonstrate changes in relative participation
of various pathways in glucose metabolism.
One must be cautious in interpretating these prelim-
inary data, the changes reported herewith may or may not
be detrimental to the animals, especially if the changes
are temporary. Furthermore, the toxicity of methylmercury
appears to be influenced by other factors such as the
composition of the media. Ganther et al. recently demons
strated that 20 ppm methylmercury in a diet containing M%
(by weight) tuna was less toxic than the same concentration
of methylmercury in corn-soya diet fed to Japanese quail.
These investigators also showed that selenium content in
the diet, comparable to that contained in the tuna diet,
decreased methylmercury toxicity in rats. Tuna meat contains
a relatively high concentration of selenium and tends to
accumulate additional selenium when mercury is present.
42
-------
70
60
1 50
~E 40
o
c
I 30
in
C
20
0)
10
l.Omg/kg BW
2.0mg/kg BW
.A /\ A
VVV
AAA
v x/ w
'\ /\A
VVV
A A /V
V V V
\ A /V
VVV'
f\/\/\
vv v
AAA
VVV
/\ A A
v v v
AAA
\/\/w\.
VVV
\/\A/\
VVV
/VVV
VAAA
S.Omg/kg BW
AAA
VV •"
AA/
VVV
AAA
VVV
AA/\
VVV
AAA
VVV
AAA
vvv
AAA
VVV
/\/\/v
VVV
/\/\/\
VVV
/\A/\
VVV
/\/\ A
vvv
0 24 48
0 24 48
0 24 48
Hours After Initial Dose of Methyl Mercuric Chloride
FIGURE 2.
Single Dose Levels of Methylmercury Chloride
and Its Effect on Whole Blood Glutathione.
These authors concluded that selenium in tuna is not
a hazard in itself and that it may lessen the danger
of mercury poisoning in man following ingestion of
mercury-contaminated tuna.
43
-------
EARLY BIOCHEMICAL EFFECTS OF 03 AND NO? AND
INFLUENCE OF VITAMIN E ON THESE EFFECTS
S.D. Lee, R.M. Danner, K.C. Butler,
D.B. Menzel and J.F. Stara
Trace amounts (ppm) of the air pollutants 03 and
N02 rapidly oxidize polyunsaturated fatty acids (Figures
1 and 2). Phenolic antioxidants retard this oxidation
(Figure 3); vitamin E decreases the acute toxicity of
both 03 and N02- On continuous exposure to 1.5 ppm of
03 the LT5Q for vitamin E depleted rats was 8.2 days,
compared with 18.5 days for continuously supplemented
rats (Figure 4). Similarly the LTso for depleted rats
exposed to 33 ppm N02 was 11.1 days versus 17 days
(Figure 5). Exposure to 0.5 ppm of 03 also accelerated
the depletion of vitamin E from erythrocytes of exposed
animals in 23 days versus 36 days for unexposed animals.
The polyunsaturated fatty acid content of lung tissue
significantly declined in rats fed a constant fatty-acid-
composition diet free of vitamin E, or exposed to N02, or
both. The 03 exposure decreased the oleic and linoleic
acid content but increased the arachidonic acid content
(Figure 6, Table 1). These changes may be complex re-
sponses of the lung to increased oxidant stress, as shown
by depression of serum reduced glutathione (Figure 7) and
by tissue sulfhydryl compound content, or may be related
to other metabolic roles of vitamin E in the biosynthesis
of polyunsaturated fatty acids.
5.4 ppm
0.6
I.Oppm
IOO.4J
ro
CM
O.Sppm
CONTROL
20
TIME (hours)
25
30
Figure 1. Oxidation of Methyl Llnolenate In
Atmospheres Containing N02: Diene
Conjutgation.
44
-------
o.r
0.4
0.2
DIFFERDiCe SPCCTR/'.
2,5 HOUR SAMPLE vs. KETHA'iOL
300 200
250
300 MI
Figure 2.
Spectra of Methyl Linolenate Exposed To
5.4 ppm of N02 (0.1 mg per ml Methanol )
45
-------
60O
K
UJ
CO
400^
z
<
CO
ZOO-
CONTRO!
BHA
60 160
TIME (minirtes)
Figure 3. Oxidation of thin films of methyl linoleate in the presence
of IS ppm of NO> TBA number equals ppm of malonaldehyde,
»*•
o
X
*
10 4
DAV J
20
Figure 4. Mortality of vitamin E-dcficicnt and supplemented rats
exposed to NOj and O>. A. O1 exposure to 1.5 ppm. B. NO.
exposure to 33 ppm. Solid lines represent vitamin E-dcficicnt
rats; dashed lines, supplemented rats.
46
-------
Figure 5.
Linoleic Acid Concentration of Lavage
Lipids From Rats Exposed to 1.6 ppm
Ozone or 3.0 ppm Nitrogen Dioxide,
12
10
Days of Exposure
20
TABLE 1. TOTAL FATTY ACID COMPOSITION OF LUNG TISSUE LIPIDS FROM
RATS EXPOSED TO 1.0 ppm 03 FOR 9 DAYS*
Mole % (+SEM) of total lung tissue fatty acids**
Fatty acid+
14:0
16:0
16:1
18:0
18:1
18:2
18:3
20:4
20:5
22:5
22:6
Supplemented
Control
1.07 + 0.11
24.68 + 0.81
4.97 + 0.49
9.73 + 0.67
24.09 + 1 .87
7.65 + 0.45
6.19 + 0.67
6.01 + 0.59
3.25 + 0.68
5.35 + 1.02
1.67 + 0.10
Deficient
Control
1.08 + 0.11
25.16 + 0.34
4.85 + 0.33
9.72 +• 0.63
23.85 + 2.58
7.55 + 0.47
6.14 + 0.98
6.01 + 0.90
3.67 + 0.51
4.77 + 0.75
2.05 + 0.54
Supplemented
03 exposed
1 .03 + 0.11
27.62 + 0.65
5.22 + 0.67
9.76 -i- 0.83
23.42 + 2.99
6.97 + 0.47
4.98 + 1.06
7.18 + 0.93
3.27 +• 0.86
4.12 +• 1.26
1.64 + 0.23
Deficient
03 exposed
0.83 + 0.04
29.22 + 0.84
4.34 + 0.20
9.73 + 0.37
20.70 + 0.47
6.14 + 0.74
3.43 + 0.21
8.87 + 0.81
2.21 + 0.16
5.80 + 1.67
3.50 + 0.43
*Minor components omitted for clarity .
+The notation for fatty acids is X:Y, where X is the number of carbon atoms
and Y the number of unsaturations.
^Number of animals in each group was six.
47
-------
CD
20 40 60
EXPOSURE TIME (DAYS)
80
Figure 6.
BLOOD GLUIATHIONE LEVELS OF RATS EXPOSED
EITHER TO FILTERED AIR OR 0.5 ppm 03
(SOLID LINES - CONTROL, INTERRUPTED LINES
03 EXPOSED).
48
-------
ALVEOLAR PROTEIN ACCUMULATION: A SENSITIVE
INDICATOR OF LOW-LEVEL OXIDANT TOXICITY
S.D. Lee, R.M. Danner, S.M. Alpert, B.B. Swartz
and T.R. Lewis
Studies of response to low doses of edematogenic gases
have been hampered by the insensitivity and the nonquanti-
tative nature of the major indicators of response. Occur-
rence of edema in rats lungs following exposure to 0.67
ppm 03 for 7 days is shown in Figure A new and more
sensitive indicator, the recovery of 131I-albumin from
the alveolar spaces 6 hr after its intravenous injection
in rats, has been applied. Significantly increased albumin
recovery was found for all concentrations of 03 at and
above 0.5 ppm, and there was no consistent histologic
finding except for slight sloughing of bronchial epithelium
at 2.5 ppm (Figure 2). Application of these methods to
studies of steroid effects revealed increased sensitivity
to 03 following administration of methylprednisolene sodium
succinate. In addition, animals treated with steroids
before exposure to 0.25 ppm 03 became tolerant to subsequent
03 challenge, whereas animals given preexposure but no
steroids did not.
;
ED
.
Figure 1. Excised rat lung exposed to 0.67 ppm
03 compared with control.
49
-------
two
1000
900
800
700
~ 600
0
"m 500
* 400
o
o 300
* 200
HI
y=13.5(e1
—
_
—
{
3 *
J
100
5
>. 9O
—
2 80
u
2 70
a
* 60
50
40
::
X
K
-
w
—
-
m
—
•-
—
-•—
—
—
__
—
mi
•^
O, Concentration
ppm>6 hr Exposure
ply* Animals Ex-
posed at 0 ppm i
•— -
J
1
•ty .-»AJU
18
0
'55C-1)+19
T
. , '.(. '. t
9
0.25
>0.20
•
-
. \'V
—
v \- v
M.
.• V WVV
!
)
0.50
' •,«
250
< 0.0 01
Figure 2.
Pulmonary alveolar protein accumulation
in response to ozone exposure (values
+_ 1 SD-No. of animals per group on
columns).
50
-------
CHRONIC EXPOSURE EFFECTS OF OZONE IN'BEAGLES
J. Stara, T. Lewis, K. Campbell
B. Johnson and D. Coffin
The biochemical effects of acute ozone intoxication
have been well documented. The effects of chronic ozone
exposure on biological systems has received little
attention, however. As a result, a study was designed
in which female beagle dogs were exposed for 18 mo to
air containing 1 to 3 ppm of ozone. The original proto-
col called for the assessment of the pulmonary and
hematological effects of chronic 03 exposure. Before
the study was completed, however, several additional
parameters (cardiovascular physiology, immune competency,
neurophysiology, neurochemistry, and pathology) were
investigated to evaluate the biological effects of 03
more completely.
Ozone was found to have a significant effect on
total lung capacity, nitrogen washout, and functional
residual capacity. The hematology studies indicated
that the mean corpuscular volume and red blood cell
fragility both decreased with increasing exposure to
ozone. The immune response has been shown to be de-
pressed in those animals exposed to ozone.
Recent electromicroscopic studies of the dog's lung
tissues, performed by Dr. 6. Freeman, e_t a_l_. under a
contract with EPA, indicate the earliest response in
the appearance of macrophages near the respiratory bron-
chiolarductal region and in adjacent alveoli. The
number of macrophages increased with increasing ozone
exposure. The dogs exposed to the higher levels of ozone
also developed squamous metaplasia and stratification
of cuboidal cells in the bronchiolar epithelium. In
addition, thickening of bronchiolar walls, narrowing
of alveolar openings, and a higher proportion of mucus-
forming cells were noted.
Another recent report, completed by Dr. R. J. Stephens
e_t aj_. under a contract with EPA, also indicates that
the epithelium of the terminal airways and proximal alveoli
are greatly changed by ozone exposure. In addition,
Type 2 cells appeared to be metabolically altered and
large accumulations of grid-like material appeared in
the alveoli. The investigators contend that the reduction
in clearance rate that results from the morphological and
structural changes in the terminal airways may be an additional
factor related to the accumulation of the grid-like material.
51
-------
Final report on this study is expected to be issued
when pathology and neurophysiology data are collected
and analyzed.
52
-------
INFLUENCE OF EXPOSURE PATTERN ON
TOXIC RESPONSE TO NITROGEN DIOXIDE
K. Campbell and L. Hall
The probable influence of exposure pattern on associ-
ated toxic response was investigated because of its
importance to the modeling and interpretation of bioeffect
studies that are designed to determine relative toxic
hazards of engine emissions when various fuels and fuel
additives are used. Two experiments have been conducted
to date to examine the comparative magnitude of selected
toxic responses resulting from inhalation exposure to N02
delivered in three different patterns: continuous, varied
and intermittent.
In Experiment A, rats and hamsters were exposed for
5 days to N02 in a constant-level pattern (31 ppm) and in
a varied-level pattern (18 hr at 25 ppm and 6 hr at 45 ppm
per day, mean concentration 26 ppm); the total dose, in
ppm-days, was 126 for varied level and 149 for constant
level. Timed mortality and pre- and post-exposure body
weight were recorded. The data indicated that hamsters
were more susceptible than rats to these exposure conditions,
and further, that hamster lethality response was more
severe in magnitude and time in the varied than in the
constant-level exposure, even though the average concentration
and "total (CxT) dose" were similar. It is suggested that
(1) this exposure-pattern influence resulted from the high
peak exposure levels used, even though for short periods,
and (2) in this CxT condition, peak concentrations are more
determinant than other co-factors (duration, average concen-
tration, or "dose"). There was no mortality and negligible
weight change among the room-air control animals. Principal
results for Experiment A are shown in Table 1.
53
-------
TABLE 1. EXPERIMENT A:
VARIED-LEVEL AND CONSTANT LEVEL EXPOSURE OF
RATS AND HAMSTERS TO NITROGEN DIOXIDE
Exposure Pattern
Effect Varied Level Constant Level
Total mortality
Rats 40 30
Hamsters 100 70
Est. LT5Q, hrs.
Rats 72 68
Hamsters 28 46
Rel. body-weight
change, %
Rats +9 - 5
Hamsters - 7 -18
In Experiment B, mice, rats and hamsters were ex-
posed to 37 ppm of N02 for 15 days at a constant-level
in an uninterrupted pattern and in an interrupted pattern
(8 hr N02 and 16 hr clean air per day); total exposure
doses were 555 and 185 ppm-days, respectively. Timed
mortality, initial and terminal body weight, terminal lung
weights, and gross lung pathology in rat lungs were recorded.
In terms of total mortality and lungrbody weight ratios,
the data indicated again that hamsters were more susceptible
than rats. Mice appeared to be more sensitive than hamsters,
so that the species would rank in decreasing order of N02
sensitivity: mice, hamsters, rats. Further, it was evident
that altering exposure pattern did indeed influence the
magnitude of toxic response, in this case to a higher degree
than would be projected by a simple CxT "total dose" relation
ship. Mortality in animals exposed continuously for as
little as 3-5 days was far greater than for those receiving
15 days, 8 hrs/day interrupted exposure. Similarly, dis-
proportionately greater lung weight increases and gross
lung pathology was observed in the continuous-exposure than
in the interrupted exposure group. Analogous relationships
of response severity to exposure pattern (interrupted cf.
continuous), with equivalent total (CxT) doses, have, inci-
dentally, also been observed in plants (Episcia cupreata)
exposed to automotive engine emissions.
54
-------
Results for Experiment B are summarized in Table 2.
It is generally supposed that the reason for the reduced
toxicity accompanying interrupted exposure pattern is
that during intervening nontoxic periods the subjects'
defense and repair mechanisms are permitted to effect
a degree of recovery, or to develop a tolerance, or both,
that is denied by continuous, constant insult.
TABLE 2. EXPERIMENT B:
INTERRUPTED COMPARED WITH CONTINUOUS EXPOSURE
OF MICE, RATS AND HAMSTERS TO NITROGEN DIOXIDE
Exposure Pattern
T n + a v»»» nr
Effect
Total mortality, %
hours
Interrupted
Mice
0
NA
Rats
0
NA
Hams
0
NA
Continuous
Mice
85
76
Rats
40
NA
Hams
72
51
Body weight
change, % +3 -8 -1 34 -24 +2
Relative change in
lung: body weight
ratio, % 58 20 37 371 97 146
Relative gross lung
abnormality +. ++
The data from both studies suggest that altering ex-
posure patterns (variations in concentration or signifi-
cant interruptions in exposure) would likely affect the
variability and patterns of biologic responses in test-
exposure systems; this implies the need for control
criteria in generating and processing fuel emissions
atmospheres and for adequate atmosphere characterization
to define the exposure. Study B data inply that use of a
protocol involving an interrupted exposure pattern would
require much higher concentrations, or greatly extended
experimental periods, or both, to achieve detection and
to permit comparisons of toxic response.. It might also
be inferred that for some reported toxic responses based
on continuous exposure a "safety-factor" could exist
because, and to the extent that, "real-life" exposures
tend to simulate the interrupted-exposure pattern situation
55
-------
HEXACHLOROPHENE TERATOGENICITY
C. Kimmel, W. Moore, D. Hysell, and 0. Stara
Pregnant animals were anesthetized on day 7 and
approximately 73 mg of hexacholorphene suspended in
water and Tween 80 was Inserted Into the vagina. Next
a gauze plug was inserted and the vagina partially closed
with silk suture. On days 8, 9, and 10, additional
doses (for a total dose of 300 mg/kg) were given intra-
vaginally, and on day 11, the plugs were removed. Animals
were killed on day 20, and examined for teratological
changes. The hexachlorophene-Tween 80 mixture produced
a significant number of resorptions and malformations
(Table 1).
TABLE 1. TOXIC EFFECTS OF HEXACHLOROPHENE AFTER INTRAVAGINAL
ADMINISTRATION
Treatment
Hexachlorophene*
Starch or watert
control
No. of
maternal
animals
12
12
Maternal
mortality
2/12
0/12
No. of
im-
plants
123
158
% dead
or
res orbed
33
8
mal-
formed
40
4
*The hexachlorophene was administered as a 45% solution in distilled
water + Tween 80. Dosage = 90 mg., or 300 mg/kg.
tControls were treated with a starch paste in water + Tween 80, or
with water + Tween 80 alone.
An example of the gross external malformations to-
gether with a normal fetus (for comparison)are shown
in Figure 1.
56
-------
Figure 1 .
Abnormalities found following vaginal treatment
of pregnant rat. A. Normal fetus B. Abnormal
fetus showing microophtholmia and small size
C. Skeletal defects, wavy ribs.
The microscopic abnormalities rioted consisted of
ocular malformations ranging from microophthalmia to
anophthalmia with intermediate changes including
dysplasia of lens, retina, and optic nerve (Figures 2,
3 and 4).
;«
1
Figure 2.
Normal fetal eye showing lens, retina,
and optic nerve.
57
-------
;®
.. K7**
Figure 3.
Figure 4.
Fetal eye showing dysplacia of lens and retina
and microophthalmia (same magnification as
Figure 2).
Anophthalmia.
The only
vestige of
ocular develop
ment is the
orbital cleft.
58
-------
Cleft palate was also noted microscopically. The
vaginal infections seen late in pregnancy (days 12 to
16) in hexachlorophene-treated animals were associated
with gram-negative bacilli. Control animals did not
exhibit any noticeable infection (Figures 5 and 6).
Figure 5. Normal rat vagina.
Figure 6. Hexachlorophene-treated
vagina showing severe
epithelial ulcer and
subacute connective
tissue response.
59
-------
TOXICOLOGIC ASSESSMENT OF MOBILE EMISSIONS
(TAME) STUDIES
-------
TOXICOLICAL ASSESSMENT OF MOBILE EMISSIONS (TAME):
OVERALL STUDY APPROACH AND OBJECTIVES
J. F. Stara
Clean Air Act Amendments of 1970 have charged the EPA
with the determination of possible health hazards related
to the use of various fuels and fuel additives. The
results of these studies are to be used as guidelines
for the possible regulation of fuels and their additives.
The objects of this project were:
1. To provide a comparative study of the chemical
and physical nature of the emissions of a fuel
composition with and without the presence of a
fuel additive.
2. To assess comparatively the potential toxico-
logical hazards resulting from the use of the
fuel and fuel additive.
3. To develop a working test system and a definitive
toxicologic model by which harmful biological
effects can be satisfactorily evaluated.
Such a comparative study necessitates a high degree of
control, consistency, and repeatability in all three
major systems: generation and delivery, pollutant
characterization, and bioeffects determination.
The need for strict evaluation of fuel additives is
a necessity in light of the number and widespread use of
additives in all types of modern fuels. The major classes
of fuel additives are:
A. AUTOMOTIVE
1. Anti-Knock
2. Anti-Oxidants
3. Metal Deactivators
4. Detergents and Dispersants
5. Ignition Improvers
63
-------
B. DIESEL
1. Ignition Improvers
2. Detergents & Dispersants
3. Dyes
4. Anti-Oxidants
5. Smoke Suppressants
6. Anti-Static
C. AVIATION
1. Anti-Knock
2. Metal Deactivators
3. Anti-Oxidants
D. JET FUELS
1. Anti-Static
2. Smoke Suppressants
3. De-icers
E. TURBINE JET FUEL
1. Smoke Suppressants
2. Anti-Static
To ensure that satisfactory information can be sub-
mitted to EPA Headquarters, the following investigative
steps have been taken as a part of ETRL standard-procedure
for definitive toxicologic evaluation of an additive such
as methyl manganese tricarbonyl (MMT):
1. Ingestion exposure of the additive
2. Vapor inhalation exposure
3. Simple combustion exposure primarily to provide
aerometry data of what chemical forms can be
expected to be found in the automotive emissions
4. Simple combustion in mixture with fuel
5. Exposure of large numbers of animals to the complex,
whole, automotive engine emissions of the reference
fuel and the fuel with additive mixture at the
proposed or marketed concentration.
64
-------
The basic design of the toxicological study of complex,
whole engine emissions, which represent realistic atmo-
sphere found in the environment, is as follows: A fuel
additive is the test variable, although different fuel
types, engine types, and operating modes, or emission
control devices are also planned to be studied. Precise
characterization and quantification of the atmospheric
pollutants is an integral part of each experimental design.
During the past calendar year the following full-scale
automobile exhaust experiments were conducted:
TAME A. A control study using a reference gasoline without
additives. Carburetor settings were provided and
sealed at the factory.
TAME B. Another control study using updated engine specifi-
cations (maximum idle vacuum).
TAME C. A control experiment to evaluate the repeatability
of A and to serve as a baseline for D.
TAME D. A test study operating all systems as a replicate
of C with the exception that 0.37 g/gal. of an
anti-knock compound, MMT, was added to the refer-
ence fuel.
TAME E. A control study for D and F using reference fuel
and no additives.
TAME F. A test study in which 0.25 g/gal. MMT is added to
the fuel.
65
-------
DESIGN AND SYSTEM PERFORMANCE FOR
MOBILE EMISSIONS BIOEFFECT STUDIES AT ETRL
R.G. Hinners, O.K. Burkart and R. Iltis
During 1972, six studies were conducted exposing
animals to the exhaust emissions from a 1972 Chevrolet
350 C.I.D. automobile engine, which was operated for
approximately 2400 hours. The existing exhaust dilution
and exposure chamber system (Figures 1-3) was utilized
for a series of multi-disciplinary studies with several
biological models to assess comparative toxicity and
emissions characterization resulting from control fuel
(Indolene) and a pertinent test additive compound (MMT).
AIR
PURIFIER
CONDITIONING
> PURIFICATION*
AUTOMATIC
CTCIE
CONTROLLER
t
fNCINE
DYNAMOMETER
t
DILUTION AUTOMATIC
CVCTCM "* CO "*110
SY5TSM CONTROLLER
t
IRRADIATION IRRADIATED
CHAMBER EXHAUST \
\
NON- S INSTRUMENT
EXHAUST ^ ANALYSIS
/ I
*l* SYSTEM
t
COMPUTER
POUUTANT ANIMAL EXPOSURE MONITORING AND
OCNERATION CHAMBERS RECORDING
FIGURE 1. TOXICITY ASSESSMENT MOBILE EMISSIONS (TAME)
FLOW DIAGRAM
66
-------
Figure 2. Engine-dynamometer room
Figure 3. Typical irradiation chamber
67
-------
The "TAME Average Generation Conditions Summary"
(Table 1) provides relevant information regarding the
studies, such as fuel, tuning and dilution ratios. A
clean engine was operated for 150 hours (3000 miles)
on a modified 7-mode California Cycle using the refer-
ence fuel (Indolene) to stabilize engine operation and
pollutant emission factors. TAME A represents a 7-day
run after the break-in period with the carburetor set
as tuned by the factory. TAME B is also a 7-day
continuous run with the carburetor tuned for maximum
vacuum to assist in obtaining identical engine perform-
ance for future studies.
TABLE 1. TAME AVERAGE GENERATION CONDITIONS SUMMARY
CONDITION TAME A
FUEL
TUNING
EilGINE
D!G. MRS.
STUDY HRS.
FUEL CO.'ISP. LB/HR
EXH. CO. PPM
EXH. THC. PPM 1580"
TAME B
REF. ONLY
FACTORY SET
•B'
153-254
101
N.A.
1500
REF. ONLY
MAX. VAC.
'B'
279-450
171
7.2
10000
•2
2000
EHX. NOX. PPM
CO: THC RATIO
CO: KOX RATIO
THC: KOX RATIO
EXH. OXYGEN. X
AIR/FUEL RATIO
ROTOR CAP
ADJUSTMENTS
DILUTION RATIOS**
435
0.95
3.45
3.63
NA
NA
0-1 37R
HONE
16/1
600
5.00
16.7
3.33
NA
NA
0-1 37R
HONE
102/1
*z
*2
TAME C
REF. ONLY
LEAN (CO)
•B'
521-696
175
6.9
1705
1965
347
0.87
4.91
5.66
NA
NA
D-137R
NONE
18.2/1
TAKE D
REF. * 3/8WT
TAME C (CO)
•B1
779-im
336
*l
*3 6.6
2135/1925
2626/3429
335/231
0.81/0.56
6.37/8.33
7.84/14.8
NA
NA
D-13/R
(2SP. 9 CYCLE)
19.2/1
TAME E
REF. ONLY
TAME D (CO)
'B'
1354-1698
344
7.1
1924
2025
475
0.95
4.05
4.26
2.30
14.7/1*5
0-137
(3 CARS, 5 CYCLE)
19.0/1
TAME f
REF, + 1/4MMT
TAME E (CO)
•B'
1792-2131
339
7.4
2294
2236
457
1.03
5.02
4.89
2.64
14.8/1
D-137
(2 CARB.16 CYCLE)
18.4/1
Cycle speeds consistently 1w due to fuel contamination problems.
These values estimated from chamber data and dilution ratios.
1st week AVGS./Overall AVGS. - Experienced fuel contamination 2nd week of study. .
•' Dilution by RATIO of Average engine CO to Average Chamber CO. (Data for Highest Chasnber Concentrations;
*s A/F ratio average for Study using average cycle CO & oxygen and correcting for unburned HC.
*i
*2
«3
*"•
68
-------
A bar graph (Figure 4) illustrates the increase in
CO during study B with the engine tuned for maximum
vacuum to provide reproducibi1ity, rather than at the
factory setting of lean CO. Study D is a replicate of
Study C plus the additive MMT. Study F repeats Study E
and includes 0.25 g MMT per gallon of gasoline.
lO.OOO-i
9.000-
B.OOO-
7,000-
6,000-
5,000 -
4,000 -
3,000
2,000
1,000 -
FIGURE 4. Engine "B" average gaseous emissions
comparison for TAME studies A,B,C,D,E
and F.
69
-------
Curves of the average daily gaseous emissions for Study C
(7 days-Indolene only) and Study D (14 days-Indolene and
additive) are shown in Figure 5. The large increase in
total HC and decrease in NOX during the second week of
Study D is attributed to fuel contamination by water
rather than the MMT additive.
Figure 5.
Engine "B" average daily gaseous
emissions comparison for studies
C and D.
70
-------
The average gaseous emissions curves for TAME E (Indolene)
and TAME F (Indolene and additive) for 14 days of contin-
uous engine performance are shown (Figure 6). In these
studies, the engine was tuned at the start to match the
CO output from the preceeding study, and illustrates the
degree of engine reproducibility obtained. As the engine
hours increase, CO also increases and the emissions of
studies E and F are as comparable as can reasonably be
expected for a 2-wk continuous run.
5000 -i O-
4000-
3300-
2000-
1000-
LEC-EHP
TAME "I
TAME "
D CO
A THC
o NO*
MR/FUEl
14.7
14.8
nmrrm! RATIO
19.0
18.4
Figure 6.
Engine "B" average daily gaseous
emissions comparison for studies
E and F.
71
-------
A new air dilution system for mixing raw exhaust with
CBR filtered and temperature controlled air has just been
installed. This has eliminated the water cooled heat ex-
changer and surge tank, which is believed responsible for
some particulate loss in the former system. As may be
seen in the schematic drawing (Figure 7) the raw, hot
exhaust is introduced into the cooling air dilution tube
at an orifice plate and then flows into a large mixing
chamber. The diluted exhaust is then directed back into
the existing distribution piping that feeds the various
irradiation and animal exposure chambers.
MOTORIZED VALVE
BLEED
(TO nTH.
POSITIVE
SHUTOfF
(GATE)'
PRESSURE SENSOR
ff
MOTORIZED
DAXPER
AIR
-£*-
PRIMARY AIR
(CONSTANT)
FLOW
DENSITY
DISTRIBUTION SYSTEM
TO
IRRADIATION &
EXPOSURE CHAMBERS
FLO" _
DENSITY
Co
TAILPIPE
l-D
UKII-lLt fLflft- »||L^
if
T—^—^—s—s—r
633 C.F. CHAMBER
^ ^ ^ ^ _
20 FT. DILUTION TUBE
Figure 7. Schematic of air cooling and dilution
utilizing constant dilution air flow
72
-------
Future experiments will utilize the exhaust emissions
from a Lister SR-2, 4-cycle, direct-injected, and
naturally aspirated type diesel engine, in conjunction
with the new air cooling dilution system. Future planning
includes operating a 1973 Chevrolet 350 C.I.D. engine with
added controls as now marketed, also a 1975 engine proto-
type with catalytic converter and possibly a Wankel rotary
engine if the animal exposure time can be appropriately
scheduled.
Table 1. AEROMETRIC CHARACTERIZATION OF EXHAUST EMISSIONS
Pollutant component** Analytic method Automatic Manual Uhere determined'
Carbon monoxide (CO)
Total hydrocarbons
(THC), as CH4
Nitrogen oxides (NOX
includes NO and N02)*'
Ci to Cc hydrocarbons
(several compounds)
Cg to CIQ aromatic
hydrocarbons (several
compounds)
Aldehydes, total
Participates, total
mass
Particulate size
distribution:
Aerodynami c
Photonomeric
Particulate com-
posi tion
Ozone, "oxidant" **
Nondispersive X
infra-red spec-
troscopy
Flame ionization
spectroscopy X
Colorimetry using
Saltzman reagent X
Gas chromatography
Gas chromatography
MBTH according to
Mauser
Filtration gravimetry;
optical density (Sinclair-Phoenix)
Stage impaction
(Anderson)
Photoelectronic
(Royco)
Infra-red and ultra-
violet spectrophotometry
Coulometry (mast) X
lodometric colorimetry
X
X
X
X
X
X
EPM, EC
EPM, EC
EPM, EC
EC
EC
EC
EC
EC
EC
EC
EC
EC
*EPM - Exhaust or primary exhaust:—air mixture; tu - exposure cnamoer~ ~~
**NOx and ozone methods to be replaced by automatic chemi1uminescence instruments.
73
-------
ATMOSPHERIC CHARACTERIZATION IN AUTO EXHAUST EMISSIONS
M. Malanchuk, G. Contner and R. Slater
In conducting biological effects studies of mobile
emissions, it was necessary to evaluate the atmospheres
at various points in the engine-to-exposure chamber
system for gases, vapors, and particulates.
The methods employed for measuring component concen-
trations are summarized in Table 1.
74
-------
The average values over the entire period of the
exposure run for the six different runs are given in
Table 2. The measurements are listed for those chambers
in the RH (nonirradiated, "high" concentration) group
and in the IH (irradiated, "high" concentration) atmo-
sphere chambers.
TABLE 2. SUMMARY OF EXPOSURE CHAMBER AEROMETRY TAME STUDIES
Pollutant
CO
THC
NO
N02
Aldehydes
GC-HC's: CrC5
C6-C10
Participates
ug/m3 (Total)
Parti cul ate
Ratio
Treatment
RH
IH
RH
IH
RH
IH
RH
IH
RH
IH
RH
IH
RH
IH
RH
IH
IH/RH
A
93
96
98
102
23.7
18.9
3.6
8.6
311
433
1.4
B
99
99
19.6
19.4
5.8
5.1
0.3
2.0
38
75
2.0
C
92
92
109
95
17.4
10.8
3.2
9.2
5.2
17.2
164
1726
10.5
D
101
98
182
154
10.8
4.5
1.8
5.8
14.1
36.4
556
3293
5.9
E
101
101
99
81
24.3
18.7
3.4
8.8
2.8
6.5
6.8
5.8
2.1
1.9
164
980
6.0
F
125
118
106
87
21.4
13.0
2.9
7.6
3.3
7.3
9.6
6.9
2.7
2.1
387
1290
3.3
*Control air levels for CO and HC range 3-6 ppm
Concentrations in ppm except as noted
Although various comparisons can be made among the
several runs, TAME-A to TAME-F, with respect to atmo-
spheric compositions, a most useful comparison can be
made between TAME-E and TAME-F. During those two exposure
studies, the system conditions were closely controlled so
that the results could be reliably compared for the effect
on exhaust emissions of introducing additives into the
gasoline. A quick appraisal of the close control of engine
operation can be seen in the consistent values for CO, THC,
and NOX concentrations over the length of the runs of TAME E and
TAME F in Figures 4 and 6 of the previous report.
75
-------
TAME E used the reference fuel, Indolene. TAME F
used the reference fuel to which methylcyclopentadienyl
manganese tricarbonyl (MMT) at the ratio of 0.25 g (as Mn)
MMT per gallon of fuel was added.
The particulate concentrations produced in various
parts of the system can be compared in Table 3.
TABLE 3. PARTICULATE CONCENTRATIONS IN
TAME E AND TAME F STUDIES
Heat Exchanger, Effluent
Surge Tank
PARTICULATE, mg/M'
TAME E
2.90
2.55
TAME F
5.01
Exposure Chamber #6
(Nonirradiated)
0.17
0.39
Irradiation Chamber #3
Exposure Chamber #15
(Irradiated)
0.15
0.66
0.31
.1.31
The TAME F/TAME E pairs of values show a consistent
2:1 ratio. It appears that the presence of the MMT in
the fuel, which was the one major variable in the two
studies, was primarily responsible for the considerable
increase in particulate emissions in TAME F. Irradiation
chamber #3 was the source of irradiation treatment of the
atmosphere introduced into exposure chamber #15.
Key components analyzed in TAME E and F were calcu-
lated to a g/mile basis for comparison with other data
presented as typical for a 1971 Ford-8 operated on a
dynamometer, using Shell no-lead gasoline (John Sigsby, Jr,
Conference on Health Intelligence for Fuels, RTP, 05-07
January 1973). For that purpose, the TAME values of ppm
were converted to g/mile by assuming that the modified
California cycle used with engine "B" of the TAME studies
resulted in an average exhaust flow of 1 nwmin and an
average of 22 mpg equivalent road speed. The values are
listed in Table 4.
76
-------
TABLE 4. GASEOUS EMISSIONS OF AUTOMOBILE ENGINES
Component
THC
CO
NOX
Ethylene
Propylene
Butene
TAME E
3.46
5.95
1 .57
0.083
0.055
0.013
TAME F
3.84
7.02
1.51
0.166
0.125
0.021
'71 Ford Operated on
Dynamometer using Shell
No-Lead Gasoline, Hot
Start
1.81
15.60
4.06
0.212
0.117
0.026
Acetylene
0.053
0.106
0.158
Benzene
Toluene
o-, p-Xylene
Butylbenzene
i-Butane
n-Butane
i-Pentane
n-Pentane
0.041
0.071
0.076
0.028
0.009
0.055
0.072
0.042
0.105
0.159
0.160
0.061
0.009
0.046
0.071
0.043
0.142
0.142
0.050
0.031
0.005
0.077
0.085
0.019
From analyses of the particulate collected at the
surge tank, it was determined that 27.0% of the Mn burned
in the engine (combustion) was recovered in the particulate
from the surge tank. That may be most all the particulate
from the exhaust that is of airborne size (<10y) and,
therefore, of potential biological significance.
77
-------
PARTICULATE SAMPLING PROCEDURES FOR AUTO EXHAUST EMISSIONS
M. Malanchuk and A. Cohen
Group samples of auto exhaust atmospheres were taken
Curing the TAME-F study) to explore the adequacy of
currently widely-used procedures for particulate evaluation
One group of samples were collected on glass fiber
filters for particulate mass measurements. Another group
of samples were collected by an Andersen sampler for
particle size distributions.
The filter sampling conditions consisted of sampling
from an exposure chamber (#16) containing irradiated
exhaust emissions that had been diluted with clean air
by a factor of about 24-25 before the irradiation treat-
ment, The identical sampling rate, 3 cfmf was used for
15-, 30-, 60-, and 120-minute collections on 142-mm glass
fiber filters. The initial weighings (immediately after
the sampling) established sample weights that indicated
concentration values in a logarithmic relationship to the
sampling times (Figure 1).
Figure 1. Particulate Concentrations Measured in the
Irradiated Atmosphere of Exposure Chamber #16
10
8
6
5
4
CO
i
I
50 100
Sampling Time, min.
The atmospheric particulate concentration calculated
from the 15-min. sampling was more than twice the value
for the 120-min. sampling although the concentration values
should be the same, since the same source of atmosphere was
sampled. Later (by several days) weights of the self-same
filter samples showed a progressive tendency for the samples
to lose weight (the shorter-term samples lost at a greater
rate than the longer-term samples); the recalculated concen-
trations approached a more common value.
78
-------
A different group of participate samples were
from the surge tank unit by an Andersen sampler.
rate was also 3 cfm. Collection times were 15, 30
75 minutes. Glass fiber filters were used on each
the sampler to retain the impacted particles with
efficiency. Weighings were amde as soon as possib
the sampling was concluded. Calibration values de
for the increased flow rate were used to plot the
on the log-probability scale (Figure 2).
collected
The flow
, 60, and
stage of
maximum
le after
termined
wei ghts
CUMULATIVE MAU PERCENT ft * PARTlCUE DlAMC
It M M MMHMM M II W
|U
m ;
Figure 2.
Effect of Sampling Time Upon Particle
Size Distribution Measured in Auto
Exhaust emissions
79
-------
The parallel relationships of the plots emphasize
the similarity of distributions. The broken line
characteristic indicates a skewed effect upon the normal
distribution picture. The equivalent mass median
diameter (EMMD) for these (uncorrected for normal dis-
tribution appearance) samples show decreasing values of
such diameters with longer sampling times - from 0.41 y
for the 15-min. sample to 0.11 y for the 120-min. sample.
From the data obtained (pictures evident in the
figures) the filter and the Andersen sampler measurements,
it is obvious that modifications or alternatives in the
procedures, or equipment, or both should be used to attain
greater accuracy in such measurements (as made on systems
like auto exhaust emissions).
80
-------
DIFFERENCES IN FUEL EMISSION COMPONENTS IN
TAME E AND F: TENTATIVE INFERENCES CONCERNING
INFLUENCE OF A TEST GASOLINE ADDITIVE AND OF
GENERATION VARIABLES
K. Campbell, M. Malanchuk, R. Slater, G. Contner
R. Iltis, J. Burkart, R. Hinners, Y. Yang and J. Stara
Comparisons were made concerning the composition of
irradiated (I) and nonirradiated (raw, R) automotive
engine exhaust resulting from the use, in two sequential
experiments, of reference gasoline* in one study (TAME-E)
and the same reference gasoline plus a test additive"1"
(TAME-F). Generation, control and distribution systems,
and atmospheric characterization are described elsewhere
(Hinners, ejt
-------
Chamber atmospheric monitoring data for several
components in TAME E and TAME F are compared in Tables
2 and 3. The direct ratio of F:E means are shown to
express magnitude of differences; also shown are the
results of simple student's t-tests of statistical
significance. It can be seen that for some emission
components differences between E and F are larger in
proportion than might simply be expected in the light
of relatively small differences in the control criteria
and that several of the larger proportional differences
are supported by statistical significance.
TABLE 2. COMPARISON OF ENGINE EMISSION
COMPONENTS IN TAME E AND F
Component Atmosphere
CO, ppm
Tot. hydro-
carbons, ppm
NO, ppm
NO?, ppm
Tot. alde-
hydes, ppm
Aliphatics,++
ppm
Olefinics,"1"*"
ppm
Aromatics,++
ppm
Tot.particu-
late,ug/m3
Particulate
Mn, ug/m3
R
I
R
I
R
I
R
I
R
I
R
I
R
I
R
I
R
I
R
I
TAME-E
102.6
108.1
101.4
88.8
24.6
20.7
3.3
8.6
3.1
5.4
1.42
1 .26
6.03
5.40
2.49
2.11
173
658
0.10
0.26
TAME-F
126.9
123.6
107.6
99.6
22.5
15.9
2.8
7.5
4.0
5.8
1.34
1.25
8.28
6.90
2.67
2.35
388
1311
99
90
RATIO
F/E
1 .24
1.14
1.06
1.12
.92
.77
.87
.87
1.30
1.07
0.94
0.99
1 .37
1.28
1 .07
1.11
2.24
1 .99
•
t-Test+
***
**
NS
*
NS
**
?
NS
*
NS
No test
ii
II
ii
ii
***
***
Obvious signif.
Obvious signif.
+NS = not significant, p >0.1;? = questionable significance,
0.1>p>0.05; *, **, and ***, significant at p <0.05, p< 0.01, and
p<0.001 , respectively.
++See Table 3 for components whose concentrations are summed herein,
82
-------
TABLE 3. COMPARISON OF EXHAUST HYDROCARBON
COMPONENTS IN TAME E AND F+
COMPONENT
Al iphatics:
n-Butane
i-Butane
n-Pentane
i-Pentane
Olefinics:
Acetylene
Ethyl ene
Propylene
Butene-1
Isobutylene
Butadiene
Aromatics:
Benzene
Toluene
o ,m-Xylene
p-Xyl ene
Bu-Benzene
TAME
R
0.50
.08
.31
.52
1.54
2.37
1.06
.16
.59
.30
.56
.88
.57
.25
.24
E
I
0.44
.09
.29
.44
1.55
2.27
.88
.13
.38
.18
.57
.76
.42
.18
.16
TAME
R
0.42
.08
.32
.52
2.15
3.14
1.58
.20
.77
.44
.72
.92
.58
.22
.24
F
I
0.41
.07
.30
.47
2.03
2.84
1.15
.14
.51
.23
.71
.83
.46
.17
.18
RATIO,
R
0.84
.95
1.04
.98
1.39
1 .32
1 .48
1.22
1 .32
1 .46
1.29
1.04
1.01
.88
1 .02
F/E
I
0.92
.77
1.06
1 .06
1.31
1.25
1.30
1.10
1.32
1.29
1 .25
1 .07
1 .05
.93
1 .13
t
R
NS
NS
NS
NS
**
*
**
NS
NS
*
*
NS
NS
NS
NS
-TEST
I
NS
7
NS
NS
*
7
?
NS
*
*
*
NS
NS
NS
NS
Concentrations are in ppm; statistical designations as in Table 2,
83
-------
No attempt has been made to adjust TAME F data in
proportion to control criteria differences, nor to
attribute environmental significance to differences noted.
In some cases relatively small differences in means were
statistically significant because of small data variation,
and may not be of chemical significance to the environ-
ment. On the other hand, true differences of some magni-
tude may fail statistically by virtue of great variability.
Further experimentation and additional judgements are
considered necessary for a conclusive assessment concerning
the true effect of the additive and its environmental
importance.
However, tentative inferences from the preliminary
data include:
1. That of the components monitored here, the
following are the most worthy of further attention
as being affected by this additive and which might
also be of biological importance:
(a) Total and manganese particulate
(b) Certain olefinic hydrocarbons
(c) Benzene
(d) Other exhaust species in low concentrations
to be considered are:
(1) Phenols
(2) Polynuclear aromatics
Nitroorganics
Epoxides
Long chain aliphatics
Amines
Sulfonates
Azoarenes
2. That an additive should and may be tested in a
system carefully engineered, controlled, and monitored,
and that the impact of an additive may be through its
influence on other exhaust components than merely its
own products.
3. It is also quite evident that there are numerous
engine-related and operational factors to be controlled,
monitored and assessed in the conduct and interpretation
of such engine emission studies.
84
-------
The data of these studies also reflect the influence
of irradiation-induced photochemical reactions on exhaust
composition in this facility. Although because of quanity
and relationships of hydrocarbons and nitrogen oxides there
has been negligible, if any, oxidant formation in this test
series, the following effects do appear significant.
1. Moderate reduction of NO and considerable
increase of
2. Considerable increases in total aldehydes and
total particulate.
3. Slight to moderate decreases in specific hydro-
carbons
Determination of effects of fuel factors on emissions
composition is important in suggesting areas of environ-
mental impact and health concern, but the establishment
and evaluation of standards and regulations concerning
fuel products will likely also require definitive demon-
stration of associated effects (comparative toxicity) in
billogical systems exposed to these emissions, such as
increased body burden and functional and pathologic
alterations.
85
-------
CHANGES IN PATHOLOGY OF RATS AND HAMSTERS FOLLOWING
INHALATION EXPOSURE TO MOBILE EMISSIONS
D. Hysell and W. Moore
In TAME C and D (the latter with MMT) sufficient
animals were maintained In clean air, CO control, and
high raw (RH) and high Irradiated (IH) exhaust chambers
so that five rats and five hamsters from each exposure
group could be sacrificed on days 2, 4, and 6 for patho-
logic evaluation. In addition, in TAME D, sufficient
rats were maintained in clean air and in RH and IH ex-
haust so that tissues from 18 rats in each group could
be chemically analyzed for tissue Mn; of these, tissues
were saved from six rats of each group for pathologic
evaluation. In TAME E and F (with MMT), the numbers were
increased so that animals could be examined on days 1-5
as well as 20 rats per each exposure group for the 2-wk
exposure period. No gross abnormalities were noted in
any animals except for chronic respiratory disease (CRD)
in a significant number of rats. Tissues collected for
histology were larynx and trachea, lung, Hver, and kidneys.
Tissues collected for chemical analysis from the rats
exposed for 2 wk, were brain, heart, lung, liver, and
kidney. One eyeball was saved from each 1-to 5-day-ex-
posed hamster and rat 1n TAME E and F and processed for
cornea! mitotic rate determination.
Microscopic evaluation of tissues from TAME C and D
has been completed. In those rats sacrificed on days 2,
4, and 6, no abnormalities were noted other than CRD.
In TAME D, the rats maintained for 2 wk all showed rather
severe CRD, but in all the IH exhaust animals there was
a marked acute purulent bronchopneumonia superimposed.
One third of the RH exhaust group showed these changes.
In the hamsters from both TAME C and D, there were rather
marked changes 1n pulmonary tissue compared with control
animals (Figure 1).
As early as day 2, the exhaust animals manifested a
rather prominent increase in leukocytes and macrophages in
alveoli at the level of the terminal brochioles (Figure 2).
By day 6, there appeared to be thickened alveolar
septae, a possible Increase in Type II alveolar septal cells
and early epitheliazation of more peripheral portions of
respiratory ducts (Figure 3). These changes occurred in
both RH and IH exhaust groups but affected a higher per-
centage of the latter. No abnormalities relatable to
the Mn additive were noted. If anything, pulmonary changes
appeared more severe in TAME C.
86
-------
Figure 1. Normal hamster lung
Figure 2.
Hamster lung after 2 days exposure to
exhaust in TAME study.
87
-------
figure 3.
Hamster lung after 6 days exposure
to exhaust in TAME study.
The results of cornea! mitotic rate determination
in hamsters from TAME E are shown in Figure 4. As may
be noted there is an early and marked cyclical response
in the IH exhaust group. The RH group also shows a
response which is somewhat delayed. Determinations in
the rats (not presented here) show an identical pattern
Microscopic evaluation of tissue
TAME E and F and cornea! mitotic rate
from TAME F are not yet completed.
specimens from
determination
-------
CORNEAL MITOTIC RATE
o
-------
EFFECTS OF MOBILE EMISSIONS ON BODY WEIGHT AND
TISSUE LEVELS OF Mn IN RATS
R. Miller, W. Moore, and D. Hysell
Groups of 18-20 animals were exposed for 14 days to
each of the atmospheres in TAME D, E, and F. During the
exposure, the animals were weighed at intervals of time,
and the data are presented in Figures 1, 2, and 3. Al-
though some of the body-weight curves for different types
of exposure were not significantly different, it should
be noted that there is a consistent trend in the weight
data. Exposure to the raw (RH) and irradiated (IH) ex-
hause atmospheres consistently depressed the weight gains
which indicates that these atmospheres, in some unknown
way, influenced the growth rate of these animals. The
irradiated atmosphere in each experiment had a greater
effect than the nonirradiated exhaust atmosphere.
300r-
6 8 10 12
DAYS, POST EXPOSURE
14
Figure 1. Weight of rats after exposure to auto emissions
when 0.37 g MMT added to reference fuel, TAME D.
90
-------
340r-
320
300
o 280
260
o--CA
jj^^^^j^^j^^j^^f^^i^^^^t^^i^^it^^i^^t
2 4 6 8 10 12 14
DAYS, POST EXPOSURE
Figure 2. Weight of rats after exposure to auto emissions with
reference fuel only, TAME E.
400
380
360
o
~-340
320
I L I J I I i I
2 4 6 8 10 12 14 16
DAYS, POST EXPOSURE
Figure 3. Weight of rats after exposure to auto emissions when
0.25 g MMT added to reference fuel, TAME F.
91
-------
At the end of the experiment (14-15 days), the ani-
mals were sacrificed and selected tissues taken for Mn
analysis by atomic absorption spectrophotometry. The
Mn content for tissues taken in TAME D, E, and F are
given in Table 1. There was some variation in the tissue
TABLE 1. MANGANESE TISSUE CONCENTRATIONS FOLLOWING INHALATION EXPOSURE TO
AUTO EXHAUSTS FROM INDOLENE WITH AND WITHOUT MANGANESE
Experiment
Lung
TAME E
F
D
Kidney
TAME E
F
D
Liver
TAME E
TAME F
TAME D
Brain
TAME E
F
D
Type of
fuel
Indolene
Indolene
0.25 g Mn
Indolene
0.37g Mn
Indolene
Indolene
0.25 g Mn
Indolene
0.37 g Mn
Indolene
Indolene
0.25 g Mn
Indolene
0.37 g Mn
Indolene
Indolene
0.25 g Mn
Indolene
0.37 g Mn
Mn
Clean Air
2.86
3.19
5.98
3.15
4.13
4.85
6.99
7.58
8.19
4.49
5.03
5.42
vg/g Dry Sample
Nonirradiated
exhaust (RH)
1.80
5.47
8.15
3.03
4.49
6.57
6.65
7.57
7.93
6.16
5.65
5.18
Irradiated
exhaust (IH)
2.16
3.98
7.37
3.58
3.97
6.46
8.41
8.22
7.72
6.67
4.77
3.23
levels of Mn among the control groups when the different
TAME studies were compared; for example, all the Mn levels
for the control tissues in TAME E are lower than TAME F,
92
-------
and the control values for TAME F are lower than TAME
D. These differences may be attributed to either slight
deviations in chemical analysis or to slight differences
in the tissue Mn levels of these three different rat
populations. The rats used for each TAME experiment
were received approximately 10-14 days before onset of
the study, and thus, the animals did not have suffi-
cient time for their Mn tissue levels to equilibrate with
Mn levels in our rat diet. It has been shown that the
concentration of Mn in the tissues is directly related
to the level of Mn in the diet. Some of the lung and
kidney samples for TAME D and F showed increased Mn con-
centrations over the control values. These studies do
not, however, Vesolve the question of whether or not
Increased atmospheric levels of Mn would significantly
effect the total body burden.
93
-------
DETECTION OF EARLY BIOCHEMICAL ALTERATION
IN HAMSTERS EXPOSED TO AUTO EXHAUST GASES
S.D. Lee, R.M. Danner, L. McMillan,
K.C. Butler, W. Moore, and J. Stara
Recently, we have demonstrated oxidative destruc-
tion of polyunsaturated fatty add films exposed to 0-
and N02 In terms of gravimetric change, spectrophoto-
metrlc change, and malonaldehyde formation. Furthermore,
these changes were shown to be reaction-time and con-
centration dependent. Thus, 1n this study, polyunsatu-
rated fatty acid films were exposed to irradiated auto
exhaust to investigate whether similar alteration would
take place or not.
Figure 1 shows the gravimetric changes observed with
methyl esters of linoleic and linolenic acid in TAME F.
Approximately 7% and 13% Increases were observed in
linoleic and Hnolenic acid films, respectively, 1n a
4-hour period. A comparison of relative changes in the
two polyunsaturated fatty acid esters in two Indolene
and Indolene + Mn-carbonyl studies is made in Figures
2 and 3. These data Indicate that a greater change
occurred in the studies in which Mn-carbonyl was used
as the fuel additive. However, a positive correlation
between these changes and Mn-carbonyl could not be made,
as yet.
There was an apparent increase in blood glutathlone
level In hamsters exposed to raw (RH)and irradiated (IH)
exhausts and an increase In the group that was exposed
to IH auto exhaust (Figure 4). A marked increase in GSH
was observed 1n the experiments in which Mn-carbonyl
was used as the fuel additives; however, a concommitant
Increase was observed in the CO control. For this reason,
further studies are necessary to clarify the above findings
Lactic dehydrogenase appeared to have altered with
a decreasing trend in Mn-carbonyl studies and perhaps
an Increasing trend In a control study (Figure 5). No
appreciable difference or changes were observed in leucl-
neaminopeptidase and glucose-6-phosphate dehydrogenase.
94
-------
25
20
O
15
10
IM
- 5
18'-3 Exposed
3
HOURS
Figure 1. Relative changes in weight in
Methyl esters to high irradiated
exhaust and clean air in TAME F.
Figure 2. Gravimetric change in fatty
acid (18:2) in. vitro.
95
-------
TAME F
Figure 3. Gravimetric change 1n fatty
add (18:3) in vitro.
to
so
""rm
CONTROL
I I I
C O If In fs F,, C S ls I,, Fj F,t C O Ij 1)1 FS FH
CO CONTIOl »AW litAOIAItO
Figure 4. Comparison of blood glutathione
levels of hamsters exposed in
TAME.
96
-------
COMPARISON OF SERUM LAP LEVELS OF HAMSTERS EXPOSED IN TAME
50 i-
40
JZL
30
20
CONTROL
n
I I I I I I I I I I
C D E5 E12 F5 F]2
CO CONTROL
C D E5 E12 F5 F12
RAW
C D E5 E)2 F5 F12
IRRADIATED
Figure 5. Comparison of serum LDH-P levels of hamsters in
TAME.
97
-------
DISTRIBUTION, EXCRETION, AND BIOTRANSFORMATION
OF PARTICULATE POLYCYCLIC HYDROCARBONS
L. Hall, I. Washington, H. Ball, J. Adams and K. Campbell
Several compounds resulting from automobile and diesel
motor fuel emissions are known to be carcinogenic in ex-
perimental aii mals. The determination of the biological
fate of these compounds following inhalation of total ex-
haust emissions is of utmost importance since biotrans-
formation of arenes like benzo(a)pyrene has been implicated
mechanistically in the initiation of carcinogenesis through
epoxide formation. Experiments in whole animals, however,
show a decrease in tumor incidence with induction (Conney,
A.H. and Burns, J.J., 1972, Science 178: 576-586). The
question of whether this biotransformation is a true detoxi-
fication is unresolved at this time.
Preliminary investigations have begun for assessing
the interaction of whole exhaust emission with the micro-
somal enzyme system that metabolizes the polycyclic hydro-
carbon. Biotransformation by aryl hydrocarbon hydrolase
(AHH), a cytochrome P-450 mixed function oxidase, results
in metabolites, principally phenol and quinoids,with greater
water solubility that are more readily excreted when compared
with the lipid soluble parent compounds. Increased excretion
results in a decrease in the biological residence time. Lung
AHH activity has been assessed in old male hamsters (10-14
months) following exposure to diluted auto exhaust. The
activity was assayed spectrof1uorophotometrically (as de-
scribed by Sunderman, F.W., Jr., 1967, Cancer Research 27:
950-955) on whole lung homogenates. Figure 1 shows the~A~HH
activity after 5 days continuous exposure to CO (100 ppm),
raw exhaust (100 ppm CO), and irradiated exhaust (100 ppm CO).
No statistical difference was found between control (0.0145 +_
0.005) (mean +_ S.D. relative fluorescence/minute/mg lung
protein) and the CO animals (0.016 + 0.002). However, raw
(0.009 + 0.003) and irradiated (0.00~6 + 0.001) are different
from control and from each other.
Figure 2 shows the temporal effects of exposure on AHH
activity. A significant decrease (62%) occurred in 2 days.
After 13 days of exposure, the activity is still depressed
although variability has increased, which suggests perhaps
that some animals may be recovering. This facet needs to be
resolved.
98
-------
The goal of our research is to discriminate between
effects of different fuels and fuel additives. In Figure 3
the effects of a fuel additive (MMT) can be compared with
effects of a reference fuel following 5 days continuous
exposure. No statistical difference was observed between
controls. The raw exhaust exposures, however, are different
at the P = 0.06 level and the irradiated exposures are
different at the P = 0.085 level. Since small numbers of
animals are used in the comparison (five hamster/group),
the true significance of this difference is not known
although the data suggest a difference between exposures.
Further research is intended to resolve this question.
.020
.016
.012
.008
.004
f
f
*
CONTROL
CO
RAW
IRR.
Figure 1.
Effect of auto exhaust pollutants
on hamster lung aryl hydrocarbon
hydroxylase (AHH); comparison of
different atmospheres.
99
-------
.020
.015
.010
.005
* •
DAYS
10
12 U
Figure 2
Temporal effect of exposure to
auto exhausts on AHH activity
in hamsters.
TOO
-------
018-i
.016-
.014-
5 .012-
HI
o
•
? .010-
« .008 -
o
S .006-
.004-
.002-
'
.
•
LEGEND
1 1 - INDOLENE
E7D - INDOLENE +
'
\
%
^
//
i
MEAN 1 b.D.
,1 I
^ 'I
Y/ *'*',
y/ '/'
% l
y/. i
CONTROL
RAW
IRRADIATED
Figure 3.
Comparison of hamster lung
aryl hydrocarbon hydroxylase
(AHH) activity following
exposure to indolene and
indolene + MMT emissions.
101
-------
EFFECT OF ATMOSPHERIC POLLUTANTS ON PULMONARY
DEPOSITION AND CLEARANCE
L. Hall, M. Malanchuk, A. Cohen, J. Adams,
K. Campbell and J. Stara
An investigation is being conducted on pulmonary
deposition and clearance as affected by exposure to fuel
and fuel additive combustion products. With a surface
area of approximately 70 m2, the respiratory tract is a
major route of entry for environmental pollutants and a
major target organ for toxic effects. Effects on the
self-cleaning processes (phagocytosis, mucociliary trans-
port, and solubilization) of the lungs play a prominent
role in the development of chronic lung disease and
carcinogenesis.
In order to assess the integrity of this system, the
method as described by Ferin, J. (1971) AIHA 32., 157-162)
was used. After 13 days, continuous exposure to irradiated
auto exhaust, the mice (10-14 wks) were exposed to the test
challenge (TiOo) at 10-15 mg/m3 for seven hours, generated
with a Wright Dust Feed. The mice are sacrificed at 0, 8
and 25 days post-treatment and the titanium lung burden
determined spectrophotometrically with 4,4'diantipyrylmethane
Figure 1 shows the titanium lung burden in mice exposed
to auto exhaust. Although no definite conclusions can be
made because of limited sample size (2 animals/group), the
data suggest a difference between control and exposed animals
The apparently increased deposition and lung clearance in
exposed mice is being tested in larger groups of animals.
The method appears to be a satisfactory tool for studies of
effects on pulmonary physiology and lung defense mechanism.
102
-------
1.0
0.9
0.8
$0.7
J 0.5
o>
:§ °-4
«0.3
0.2
0.1
-o— Control
-«-- Irradiated Exhaust
ii_i i t i i j I
12
16
20
Days
Figure 1.
Retention of titanium dioxide test
aerosol in mice exposed to irradiated
auto exhaust.
103
-------
SERUM ALPHA-1-ANTITRYPSIN IN ANIMALS EXPOSED
TO FUEL COMBUSTION EMISSION
L. Hall, I. Washington, K. Ball,
J. Adams and K. Campbell
Alpha-1-antitrypsin (AT), a low molecular weight
glycoprotein, Inhibits the activity of a variety of
proteolytlc enzymes. It Is believed to be synthesized
at least by the liver (Sharp, H.L., 1971, Hosp. Practice
6^:83-96). A deficiency, genetically transmitted, 1s
associated with panacinar emphysema 1n adults and cirrhosis
in children. Increased serum liters have been associated
experimentally with N02 exposure, surgical procedures,
and Injections of turpentine (Ihrlg, J. et al., 1971, Amer.
Rev. Resp. D1s. 103, 377-388).
Because of the sequelae associated with decreased
titers of this protein, Investigations have begun to
assess the effect of auto exhaust on this system. Serum
from old, male hamsters (10-14 months) exposed to raw
and Irradiated exhaust for 0-13 days was collected and
assayed for Inhibitor activity as described (Eriksson, S.,
1964, Acta Med. Scand., 175, 197). Although the sample
size was -limited and rather large control variation was
seen, comparison of time-paired controls and exposed ani-
mals suggests a decrease in serum AT following exhaust ex-
posure (Table 1). Additional studies are in progress.
TABLE 1. EFFECT OF IRRADIATED AUTO EXHAUST ON
SERUM ALPHA-1-ANTITRYPSIN (AT) IN
HAMSTERS
Exposure Time Control*
Days mg AT/ml
1
2
3
1
1
1
.40
.21
.10
(2)
(3)
(2)
Exposed
mg AT/ml
1
1
1
.20
.09
.08
(1)
(3)
(2)
Percent
Decrease
14
9
1
.3
.9
.8
* Duplicate analysis reported as mg of Trypsin
inhibitor/ml Serum. Brackets represent sample
size.
104
-------
CHANGES IN BLOOD AND WEIGHT PARAMETERS IN AGED RATS
EXPOSED TO FUEL AND FUEL ADDITIVE EMISSIONS
M. J. Wiester
The purpose of this study was to gain information
about the change in general health of a homogenous group
of aged animals as a result of fuel emission exposure.
Attempts were made to identify a dose response to the
atmospheres as well as to estimate quantitatively if
differences could be detected between the toxicity of
emissions from pure gasoline with that from gasoline
plus an additive, methylcyclopentadienyl manganese tri-
carbonyl (MMT).
Charles River retired male breeder rats (+_ 600 g)
were exposed to selected "TAME" atmospheres for continuous
periods of 7 and 13 days. At the termination of the ex-
posure, animals-were tested for arterial blood pOg, pC02»
pH, COHb, WBC, Hbg, and HCT. Body weight change was also
noted. On the day of bleeding, the animals were lightly
anesthetized (30 mg/kg pentobarbital I.P.) and catheters
(50 PE) were surgically inserted in femoral arteries.
Rats were returned to their respective atmospheres and
allowed to recover from the anesthesia under continued
exposure for 3 hours. Arterial blood was collected under
anaerobic conditions from quiet unanesthetized animals
while equilibrated with their atmospheres. Plastic glove
bags were utilized for this procedure. Blood gases and
pH were read on a Radiometer Copenhagen, and WBC's on a
Coulter Counter. The cyanmethemoglobin method was used for
hemoglobin analysis and a standard NH/jOH spectrophotometric
method was used for COHb.
Table 1 lists the primary pollutant concentrations for
each atmosphere to which rats were subjected. There were
four exposure periods, two in which Indolene was burned
and two with Indolene + MMT. Animal data from similar ex-
posures were combined and the significance of the differences
were determined by mean of the student t test (Table 2).
Results indicate that there were harmful effects caused
by exposure to dilute emission atmospheres when the additive
was used. After 1 week of continuous exposure, weight loss,
anoxia, and C02 retention in three animals was significantly
different from control animals as well as from their Indolene
exposed counter parts. After 13 days, even the blood pH was
significantly decreased. Exposure to the more concentrated
105
-------
atmospheres produced more pronounced harmful effects
with respect to weight loss, anoxia, C02 retention,
blood acidity, and dehydration. A comparison of weight
loss for all animals is shown in Figure 1. Increased
hemoglobin and hematocrit levels do not appear to be a
response to Increased carbosy hemoglobin levels. De-
hydration would be a factor contributing to the Increased
weight loss seen in the High Irradiated + MMT atmosphere.
No effects seen could be directly attributed to CO. Un-
defined interaction of components 1n the complex atmos-
pheres, however, could be a modifying factor. None of
the animals showed significant changes 1n their WBC levels
that might indicate that bacterial Infections were not
responsible for effects seen.
In summary, rats exposed for 13 continuous days to
concentrated atmospheres of fuel emissions show marked
harmful effects when MMT 1s added to the fuel.
TABLE 1 . POLLUTANT CONCENTRATIONS IN TREATMENT TEST ATMOSPHERES
oAii,,+*n+e rAn+wO co Low Hfid Low Med High High Irr.
Pollutants Control Control Irr Irf +
CO, ppm
HXC?, ppm
NO, ppm
N0« , ppm
5.4
6.0
0.125
0.075
102.0
6.0
0.1
0.01
48.0
44.0
7.11
4.78
53.0
63.0
5.05
3.8
96.0
95.0
16.6
8.8
95.0
121.0
9.7
6.1
Particulates
ug/m3 22.0 - 3.42 1432.0 1698.0 2135.0
Manganese,
pg/m3 0.3 51.0
106
-------
TABLE 2. PHYSIOLOGICAL EFFECTS IN RATS AFTER EXPOSURE TO FUEL EMISSIONS
TREATMENT
days of exposure
A weight (g)
;f .S.D.
Slqnificance
p02 millg
+ S.D.
Significance
pC02 n™iig
IS.D.
PH
+S.D.
Sfqnlffcance
Hemoglobin gt
+S.D.
S1qni ficance
Hematocrft %
iS.D.
Significance
UBC 1000/im3
IS.D.
Significance
COJIB gX
iS.D.
Significance
CONTROL
7 n
-9 .3
23 22
85.9 91.?
9.6 8.5
30.9 37.1
4.9 4.1
7. 401 7.424
.028 .046
16.0 15.6
.98 1
, 43 44
3 3
10800 .12300
4.6 5.2
. 4. 2
5.0 5.0,
CARBON MONOXIDE
CONTROL
7 13
-a -a
17 8
80.3 09.8
9.3 12
30.6 37.5
5.4 4.3
7.376 7.121
.006 .053
15.9 16.2
1.4 1.3
Aa
44 45
4 3.
10800 11 GOO
4 4
18 15
7 4
LOW MED IRRAD
7 13
-6 -4
26 16
90.7 90.8
6.5 4
33.5 39.8
4.3 2.7
7.409 7.416
.028 .020
16.4 16.8
1.5 .04
45 46
4 .6
16420 17400
4.8 1.2
12 8
6 6
LOW MED IRRAO
MMT
7 13
-43 -42
37 45
Af.TC Ac.tc
82.5 74.7
12.6 17.5
fc AOjtb
39 43.2
4.6 14
Yd A3
7.397 7.300
.03 .1
Aa
16.4 16.3
1.65 1.2
45 45
3.8 4
A3,
10440 13250
3 5
8 5
7 4
HIGH IRRAD
7 13
-41 -2
40 20-91
if
78.9 80
7.9 3.6
A6. AC,
37 42
4 5
AC
7.416 7.433
.026 .017
16.9 16.9
.94 .72
AC AC
45 47
2.3 2.5
11000 14800
5 4
26 20
•5 6
HIGH IRRAD
MMT
7 13
-60 -94
31 66
if.rb le.te
70.3 65.9
12 26
Ad, Ac.Ta
41 45
10 12
AC AC _
7.380 7.368
.0460 .082
Ad Ab.ra
17 17
.91 .9
Af,«e Af,«c
47 50
2.3 1.9
•d,7c «e,Tc
10300 14000
3.8 3.2
10 19
6 4.
Legend Significance
a.
b.
c.
d.
e.
f.
t.90
t.95
t.975
t.99
t.9Q5
A « dlff. from controls
T • dlff. from pure Indolene atmosphere (no MMT)
• • dlff. from CO control
No mark indicates differences not significant or
comparisons were not made.
-------
20
o
*
o
e
s
o
20
40
•0
100
• CONTROL
CARBON MONOXIDE CONTROL
LOW MEDIUM IRRADIATED
LOW MEDIUM IRRADIATED WITH MMT
HIGH IRRADIATED
HIGH IRRADIATED WITH MMT
13
Figure 1.
Effect of exposure to fuel
emission on body weight of
old male rats.
108
-------
ACTIVITY WHEEL RUNNING OF MICE RELATED TO AUTOMOTIVE
FUEL EXHAUST. I. EFFECTS OF EXHAUST LEVEL
M.I. Gage, Y.Y. Yang, A.L. Cohen and J.F. Stara
Evidence from a number of sources indicated that
exposure to emissions from automotive and diesel fuels
altered the functioning of the nervous system and
behavior of animals. Most of the reported behavioral
changes have been decreases in animal activity as a
function of exposure to automobile engine exhaust, or
constituents of exhaust in ambient air or laboratory
created atmospheres. The present research was under-
taken to evaluate the use of activity wheel running
of mice in discriminating the relative severity of
effects of exposure to exhaust emissions when the com-
position or gasoline is changed by using different
brands or additives, or when emission control devices
are added to the engines. This evaluation differed
from prior studies in the use of 1972 engines tuned
either lean or rich to alter relative pollutant levels
and in the sim<aneous exposure of different groups
of subjects to multiple levels of gasoline exhaust
containing no additives.
Albino mice (Charles River CD-I, COBS) from 10 to 12
wk of age were divided randomly into groups of three
male and three female mice. Several days before the
start of exposures, they were placed in individual stain-
less steel activity wheels 5-3/4 in. in diameter, with
small attached compartments enabling continuous access
to food and water. Groups of six mice in activity wheels
were placed on the top shelves of appropriate inhalation
exposure chambers so that one of the groups inhaled each
level of nonirradiated and irradiated exhaust, clean air,
and clean air with 100 ppm carbon monoxide (CO) during
every exposure. Exposure periods lasted from 4 to 7
consecutive days. During exposures A and C, the engine
was tuned to factory specifications. During exposure B,
the engine was retuned for maximum manifold vacuum at
idle, and total exhaust concentration in each exposure
chamber was decreased so the amount of exhaust CO nominally
matched the amount in each chamber during exposures A and C,
A microswitch was activated by a flat portion on the
shaft of each activity wheel causing a counter to increment
once with every wheel revolution. Accumulated counter
values for each wheel were recorded every 2 hours during
the exposure period.
109
-------
The mean daily counts of wheel revolutions for each
group in exposures A, B, and C are shown in Figure 1.
Separate bars represent counts for male and female mice
as a function of exhaust level and atmosphere. The
results of analysis of variance indicated a significant
effect due to treatment in exposures A (F = 4.43, df =
.9/40, p < .001) and C (F = 28.0, df = 7/32, p < .001),
not in exposure B (F = 0.95, df = 7/32, p = 0.48).
There were significant differences between the sexes in
all three exposures (F = 17.7, df = 1/40, p < .001, in
A; F = 16.1, df = 1/32, p < .001, in B; and F = 35.1,
df = 1/32, p < .001 in C), with the males generally
making fewer daily revolutions than the females. There
were significant treatment by sex interactions in exposures
A (F = 2.3, df = 9/40, p <.05) and C (F = 7.8, df = 7/32,
p <.001). A greater decrease in wheel running was seen
with increasing levels of exhaust and a slightly greater
decrease was seen with exposure to irradiated than non-
irradiated exhaust of the same concentration in exposures
A and C. The decrease in activity levels appeared within
the first day of exhaust exposure and remained faiMy
consistent throughout the duration of the exposure period.
Within the first day after termination of exhaust in-
halation the behavioral activity returned to or exceeded
the pre-exposure levels.
Wheel running of mice served to measure behavioral
consequences of exposure to automotive exhaust, confirm-
ing several previous studies. The results of the current
study were particularly interesting considering that no
attempt was made to preselect mice for high pre-exposure
activity or low variability of the measured behavior.
The observed decrease in behavior was not a consequence
of the CO in the atmosphere because, in exposures A and
C, mice exposed to 100 ppm CO had behavioral changes that
were different from those of mice exposed to exhaust
containing the same level of CO. In exposure B, no signi-
ficant activity decreases were observed; this was when
levels of CO were comparable with those in other exposures
but levels of other measured exhaust constituents were
lower in all chambers than in the lowest levels in other
exposures.
Automotive exhaust can be said to have suppressed the
wheel running activity of mice because the counts fell
and rose with the onset and offset of exposure. The
amount of suppression was a direct relation to the concen-
tration of exhaust. The fact that a behavioral measure
that did not require training and that responded rapidly
to the onset of exposure was a toxicological indicator was
important because it provided an approach to rapidly acquire
data about the effects of some environmental pollutants on
certain aspects of behavior.
110
-------
30-
n
O
n
2 20-
O
g
O
•c
r-
<
w
s
CONC
TREAT. 4
-
•
|
\l
EXPOSURE A
If
1
O 100 i 25 5
1
111
T
,
i
0 75 1 100 !
u*i co INONIRRADIATED '
1
EXPOSURE B
1 T
! TT l
1 f| i i
25 I 50 , 75 . 100
0
IRRADIATED AIR
-
-
100
CO
J.
r
-i
-
50 | 75
T
100 | 5
j
1
ij; ;
3 75 | 10
EXPOSURE C C/Q
T ?M
li
\
i
]
;
J
1
ffl IT
Hi m fn ft i
3 0 j 100 1 50 | 75 | 100 j 50 | 75 | 1OO
NONIRRADIATED IRRADIATED AIR ' CO NONIRfi ADI ATED ' IRRADIATED
EXPOSURE LEVELS AS NOMINAL CO CONCENTRATION (ppm)
Figure 1. Daily v;hee! runnino of mice as a function of inhalation atmosphere in three exhaust exposures.
Daily revolution counts for each mouse were averaned over the duration of exposure. Bars
indicate the r.oanr- and standard deviations of these individual averages for groups of three mice.
Conponrnts of exlK-ust other than CO during exposure B were lower than in the lov.-est levels of
exhaust during exposures A and C.
Ill
-------
ACTIVITY WHEEL RUNNING OF MICE RELATED TO AUTOMOBILE
FUEL EXHAUST. II. EFFECTS OF A MANGANESE ADDITIVE
M. I. Gage
The mean number of dally revolutions made by mice
in an activity wheel decreased when these mice were ex-
posed to emissions from automotive fuel exhaust. This
decrease was directly related to the concentration of
exhaust in the exposure atmosphere. The purpose of the
present experiment was to see if this behavioral measure,
which was sensitive to quantitative differences in ex-
haust concentrations, was able to show changes in per-
formance as an effect of inserting a methyl cyclopenta-
dienyl manganese tricarbonyl (MMT) additive.
Subjects and methods were similar to those used in
earlier exposures. Groups of three male and three female
albino mice were placed in activity wheels with continuous
access to food and water five days before the start of
exposures. The wheels were connected to counters that
recorded revolutions of each wheel Independently. Expo-
sures E and F lasted 2 weeks. One week before the start
of exposure F a 1-day exposure to exhaust of fuel contain-
ing MMT occurred. Activity of the mice was measured before,
during and for 5 days after the 2-week exposure. During
these periods, the room with inhalation chambers was kept
in a constant state of Illumination. In each exposure,
one group was exposed to every level of irradiated and
nonlrradiated exhaust, clean air, and clean air with
carbon monoxide (CO).
Exposure to automotive exhaust emissions decreased
activity wheel running of the mice. Results of analysis
of variance of revolution counts of male mice indicated
a significant effect due to exhaust concentration (F - 7.42,
df = 2/24, p < .005) but no effect of irradiation or
additive. Results of a similar analysis of the data
from female mice Indicated a significant effect due to
both concentration (F = 9.93, df = 2/24, p < .001) and
additive irradiation. Separate analyses of variance showed
there were no differences among groups of male or female mice
exposed to clean air or just CO polluted air in both ex-
posures. Figure 1 shows a decrease 1n revolutions as a
function of atmospheric type and level, with each animal
serving as its own control, for both exposures and sexes
112
-------
For each animal, the mean daily revolutions averaged over
the entire exposure period was divided by its own mean
daily revolutions obtained 3 days before and after ex-
posure (when it inhaled clean air) and is expressed as
percentage. The mean and standard deviation of the per-
centage change of all animals in a group were plotted as
<150
B
D
W
£
I
JlOO
i 50
E Mole
F Hole
f Female
F Femofc
jonc
0
100
50 I 75 I 100 I 50 1 75 I 100
Iicot. ] Air I (0 I Notiinadmtcd I llmdiotcd
Eshtrust level n Nominal (0 (oncenlrotitm (ppm)
Figure 1. Percent charge of wheel running as a function of inhalation atmospheres
for one exposure without (E) and one exposure with (F) a Manganese
additive in the fuel. Points represent r.eons and standard deviations
for group; of 3 mice of the percent change fron control values of
each mouse
the ordinate values. Considering each animal as its own
control, no differences appeared during the exposure period
in mice inhaling clean air or CO. However, decreases in
revolution counts appeared in all groups exposed to exhaust.
The amount of decrease was a direct function of exhaust
concentration. Little differences appeared between the
changes in male and female performance and between changes
during the different exposures. There seemed to be slightly
less of a decrease in activity of females exposed to non-
irradiated exhaust from fuel containing the MMT. Figure 2
shows the mean daily revolutions for four of the eight groups
of each sex over the course of each exposure. As in earlier
exposures, activity of mice exposed to exhaust decreased
within the first day of exposure, remained low during the
exposure period, and returned to the level of control groups
soon after the termination of exposure. Data from the aborted
1-day exhaust exposure at the start of exposure F indicated
that the behavioral results can be adequately predicted from
only a very short period of exhaust inhalation.
113
-------
*•••* Ckan Air
Nonirrcdiated Exhaust
Irradiated Exhaust
'!/"«» r> y
/ ' / v "
tfA/ /\ ft *A.A ft
V u \\ / A ' \ / A '
*l V \V *•! r * ^rf
Figure 2. Mean dally wheel running of different groups of 3 mice over the course of two exposures
uata are shown oniw for th« miVo avnnco>< »^ *t._ u.-_i—^ i *_ K«-.«iv.*
Wheel running activity of mice was suppressed by auto-
motive fuel exhaust as it had been in earlier exposures.
The amount of behavioral suppression was only minimally
altered by introducing MMT into the fuel. This change in
behavioral suppression reached significance only in the
female mice. It was not surprising that the Mn additive
exerted so little an effect in comparison to the effect of
exhaust exposure itself. The Mn did little to alter
the composition and quantity of measured gaseous constituents
of the exhaust even though it increased the particulates
measured. Results of this experiment suggest, therefore,
that measured behavioral suppression was in some way related
to the level of gaseous components of exhaust but not the
level of CO or particulates.
114
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GASOLINE EXHAUST EFFECTS ON WATER LICKING OF RATS
M.I. Gage and D. Schneider
Intake of adequate amounts of food and water is essen-
tial to the general health and well being of living organisms.
If an environmental pollutant alters food and water intake,
it will produce changes in the physiological and biochemical
measurements of an organism leading ultimately to death. A
study was undertaken to see if changes in a stereotyped motor
act related to water ingestion, that of licking at a spout,
was altered in response to exposure to automotive fuel ex-
haust emissions when food intake was in no way restricted.
Two groups of four or six male albino rats (Charles
River, CO, COBS) were tested in each of the exposures C
through F. From the time of arrival (1 to 3 weeks before
the start of exposures), until 5 days after the exposure
ended, the rats had daily access to water only during a
15-minute period when they were placed in a small operant
conditioning chamber which contained clean, filtered air.
Food was available at all other times in their home or ex-
posure cages. The rats could drink water by licking at a
spout, the tip of which was flush with one end panel, 1.5 in.
above the floor and 2.0 in. from the right side wall of the
testing chamber. The number of licks, and times of onsets
to offsets and of offsets to onsets of licks (interevent
times), were measured by an electronic contact switch passing
a small alternating current through the animal as he licked.
DUring the exposures, one group was kept in an exposure
chamber containing clean air and the second was kept in an
exposure chamber containing irradiated exhaust with nominally
100 ppm CO. They were removed from these chambers only for
the daily testing.
Without special training, the rats began licking at the
water spout and within a few days emitted from 2000 to 4000
licks during a session. Typical control performance of a
rat is presented in Figure 1. The left graph is a cumulative
record in which licks are represented cumulatively along the
ordinate and session time along the abcissa. The graph re-
starts from the baseline after every 400 licks. The licking
rate is therefore read directly as the slope of the line.
Rats licked at the rate of about 7/sec., which is similar to
rates reported by others. As a session progressed, periods
of long pauses ensued. Most rats ceased licking some time
before the 15-minute session ended. The right graph in
Figure 1 is an interevent time histogram in which time, in
milliseconds, between onset and offset and time between off-
set and onset of each lick is the abcissa and number of onsets
115
-------
352-
.2288-
-S224H
96-
32-
10
~r
12
I I
14 16
I
96
I I I I I
Figure 1.
Time in Session (Min.)
32 96 160 224
Interevent Time (Msec.)
Computerized plots of typical performance of a control rst during one fifteen minute session of licking for
wter. Left graph is * cumulative record of the licks. Right graph is an interevent time histogram of
the same licks.
or offsets occurring at that time is the ordinate. These
graphs showed that most rats had a highly stereotyped
pattern of licking which, although different from rat to
rat, was similar from day to day in the same rat. Only
in the data from some rats were two peaks in the interevent
time histogram dlscernable. Oscilloscope tracings of the
lick sensor output indicated that the time from the ordi-
nate to the first peak was the modal offset to onset time
and whereas the time from the ordinate to the second peak
was the modal onset to offset time of the licks.
The daily performance of the rats before, during,
and after exposure C (Figure 2) showed that licking was
suppressed in the animals inhaling automotive exhaust
throughout the course of exposure. The mean number of
daily licks quickly returned to or exceeded the level of
the control group when the experimental group again inhaled
clean air. During the exposure period, the mean number of
daily licks for rats in the exposed group was 2406.9, an
amount significantly different (t = 5.26, df = 6, p < .01)
from the 3304.7 mean number of daily licks for rats in the
control group. During this period, rats inhaling exhaust
gained less weight than rats breathing clean air (4.75 gm
versus 34.5 gm, t = 4.65, df = 6, p < .01). The pattern
of licking and the interevent times did not change In a
consistent way related to the treatment during exposure.
116
-------
EXPOSURE C
X
-g
— 2
—o—Control
—•— Irradiated Exhaust
J t
t i
8
-EXPOSED-
10
12
14
Days
Figure Z. Dally licking for water during exposure C. The ordlnate 1s the mean number of licks per day of each
group of four rats.
Similar results occurred for other exposures. An
analysis of the mean number of dally licks for the last
2 exposure days during exposures E and F showed a signifi-
" df = 1/20,
additive or
additive by exhaust interaction. Data were obtained for
only 2 days during the exposure
lick sensing apparatus.
cant effect of exhaust exposure (F = 5.89,
p < .05) but no significant effect of the Mn
because of failure of the
The suppression of water licking by inhalation of
automotive fuel exhaust emissions was complicated by the
failure of the exposed rats to show a weight gain equal
to that of the control rats. Water intake may have been
lower in the exposed rats because they weighed less. How-
ever, cursory examination revealed no clear relationship
between weight and number of licks. Rats in the exposed
group may have gained less weight because they drank less
water. Cause versus effect could not be elucidated. Water
intake may have been decreased by a failure of the exposed
rats to eat as much food as the control rats. Rough esti-
mates of the amount of food eaten each day showed no diff-
erence between the groups. In a related experiment,
hamsters deprived of both food and water were trained to
press a bar for food and lick for their water during daily
15-mln. sessions. There were no discernible effects of
exhaust exposure on hamster bar presses or licks. It is
clear, however, that automotive fuel exhaust emissions de-
creased the number of daily licks of rats for water. This
decrease was not altered by the addition of MMT additive to
the fuel, presumably because the additive did not greatly
alter the gaseous constituents of the exposure atmospheres.
117
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COMPARATIVE ACUTE TOXICITY OF EMISSIONS FROM AN INTERNAL
COMBUSTION ENGINE USING TWO DIFFERENT NO-LEAD GASOLINES
K.I. Campbell and L.H. Hall
Preliminary to a program of studies to assess the
comparative toxic hazard of fuel and fuel additive
emissions, a pilot study was conducted to determine
comparative susceptibility of laboratory animal species,
strains* ages and sexes to inhalation of exhaust emissions
from a small 4-cycle internal combustion engine. A
further objective was to determine the ability of a multi-
species acute-lethality test system to discriminate
possible differences in the character of emissions as a
consequence of using two different marketed gasoline fuels.
In both experiments the same engine was used, operated
as identically as possible as to engine speed (average
about 1270 rpm) and "tuning" (smooth performance). Also,
an attempt was made to maintain a constant and comparable
ratio of purified air:exhaust in the test atmosphere
delivered to the exposure chamber (average 65:1 in experi-
ment A, 74:1 in B). Two marketed gasolines were used in
this experiment; both were unleaded and low-octane ("regular")
Males and females of the following species, strains, and
ages were exposed continuously for 5 days: "old" albino
rats, inbred and random-bred; "mature" and "infantile"
random-bred albino rats; "old", "mature" and "infantile"
Syrian hamsters. Corresponding controls were exposed to
purified air. Timed mortality during exposure and pre-
and post-exposure body weights were recorded. Samples of
undiluted exhaust and of exposure chamber atmospheres were
periodically analyzed for carbon monoxide (CO), total hydro-
carbons (THC), and oxides of nitrogen (NOg and NO). Mean
concentrations (ppm) of principal pollutants for experi-
ments A and B, respectively, were as follows: CO, 1526
and 1498; TCH, 381 and 387; NO?, 0.16 and 0.15; and NO, 1.46
and 1.11.
On the basis of acute lethality data, the following
comparative susceptibilities were generally apparent: among
aged rats, the random bred strain was more susceptible than
the inbred strain; aged and mature male and female rats were
more susceptible than hamsters of comparable age and sex,
whereas the reverse was true for infantiles (hamsters more
susceptible than rats); males more susceptible than females,
with the exceptions that in aged rats and hamsters the
sexes were about equally susceptible; aged rats were the
118
-------
most susceptible, followed by mature and infantile, but
among hamsters the infants were the most susceptible.
Substantial loss in body weight occurred in most groups
and in both experiments, but the loss did not permit
differentiation of biologic or exposure factors. Ranked
susceptibilities among exposure subject types were generally
consistent in both experiments. Table 1 summarizes these
acute toxicity data for both experiments.
Finally, there was an apparently greater degree of
toxic response in experiment A than in B. However, despite
the appearance of the data on the surface, we cannot con-
clude at this time that gasoline A is more hazardous than
B. Reasons for this include incomplete quantitative
characterization of the emissions, and the possibility of
the exposure pattern influencing the severity of response;
neither of these factors is specifically related to fuel
itself.
TABLE 1.
ACUTE TOXICITY IN RATS AND HAMSTERS EXPOSED TO EMISSIONS OF SINGLE-
CYLINDER, 4-CYCLE ENGINE USING TWO DIFFERENT NO-LEAD GASOLINES
Experiment A
Total
Test Subject mortality
Rat, inbred, old, male
Rat, inbred, old, female
Rat, randombred old, male
Rat, randombred old female
Rat, randombred mature, male
Rat, randombred mature, female
Rat, randombred infantile,
both male and female
Hamster, old, male
Hamster, old, female
Hamster, mature, male
Hamster, mature, female
Hamster, infantile, both
male and female
*
100
50
100
80
100
90
100
70
80
100
20
100
Median
lethal
time*
hr
38
108
4
10
7
21
53
84
80
85
193
3
Relative
weight
change
%
ND
-22
ND
-32
ND
-16
NO
-23
-28
NA
-25
ND
Expe r imen t
Total
mortal i ty
%
ND
ND
80
50
60
40
100
0
0
0
0
100
Medi an
lethal
time*
hr
ND
ND
9
50
60
228
69
NA
NA
NA
NA
10
G
Relative
weight
change^
%
ND
ND
-27
-24
-20
-14
ND
-20
-27
-31
- 3
ND
Calculated estimate
In relation to controls
ND Not determined; NA Not applicable
NOTE: Among controls, no deaths occurred and body weight changed
by only -6 to + 13? from pre-exposure values.
119
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CHRONIC EFFECTS OF AUTO EXHAUST AND OTHER
ATMOSPHERIC POLLUTANTS IN FEMALE BEAGLES
0. Stara, G. Hueter, T. Lewis, K. Campbell,
D. Coffin, R. Hinners, M. Malanchuk, K. Busch,
W. Bloch, J. Orthoefer, M. Wiester, D. Hysell
and W. Moore
This rather large and complicated study is aimed
at assessing a population of female beagles to determine
the long-term biological effects of inhaling auto exhaust
emissions and other major air pollutants. The selection
of beagles as an experimental animal has value chiefly
because of their large size and long life span. This
makes them better suited for various physiological
studies, and the resulting degenerative changes more
closely mimic man than is the case of other animals,
such as rodents.
The study was initiated in September 1965. Female
beagles were randomly distributed at 4 mo of age, into
26 exposure chambers, four dogs per chamber. The dis-
tribution and exposure pattern was as follows: Clean
air controls (20), raw exhaust (12), irradiated exhaust
(12), SOX (12), R •«- SOX (12), NOL + N02H (12), and
NOH + NO?! O2) for a total of 104 animals. They were
exposed daily for 16 hr.
Exposure chamber design (Hinners, R.G., et al . , Arch.
Environ. Health 13: 609-615, 1966), atmospheric measure-
ments (Crider, W.L., Anal. Chem. 37: 1770-1773, 1965, and
Amer. Lab., Nov. 1969), and statistical design (Busch, K.A
and Ludmann, W.F. Presented at the 60th Annual Meeting
APCA, June 11-16, 1967, Cleveland, Ohio) of the study
have been reported in detail. Figures 1 and 2 summarize
these systems. The average concentrations of individual
pollutants are reported in Table 1. A 5-yr summary of
the exposure levels is presented in Figure 3.
120
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AIR CONDITIONING) GAS
AND PURIFICATION I GENERATION
GAS I ANIMAL EXPO- I MONITORING AND
IRRADIATION I SURE CHAMBERS I RECORDING
Figure 1. Schematic of auto exhaust study.
EXHAUST STACK
V NO
r--— ' — T 1 1 1~ — r-
i
E
2
CA
3
NO,
"so,
5
R
6
js+sq
|HSO,
-^--
-T---I — T — T — r — T~
8
NO,
9
CA
10
s°,
II
CA
12
R
13
KiO
X
\ WO
— 1
14
I
4 /-
EPISODE
AUTO EXHAUST
AEROSOL
NO, fiO'ANO
CA CONTROL AIR
It NOM-lMtAOIATCD AUTO EXHAUST
•ff «UTO EXHAUST IMJECTIO* KXKT
Figure 2. Schematic of exposure chamber supply and exhaust flow
121
-------
Based on administrative decision, all animals were
removed from the exposure atmospheres in August 1970;
at this time they had experienced 70 months of uninter-
rupted exposure. Immediately thereafter they were shipped
under a contract arrangement to the Pathology Department,
University of California, Davis, for maintenance and
eventual terminal studies. At the present time, the
study is in its seventh year. It is hoped that following
a thorough review of the data and a final set of physio-
logical measurement, the animals will be sacrificed during
FY'74 and the final report published.
During the animals exposure regime, until 1969, the
major biological parameters studied were hematology and
pulmonary function. Commencing with the fifth year of
exposure, additional parameters and measurement of stand-
ard values were added, particularly in cardiovascular
physiology, neurophysiology, blood chemistry, patterns in
body weight changes and clinical medicine. During the
exposure period, animals that died due to fighting or
anesthesia were examined for pathological lesions. Just
before their removal from the exposure atmospheres, lobec-
tomies were performed on 15 dogs (5 from irradiated exhaust,
5 from NOX and 5 from clean, air atmospheres) to determine,
using electron microscopy, possible lung tissue effects
before the animals were removed permanently from the
chambers. Even though the final set of physiological
measurements is not completed and all such observations
must be confirmed by pathological lesions, there are
indications of progressive cardiovascular and pulmonary
effects.
Table 1. COMPARISON OF SUPPLEMENTAL GAS CONCENTRATION LEVELS (ppra)
IN THE CHRONIC DOG STUDY CHAMBERS WITH AMBIENT ATMO-
SPHERIC CONCENTRATIONS IN THREE CITIES*
Gas
CO
NO
N02
S02
HC
Oxldants
Chicago
Yearly
avg.
9.1
0.072
0.050
0.125
3.0
0.029
Max. daily
avg.
22.9
0.189
0.114
0.654
5.18
0.080
Cincinnati
Yearly
avg.
5.6
0.032
0.028
0.024
2.5
0.031
Max. daily
avg.
10.6
0.358
0.069
0.067
5.37
0.089
Washington, D.C.
Yearly
avg.
4.9
0.047
0.043
0.043
_
0.025
Max. daily
avg.
14.6
0.359
0.088
0.132
_
0.109
Control
level
100
1.5-2.
0.5-1.
0.50
24-30
0.2-0.
0
0
4
*NASN air quality data, 1967
122
-------
CHAMBER ATMOSPHERE-1RR
CHAMBER ATMOSPHERE - IRR + SO.
100
ro
o.i r
.01
-
CO
. - HC
_ N0r
~ *v
•" ^^'V .' - «. 03
^- - — ' \x ^7^—"^ ~J*
v-»'
1966 I
W67 ] 1968 I 1969 1 }<37Q \W
YEAR
100
10
i.o
0.1
.01
J I I __t. L _ ..]_ .._!._ 1 II I I
I
1967 I
1968 I
YEAR
1969 I 1970 1971
CHAMBER ATMOSPHERES - R, R + S0y, SO,. NOL + NO^
100
10
iE i.o
Cu
0.1
.01
- __ — . CO
: ( IDENTICAL FOR R AND R + SO,
1 Z _HC
-
\\
====. — -^=^ ™s-^roj+*)
: ^^^*^— -~~^ ^ ST^^OX.
•- — NUL
1 1 1 1 1 1 1 I.I II 1 ' .1
19661 1967 | 1968 i 1969 | 1970 19^
YEAR
Figure 3. Five-Year Summary of Experimental Atmospheric Levels
-------
The cardiovascular results (Bloch, W.N., et al.
Arch Environ. Health 24: 342-353, 1972) indicated that
the pol 1 utant-exposed~cfogs had a higher incidence of
ECG and VCG abnormalities as well as documented or
suspected myocardial infarctions than did the dogs
exposed to clean air. As a result, these data strongly
implicate air pollutants as a factor in causing cardiac
electrophysiological damage. An example of the type
of cardiovascular data obtained is seen in Table 2.
TABLE 2. EFFECTS OF CHRONIC AUTO EXHAUST EXPOSURE
ON CARDIAC RHYTHEM IN DOGS
Type of Atmosphere
No. of
Animals
W-QRS-C Index*
Clean air 19
Clean air + SOX 11
Clean air + NOH+N02L H
Clean air 4 NOL+N02H H
Raw exhaust 11
Raw exhaust 4 SOX 10
Irradiated exhaust 11
Irradiated exhaust 4 SOV 10
Normal*
Normal
Normal
Normal
Normal
Normal
30.8% > Normal
42.0% > Normal
*Widened QRS complex.
"•"Normal index variation was 8.6%
The preliminary pulmonary function results indicate
that after 60 months of exposure, the R 4 SOX group had
a significantly greater RV/TLC (ratio of residual volume
to total lung capacity) than did the control group (CA).
Furthermore, the R 4 SOX, R, and NO{H) 4 NOn) groups
also had a higher RV/TLC than did the SOX group.
After 63 months of exposure, the nitrogen washout in
the I 4 SOX and NOn) + NO(H) groups was significantly
lower than in the CA and R 4 SOX groups. The vital
capacity of the I 4 SOX and N0(n) groups was significantly
124
-------
higher than that of the control (CA) group. In addition,
the vital capacity in the I 4 SOX group was also higher
than that found in the SOX group. The data indicate that
chronic exposure to air pollutants at realistic levels
elicits harmful effects on pulmonary function. Results
are not as yet completely conclusive, however, the data
are being reanalyzed using various statistical
ascertain their biological significance. This
will be completed after the collection of the
of data.
methods to
review
final set
Neurophysiological studies and pathology studies, of
course, are not complete at this time. Hematologic values
throughout the study have demonstrated changes; however,
these are contributable to the continuous 100 ppm carbon
monoxide level. Soon after their removal from the contam-
inated atmospheres, all blood parameters have returned -
on the whole - to normal values.
cho^l!"1*!!'1?1 *?e ?n1mals exposed to irradiated exhaust
showed a higher incidence of epiphora (Figure 4). As nay
be seen, the condition underwent remission when the
animals were removed from the exposure chambers. The
animals exposed to exhaust products also showed a higher
incidence of chronic dermatitis and weight changes
60
-40-
o
J
a
£30
|20<
S
"" JO'
123456
9[
I Control
2 Raw
3 Irradiated
4SOK
5 RawfSOx
6 Irrod.+SO.
8NO
1
November 1969
August 1970
Jun» 1971
D*mab*r 1971
Figure 4. Incidence of epiphora among female
beagles in chronic auto exhaust study
125
-------
Arrangements are being made to review thoroughly all
available data in March 1973, at which time a decision
will be made as to the termination of the experiment. It
is most important to perform detailed tissue pathology,
including light and electron-microscopy, since this is
the sole long-term study of auto exhaust and other air
pollutants in large mammals at the present time. The data,
therefore, will have an impact on future revisions of air
pollution standards. In addition, it is expected that
the study will be widely quoted; for these reasons, a
precise evaluation of the pathological effects is of
paramount importance.
126
-------
GROSS PHYTOTOXICITY OF AUTOMOTIVE FUEL COMBUSTION
EMISSIONS TO EPISCIA AND AFRICAN VIOLET
K. I. Campbell
It has been well documented that many plant species
of ornamental, agricultural, or otherwise economic
importance are adversely affected by exposure to atmo-
spheres containing internal combustion exhaust and its
components. Requirements of recent legislation include
research concerning relative hazards to public health
or welfare of emissions from motor vehicle fuels and
addi ti ves.
In partial implementation, a project entitled
"Toxicologic Assessment of Mobile Emissions ("TAME") has
been in progress at NERC-Cincinnati for the purpose of
determining comparative toxicologic potential of emissions
resulting from and as they may be affected by the use of
various fuels and additives. To evaluate their utility as
test subjects for comparative fuel-emissions testing, two
species of ornamental plants have been included along with
the primary battery of laboratory animal bioeffect systems.
Such groupings of biological systems for toxicologic studies
is increasingly being used. In serial tests, these subjects
are experimentally exposed in environmental chambers to
emissions produced by appropriate engine systems in which
reference fuel or test fuels and additives are used. Of
the six experiments conducted to date, information regard-
ing plant effects in the last two, TAME E and F, are re-
ported herein.
The generation and exposure facilities (engines;
dynamometers; exhaust dilution, distribution, and irradi-
ation facilities; controls; fuels and fuel storage; and
exposure chambers) are described elsewhere, as are the
atmospheric analytic methods and data. Study E was a test
of emissions resulting from use of reference fuel (essen-
tially no additives) only, and F was a comparative test in
which the test additive was added to the .reference fuel.
The additive compound in this test was an organic manganese
antiknock compound proposed for use in gasoline with or in
lieu of alky! lead. It was added at the rate of 0.25 gm
Mn/gallon, twice the recommended concentration.
127
-------
Two plant species, Episcia cupreata (Silver Sheen)
and African Violet (Ultra Blue in E, Bloomin' Fool in F),
comprised the experimental test unit. Since it was
desirec to determine the plants' abilities to discriminate
influence of exhaust treatment (irradiated compared with
non-irradiated), exposure level (concentration), and
pattern of exposure (continuous compared with interrupted)
on response magnitude, as well as to estimate their
possible utility in discriminating between atmospheres
differing due to the test variable (e.g., use of fuel
additive), single units were exposed to: both irradiated
and raw exhaust types at two levels (low and high,
characterized by CO concentrations of about 50 and 100 ppm,
respectively), and in three exposure patterns (intermittent,
4 hr/day x 12 days, total pollutant exposure of 48 hr;
interrupted, two 24-hr periods with an intervening 24-hr
clean-air exposure, total 48 hours; and continuous exposure,
total 48 hrs). Units were also exposed to clean air (CA)
and to carbon monoxide (CO) atmospheres as controls.
Toxic response was assessed in terms of grossly visible
foliar damage (e.g., spotting, discoloration, droop, curl,
bronzing, wilt, death, and dehydration), and degree of
severity was graded on an arbitrary subjective scale ranging
from zero (no apparent effect) up to +12 (maximal effect
for the species concerned). In addition, specimens of
representative leaves were weighed, dried, reweighed, and
frozen. For Episcia, per cent of original post-exposure
weight retained during dehydration was used as an additional
quantitative index of injury. The exposure conditions and
results are summarized in Table 1.
Generally, in both species, but more demonstrable in
Episcia (which was the more sensitive), there were differ-
entiable exhaust-exposure effects with respect to atmo-
sphere type (irradiated more effective than raw), exposure
level or total dose (magnitude of effect reasonably pro-
portional to concentration), and exposure pattern (in-
creasingly severe effect associated with intermittent,
alternate, and continuous exposure at equivalent total dose).
CA and CO exposures produced no effects.
128
-------
TABLE 1. CONDITIONS AND PLANT EFFECTS OF EXPOSURE TO GASOLINE
AND GASOLINE ADDITIVE COMBUSTION EMISSIONS*
TAME E Study
Exposure type
Pattern, level
Intermittent
CA
CO
RL
RH
IL
IH
Interrupted
CA
RL
RH
IL
IH
Continuous
CA
RL
RH
IH
Actual
Total dose.
ppm-days
11
202
106
206
92
214
11
106
206
92
214
11
106
206
92
214
Effect
Visible damage,
Episcia
0
0
+
T
2
8
0
4
N.D.
5
N.D.
N.D.
N.D.
N.D.
N.D.
12
TAME F Study
Effect
% Weight
retention,
Episcia
7.6
5.9
15.6
15.9
20.9
10.6
41.7
N.D.
29.7
N.D.
N.D.
N.D.
N.D.
N.D.
= 90
Actual
total dose,
ppm-days
12
210
128
256
101
216
12
128
256
101
216
12
128
256
101
216
Visible
Episcia
0
0
8(?)^
3(l)f
2
4
N.D.
3
10
8
11
0
12
12
12
12
Damage
0
0
0
0
0
0
N.D.
2
3
i
3
0
5
7
4
8
% Weight
retention
Episcia
4.4
3.3
36.8j<
10. (V
4.2
55.3
N.D.
5.6
38.4
29.4
62.3
2.3
53.4
71 .7
77.6
81.2
*TAME E, reference fuel only; F, reference fuel plus additive; engine, 1972 Chevrolet V-8, 350 CID;
engine dynamometer and California-cycle operation; exhaust dilution ratios: E, 19.0:1, ana t-,
+See text for explanation of pattern. CA = clean air, CO = carbon monoxide, RL and RH = nonirradiated
low and high, and IL and IH = irradiated low and high.
f Atypical. Suspect laboratory error.
Foliar damage resulting from exposure to these auto-
motive emissions appears due primarily to olefinic hydro-
carbons and nitrogen oxides constituents (e.g., ethylene
at 2-3 ppm, N02 at 3-9 ppm), particularly since ozone
and other reactive oxidants were absent or negligible.
Plant tissue specimens have not yet been assayed for
determination of manganese uptake.
In these experiments this particular plant-effects
system, although more sensitive in terms of effect magni-
tude than several animal response criteria used, appeared
unable to clearly discriminate the test variable (additive)
effect through its influence on total emissions. It seems
fair to suppose, however, that the system would discrimin-
ate larger alterations in emissions (say 40% or more,
particularly if phytopathic constitutents are involved)
and that with refinements (e.g., multiple selected species
and more sophisticated quantitative effect parameters) a
more useful and sensitive model may result for application
in future studies.
129
-------
METHODS DEVELOPMENT STUDIES
-------
THE USE OF CORNEAL MITOTIC RATE AS A MEASURE
OF OCULAR IRRITATION
D. Hysell , W. Moore and L. Garner
Ocular irritation is an often reported discomfort in
human populations exposed to smog and other atmospheric
pollutants. Since this effect is often subjective, the
development of an animal model for assessing relative
irritability of various pollutants was considered important.
Clinical symptoms like epihora, conjunctivitis, and photo-
phobia were considered to be not sensitive enough to permit
meaningful comparisons. After a review of the pertinent
scientific literature, it was believed that determination
of corneal mitotic rate might be an appropriate technique.
The methodology required that an animal be killed and the
eyeballs be extirpated as soon after death as possible.
The enucleated eye was immedately placed in an acetic acid-
alcohol fixative, stained in orcein, decolorized, and then
prepared for mounting. The latter involved separating the
dome-shaped cornea from the rest of the eye, making sure
that no pieces of conjuctiva or iris adhered to the cornea.
Four radial incisions to facilitate flattening and mounting
were made in the cornea. The cornea was mounted in glycerin
jelly with the epithelial surface uppermost. The coverslip
was ringed with finger nail polish to preclude dehydration
of the preparation. When examined under oil immersion
microscopy, the mitotic figures were readily recognized by
their staining characteristics and morphology. One hundred
oil immersion fields were examined, and the total number
of cells in mitosis were tabulated.
Corneal epithelium exhibits a marked diurnal variation
in numbers of mitotic figures. For the technique to be
useful, there should be no extreme day-to-day variations in
mitotic rates if the animals were sacrificed at essentially
the same time each day. To check this, groups of five male
hamsters, each weighing 75-100 g and maintained in an animal
room, were sacrificed at 10:00 a.m. on 5 consecutive days.
Similar groups of hamsters, maintained in stainless-steel
exposure chambers and receiving only clean air were also
examined. In contrast to the rather stable mitotic counts
from the colony room animals, the chamber-maintained animals
showed marked day-to-day variation (Figure 1).
133
-------
CORNEAL MITOTIC RATE
ANIMAL ROOM (HAMSTER)
CONTROL EXPOSURE CHAMBER
(HAMSTER)
2 3
DAYS OF EXPOSURE
Figure 1. Cornea! mitotlc rate in hamsters main-
tained in exposure chambers and animal
room.
The chamber conditions (identical to those used in
TAME studies) that might result in these variations were
24 hr of daylight (12 hr light, 12 hr dark in animal
room) and an air flow of 15 changes/hour (about 7 changes/
hour in animal room).
A second study was performed in the exposure chambers
in which the animals were maintained on a 12-hr-light,
12-hr-dark cycle and sacrificed at 10:00 a.m., but received
exposure to either 5, 10 or 15 chamber air changes/hr. As
may be seen by the results (Figure 2), maintenance of
between 5-10 air changes/hr is critical 1n reducing day-
to-day variation; however, the light cycle is also important
as the 15 air changes/hr did not show the extreme variation
seen in the first study where there was 24 hr of light/day
plus 15 air changes.
134
-------
The cyclical rather than sustained response is what
might be expected of an irritant. It has been found that
every 3-4 days a cell in the corneal germinal layers under-
goes mitosis. Therefore, if an irritant produces a peak
mitotic response, it would be followed in several days by
a low mitotic rate because of the synchronization of mito
tic cycles in a large proportion of the cells. From these
data, the technique appears to be reproducible if certain
parameters are met and yet is sensitive enough to detect
a response from an irritant as innocuous as changes in air
flow. Because of the results of this study, TAME exposure
conditions have been changed so that all animals maintained
for pathology receive 12 hr light, 12 hr dark, and no more
than 7-8 air changes/hr. This system is being used currently
in the bioeffect studies of mobile emissions.
CORNEAL MITOTIC RATE
20O|—
Q
eft
o
o 150
o
i
i
Ik
y
100
AIR CHANGES/HR
DAYS OF EXPOSURE
Figure 2
Variations in mitotic counts between hamsters
maintained under differing numbers of air flow
changes/hr.
135
-------
BLOOD PRESSURE OF MONKEYS: NON INVASIVE
M.J. Wiester and R. Iltis
Studies in our laboratory require a simple but
reliable method to measure blood pressure In monkeys
over an extended period of time. Since indwelling
catheters are time consuming and difficult to maintain,
a tail cuff method was adpoted. In applying the method,
we found that systolic pressure, as well as diastolic
pressure could be determined and, thus, mean pressure
calculated. To evaluate the accuracy of tail cuff
pressure readings, a series of experiments was carried
out in which monkey tail cuff pressures were compared
with simultaneously recorded abdominal aortic pressures,
measured directly. The blood pressure range was changed
by means of a hypertensive drug and hemorrhage.
Rhesus monkeys (5 kg) were anesthetized with penta-
barbital, and a 5 French pressure transducer (Millar
Instruments) was inserted via the femoral artery. The
output of this direct pressure measurement was amplified
and recorded (Sanborn 350). Mean pressure was measured
by means of a planimeter and at least three pressure
pulses were averaged. The system for indirect pressure
measurement includes two sensors: a pulse transducer
(Narco) fed into an AC amplifier to monitor tail artery
pulses, and a pressure transducer (Statham p23Db) fed
into a carrier amplifier (Sanborn) to measure tail artery
pressure. The outputs of the two amplifiers are fed to a
recorder. If a chart recorder is used, pressure is repre-
sented on the Y axis and pulses are superimposed (Figure 1)
If an X-Y recorder is used, pressure is fed to the
X axis and pulses to the Y axis (Figure 2). In both
recordings, the initial point where a pulse is observed
designates systolic pressure and the "less-definite" area
where pressure pulses reach a uniform amplitude indicates
diastolic pressure. In Figure 1, the pressure pulse is
diamond shaped, and in Figure 2, it is spade shaped. Mean
pressure is calculated by an emperical formula:
P (systolic) + 2 P (diastolic)
A block diagram for the indirect blood pressure system is
shown in Figure 3.
136
-------
150
100
1 50
S - Systolic
D-Diostolic
B. P. 116/78 mm Hg
12
Seconds
18
24
36
Figure 1. Typical tall-cuff blood pressure (chart) re-
cording from an unanesthetlzed rhesus monkey,
Systolic D = Diastolic
BP =131/97
0
100
150
Figure 2.
50
Blood Pressure, mm Hg
Typical tall-cuff blood pressure (X-Y) re-
cordings from an unanesthetized rhesus monkey
137
-------
Animal
Timed
Pump
-Pulse Transducer
Pressure
Transduce?
To A/D
Digital Display
Figure 3. Block diagram for tail cuff blood pressure measurement
Of the five monkeys used in this experiment, three
were given methoxamine hydrochloride I.V. (Vasoxyl from
Burroughs Wellcome & Co., N.Y.) in doses necessary to
raise blood pressure 30-50 mmHg. All monkeys were
hemorrhaged to the point of shock. Results from a typical
experiment are found in Figures 4, 5 and 6. They show
direct and indirect pressure readings for systolic, diastolic,
and calculated mean pressures. As can be seen in the diagrams,
the two types of pressure readings follow each other throughout
the pressure range.
138
-------
SYSTOLIC BLOOD PRESSURE: DIRECT AND TAIL-CUFF MEASUREMENTS
60-
40-
METHOXAMINE
HEMORRHAGE
DIRECT
TAIL-CUFF
10
20
30
40
TEST NUMBER
Figure 4. Systolic blood pressure of monkeys
direct and tall-cuff measurements.
140-
£ 120-
E
E
Ui
I 100
o
O 80
O
60
40
DIASTOLIC BLOOD PRESSURE: DIRECT AND TAIL-CUFF MEASUREMENTS
METHOXAMINE
HEMORRHAGE
DIRECT
TAIL-CUFF
—I—
10
20
30
TEST NUMBER
Figure 5.
Diastollc blood pressure of monkeys
direct and tall-cuff measurements.
139
-------
3 too
HI
o
O
O 10
MEAN BLOOD PRESSURE : DIRECT (neosured with plonimeter)
INDIRECT (p=lP±i)
_____ D«KT
METHOXAMINE
HEMORRHAGE
TAR-COW
20 30
lESTNUMMt
Figure 6. Mean blood pressure of monkey: direct
(area under curve) and tail-cuff (2D + S)
3
The experimental findings are summarized in Table 1.
Under conditions of light pentabarbltal anesthesia or
massive hemorrhage, tall cuff pressure readings closely
reflect actual blood pressure of the animal. Tail cuff
pressure usually is the lesser of the two. If methoxamine
is administered, the differences and the standard devi-
ation both increase, with the tail cuff pressure being the
lesser of the two. Hemorrhage plus the hypertensive drug
results 1n the greatest discrepancy in tall cuff pressure
recordings.
TABLE 1. DIFFERENCES OF BLOOD PRESSURE OF MONKEYS OBSERVED
IN mmHg (DIRECT PRESSURE - INDIRECT PRESSURE)
UNDER SEVERAL PHYSIOLOGICAL CONDITIONS.
TREATMENT
Light Pentabarbital
Anesthesia
Anesthesia +
Hemorrhage
Anesthesia +
Methoxamine
Anesthesia +
Methoxamine +
Hemorrhage
NO- OF
MONKEYS
5
2
3
3
NO. or
MEASURES
44
14
33
12
WOOD PRESSURE DIFFERENCES OBSERVED M mmHg
(Direct-Indirect)
SYSTOLIC
Ave. S.D. r
6.1
«.3
16.9
17.7
6.6
6.7
11.6
16.0
0.94
0.98
0.87
0.77
DIASTOUC
Av.. S.D. r
-2.4
1.5
7.2
12.3
4.5
4.4
8.8
10.3
0.96
0.98
O.80
0.83
MEAN
Ave. S.D. r
4.8
2.8
14.6
17.3
4.0
5.0
8.1
14.0
0.97
0.98
0.87
0.79
S.D. = Standard Deviation of Difference in Readingi
r = Correlation Coefficient
A negative >ign indicate* lower readings tar the direct •ea»ureaent.
140
-------
The study demonstrates the reliability of monkey tail cuff
blood pressure recordings. It is concluded that under
conditions where blood vessels are under normal or intense
neural influence, tail cuff pressure measurements are
comparable with abdominal aortic pressure. The difference
is increased, however, when a vasoactive drug is administered
The tail cuff
on unanesthetized
(Figure 6).
measurements are quickly and easily done
chaired monkeys as shown in the photograph
Figure 6.
Equipment for tail cuff blood
pressure measurements of monkeys.
fa method prepared for the cadmium-
cardiovascular effect study in
primates.)
141
-------
CORRELATION OF EVOKED POTENTIAL AND SPINAL CORD
RESPONSES AS A METHOD FOR EVALUATING BIOLOGICAL
EFFECTS OF ENVIRONMENTAL POLLUTANTS
J. P. Lewkowski
Unlike the visual evoked potential, which may be used
as an indication of general brain function, the spinal cord
is unique in that different types of reflex arcs may be
studied in relative isolation from the influence of other
reflexes. These reflex arcs consist of several types of
cells that elicit facilitory or inhibitory effects by
releasing different transmitter agents. These trans-
mitter agents then either depolarize or hyperpolarize
specific motoneurons. For a given population of moto-
neurons, the net effect is to inhibit or to facilitate a
given reflex arc. For example, afferent Group I fiber
stimulation of one head of a muscle has been shown to
facilitate its synergists and inhibit its antagonists.
Facilitation is maximal when conditioning and test stimuli
are synchronous. Inhibitory effects are increased up to
a conditioning-test stimulus interval of 0.5/sec.
As a result, facilitation and inhibition of spinal
reflexes may be studied in isolation by stimulating and
recording from the appropriate nerves. One may then
determine the effects of toxicological agents by measuring
the change in amplitude or waveform of the control test
reflex. Furthermore, since many of the transmitters in
these reflex arcs are now known, then this technique may
be used to determine the effects of toxicological agents
on the release of a single particular transmitter. Because
the reflex arc 1s isolated, the effects will not be masked
by complimentary inhibitory and facilitory influences. Thus,
any changes may become evident a lower doses.
Furthermore, the visual evoked potential work and
spinal work can be easily correlated. If the visual evoked
potential is affected by a pollutant such as lead, then
the spinal work may indicate that the release of a particular
transmitter agent such as acetycholine is affected by this
pollutant. As a result, one may conclude that the cholinergic
component of the visual evoked potential may have been
affected. The various transmitters responsible for the complex
waveform of the evoked potential may thus be more easily
elucidated.
These methods present potentially extremely useful tools
for in vivo screening of central nervous system effects of
a wi<7e~ range of environmental pollutants.
142
-------
ESTIMATION OF RELATIVE TOXICITY:
A PROPOSED TREATMENT OF BIOEFFECTS DATA
Y. Y. Yang
In every dose-response situation, two components must
be considered: the stimulus (for example, a vitamin, a
drug, a metal test, or a physical force) and the subject
(for example, an animal, a plant, a human volunteer, or a
metal sheet). If the characteristic response is quanta! ,
occurrence or nonoccurrence will depend upon the intensity
of the stimulus. The resultant response is usually ex-
pressed in terms of the median lethal dose or the median
effective dose.
If two series of quanta! response data are compared,
their behavior of response will be expressed by the rela-
tive potency. The estimation of the relative potency is
possible only when the parallelism of two probit regression
lines is true. In many types of investigations, however,
the parallelism may not hold true and the estimation of
relative potency is of no practical use. Instead of esti-
mating relative potency, estimating relative toxicity is
proposed when such a difficulty arises, i.e., to examine
relative toxicity at different probit units. There are two
important applications of this method: (a) to provide back-
ground information to investigators for further studies, and
(b) to establish the optimal dose of the stimulus applied to
the subject.
In Figure 1, for example, the two probit regression
lines are parallel; hence, the relative potency can easily
be estimated. In Figure 2, however, the probit regression
lines are not parallel and the relative potency cannot be
estimated. In this case, the investigator has to investigate
what caused this phenomenon. If he is convinced that the
phenomenon is true bioeffect, then he can obtain the optimal
dose from the results.
The relative toxicity and its 95% confidence limits are
calculated at different probit points corresponding to their
expected response rates. Observe the relative toxicity (R)
and its confidence limits in Table 1. In cases where confi-
dence limits include unity, the young rats and infants had
the same degree of relative toxicity to the treatment. If
the relative toxicity is less (or more) than one, and its
confidence limits do not contain unity, then the young rats
responded to the treatment with less (or more) toxicity than
the infant. Based on these results, the investigator can
choose the optimal dose.
143
-------
From Table 1, one can choose a value of relative toxicity
with a satisfactory response rate; with this rate one can
establish the optimal dose from Figure 2.
z
95
4-
/
; -
E^$0
E=tJI
_^e
^P
. --;
-3
-------
Female Rats
/.
: . t -: --;•
Infant Rats
1.6 l;8 2,0
.9 1.0 1.2
-lo*-(hr«>
Figure 2.
Probit regression lines for mortality of
rats exposed to auto exhaust.
(Kampbell, 1972, unpublished results).
145
-------
TABLE 1. RELATIVE TOXICITY (R), COMPARING
YOUNG RATS WITH INFANT RATS, AND
ITS .95 CONFIDENCE LIMITS (R (L)
AND R (U) ) AT DIFFERENT EXPECTED
MORTALITY PERCENTILES
R(L) R R(U)
5
10
15
20
25
8.25
6.25
5.18
4.43
3.87
9.41
7.05
5.81
4.95
4.31
10.90
8.03
6.57
5.56
4.83
50 2.24 2.50 2.78
75 1.29 1.47 1.65
80 1.12 1.28 1.44
85 .95 1.09 1.24
90 .77 .90 1.03
95 .57 .67 .78
146
-------
THE APPROACH TO DATA ANALYSIS IN ETRL
R. Iltis
A. Analysis of Biological Responses
The Environmental Toxicology Research Laboratory
is analyzing the biological data obtained through its
experiments with mathematical modelling.
In this approach the biological systems are looked
upon as functional compartments, each one having an
input and output.
B. Objective
The fundamental objective is to relate mathematic-
ally the characteristic effect of a pollutnat (input)
to the biological effect (output).
In case of experiments that require a long period
of measurement, the objective is limited to establish-
ment of a trend.
C. Purpose and Method
The purpose of mathematical modelling is the pre-
diction of effects and the development of a suitable
experiment for meaningful and logical answer.
Methods used are linear programming, "ranking" of
data and by relating biological processes to analogus
electrical systems that are then simulated on an
analog computer.
As an example in the usage of the analog computer
is simulation of a dose-response curve. The method
used is that of R.G. Bickel*. Collected data is
plotted on a graph paper. A fourth order system is
used to describe the model. By adjusting respective
coefficient potentiometer on the computer, the shape
of the generated curve is changed until the error between
the data and the generated curve is minimized.
*USAF School of Aerospace Medicine, "Simulation" Nov. 69
147
-------
The settings of the coefficients represent the
rate constant of each biological compartment. This
technique has been used for simulation and curve
fitting of data of the Cd toxicity.
D. Additional Work
1. Additional work done at ETRL is the use of
"ranking" method to evaluate TAME data as a
function of pollutants due to fuel emission.
2. A study on applicability of mathematical modelling
to research program of the ETRL has been completed
in cooperation of Dr. Carl Evert of the University
of Cincinnati.
3. A computer program and method of analysis has
been developed to analyze neurophysiological
response pulses through FAST-FOURIER TRANSFORM
and pattern recognition method. Data analysis
and computer program was completed for studies
of renal functions in pigs and monkeys.
148
-------
PUBLICATIONS
Alpert, S.M., Schwartz, B.B., Lee, S.D. and Lewis, T.R.
Alveolar Protein Accumulation. Arch. Intern. Med. 128:
69-73, 1971.
Alpert, S.M. and Lewis, T.R. Ozone Tolerance Studies
Utilizing Uni-Lateral Lung Exposure. _J_. of Appl. Physio!
•51. 9/1 -3 9/1 C 1Q71
3. Asar, C.F., Gumpenz, E.P., Nicholson, F.S. and Moore, W.
A Practical Method for the Production Breeding of
Chinese Hamsters. Cricetulus g r i s e u s . Vol. 22, 1972.
4. Barkley, N., Busch, K.A., Crider, W.L. and Malanchuk, M.
The Concentration of Lead in Automobile Exhaust Exposure
Chambers. Amer. Indust. Hygiene Assoc. J.. Vol. 33,
No. 10: 678-683, Oct. 1972.
5. Bloch, W.N., Jr., Lewis, T.R., Busch, K.A., Orthoefer,
J.G. and Stara, J.F. Cardiovascular Status of Female
Beagles Chronically Exposed to Air Pollutants. Arch.
Environ. Health 24: 342-353, 1972
6. Bloch, W.N., Jr., and Lewis, T.R. A New Glue Holds
Rigid Cup ECG Skin Electrodes for 24 Hours and Longer.
J. of Appl . Physio! . 3_0: 893-394, 1971.
7. Campbell, K.I., Busey, W.M., Weaver, N.K., Taylor, J.A.
and Krumm, A.A. Biological Effects in Animals Chronically
Exposed to Lead Chlorobromide Atmospheres. JAVMA 159:
1523-1529, 1971 .
8. Campbell, K.I., Emik, L.O., Clarke, G.L. and Plata, R.
Inhalation Toxicity of Peroxyacetyl Nitrate. Depression
of Voluntary Activity in Mice. Arch. Environ.Health 23:
335-342, 1971.
9. Crider, W.L. Hydrogen-Air Flame Chemi1unescence of
Some Organic Halides. Anal . Chem. 41_(3), 539, 1969.
10. Dowel! , A.R., Lohrbauer, L.A., Hurst, D. and Lee, S.D.
Rabbit Alveolar Macrophage Damage Caused by Vivo Inhalation
Arch. Environ. Health 21: , 1970
11. Emik, L.O., Plata, R.L., Campbell, K.I. and Clarke, G.L.
Biological Effects of Urban Air Pollution. Arch. Environ.
Health 23: 335-342, 1971.
149
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12.
13.
14.
15.
16.
18.
19.
20.
21.
22.
Freeman, G., Stephens, R. L., Coffin, D. L. and
Stara, J. F. Light and Electron Microscopy of
Dogs' Lungs After Long-Term Exposure to Ozone.
In print.
Hall, L.L., Smith, F.A. and Hodge, H.C. Plasma
Fluoride Levels in Rabbits Acutely Poisoned with
Sodium Fluoride. Proc. Soc. Exp. Biol. and Med. 139:
1007-1009, 1972.
Hall, L
Report.
L. Letter to the
September, 1971.
Editor, Univ. of Rochester
Hemphill, F.E., Kaeberle, M.L. and
Suppression of Mouse Resistance to
Science 172: 1031-1032, 1971.
Buck, M.B.
Salmonella
Lead
Typhimurim
Hysell, O.K., DelGreco, F
The nonconditioned Macaca
Histopathologic
1970.
L., Friedman, H.M., Neves, A.J.
Mulatta: A Clinical Pathologic
and Necropsy Survey. USAMRL Report 846,
17. flysell, O.K., Neves, A.J. Hydroephorosis in the Goat
Due to Neoplasia: A Case Report. USARRL Report 850,
^ ******
1970,
Hysell, O.K., Delgreco, F.L., Janik, W.L., Morrissey,
R.L. A Case of Multiple Parasitism in a Sooty Mangabey
(Cercocebus Torquatys Atys.) USAMRL Report 853, 1970.
Kimmel, C.A., Moore, W., Jr.,
chlorophene Teratogenicity in
1972.
Stara,
Rats.
J.F. :
Lancet
Hexa-
II: 1251
Lee, S.D., Ramirez, J.R., Schwartz, B.B. and Dowell, A.R
Biochemical Composition of Human Pulmonary Washings.
Arch. Intern. Med. 127: 395-400, 1971.
Lee, S.D., Butler, K.C., Danner, R.M., McMillan, L.,
Moore, W. and Stara, J.F. Radiorespirometry in the
Study of Biological Effects of Environmental Pollutants.
American Laboratory, December 1972, p 8-14.
Lee, S.D., Menzel, D.R. and Rohen, J.N. Vitamin E:
The Biological and Environmental Antioxidant. Agri-
cultural and Food Chemistry. 20: 481-486, 1972.
150
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23. Lewis, T.R., Moorman, W.J., Ludman, W.F. and Campbell,
K.I. Toxicity of Long-Term Exposure to Oxides of
Sulfur. Arch. Environ. Health 26 (1): 16-21 (Jan) 1973.
24. Lewis, T.R., Amdur, A.O., Fritzhand, M.D. and Campbell,
K.I. Toxicology of Atmospheric Sulfur Dioxide Decay
Products. Publication No. AP-111 , EPA (July) 1972.
25. Malanchuk, M. Thermal Analysis of Sodium Metabisulfite.
Anal. Chimica Acta. 5486, 1970.
26. Moore, W. Assessment of Absorption, Metalbolic Fate,
Excretion and Acute and Subacute Toxicity of Trace
Metals, (Cadmium, Chromium, Manganese, Nickel and
Vanadium). Submitted for clearance.
27. Moore, W., Stara, J.F., Crocker, W,C., Malanchuk, M.,
Miller, R.G. and Yang, Y.Y. Comparison of "HSmcadmium
Retention in Rats Following Different Routes of Admin-
istration. Envi ron . Research, 1973.
28. Moore, W., Stara, J.F. and Crocker, W.C. Gastrointestinal
Absorption of Different Compounds of l'5niCadmium and the
Effect of Different Concnetrations in the Rat. Environ.
Research, 1973.
29. Moorman, W.J., Orthoefer, J.G. and Lewis, T.R. A Rapid
Method for the Measurement of Blood Pressure and the
Collection of Arterial Blood Samples in the Awake Beagle.
Submitted for clearance.
30. Slater, R.W., Crider, W.L. and Barkley, N.P. Flame
Luminescence Detection in Gas Chromatography with
Halide Backgrounds. In print.
31. Skoryna, S.C., Hong, K.C., Tanaka, Y. and Stara, J.F.
Inhibition of Radiostrontium Absorption by Chemically
and Enzymatically Degraded Products of Alginates.
Proceedings of 2nd Int. Conf. on Strontium Metabolism,
Glasgow, Scotland, August, 1972, p 39-51.
32. Stara, J.F., Wolfangel, R.G., Bruckner, B.H. and Moore, W.
Gastrointestinal Absorption, Distribution and Excretion
of Radionucl ides. Permagon Press - Oxford and New York,
1970.
33. Stara, J.F., Nelson, N.S. and Hoar, R.M. Concentration,
Distribution and Effects of Radioiodine in Embryonic
Guinea Pigs During Early Organogenesis. In Radiation
Biology of_ the Fetal and Juvenile Mammal , in Proceedings
of the 9th Annual Hanford Biology Symposium at Richland,
Washington, May, 1970.
151
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34.
35.
36.
37.
38.
39
40,
Stara, J,F., Nelson, N.S., Krieger, H.L. and Kahn, B.
Gastrointestinal Absorption and Tissue Retention of
Radioruthenium. Intestinal Absorption of Metal Ions,
Trace Elements and Radionuclides. Permagon Press, Oxford
and New York, 1970.
Stara, J.F. and Nelson, N.S
of Radionuclides in Mammals
20: 113-137, 1970.
Comparative Metabolism
A Review. Health Physics
Stara, J.F. Effects of Environmental Pollution on
Domestic Animals. Proceedings of 7th Annual Meeting
U.S. Health Assoc., Oklahoma City, Oct. 1971, pp
325-328.
Stara, J.F. Methods and Techniques for Acute and Sub-
acute Inhalation Studies. Task Force Report, Appendix
B, NCTR Research and Support Program Document, June, 1972
Trams, E.G., tauter, C.J., Brown, E. and Young, 0.
Cerobral Cortical Metabolism After Chronic Exposure
Ozone. Arch. Environ. Health 24: 153-159, 1972.
to
Wiester, M.J., Bonventre, P.F. and Grupp, G. Estimate
of Myocardial Damage Induced by Diphtheria Toxin. J_. of
Lab. and Clinical Medicine, Vol. 81, No. 2, February 1973
Wolfangel, R.G. and Stara, J.F.
of the In Situ Distribution of 13
in Cats Using the Scintillation Camera
Med. 10: 697-701, 1970.
Sequential
'I-Labeled
Determination
Tetracycline
of Nuclear
PRESENTATIONS AND INTERNAL REPORTS
1. Campbell, K.I., Busey, W.M., Weaver, N.K., Taylor, J.A.
and Krumm, A.A. Biological Effects in Animals Chronically
Exposed to Lead Chlorobromide Atmospheres. JAVMA 159:
1523-1529, 1971.
2. Campbell, K.I. Automobile and Diesel Engine Exhaust
Facility at NERC, Cincinnati, Presented at Symposium
Interact, University of Cincinnati, December 1972.
3.
4.
Campbell, K.I. Effects of Air
presented at NADL, USDA, Ames,
"Air Pollution Toxicology -- A
Pollutants.
Iowa, March
Summary."
Seminar
1972. Handout
Engel, R.E. and Stara, J.F.
in Air Pollution Research.
Meeting of AVMA, June 23-26,
The Role of the Veterinarian
Presented at the 107th Annual
1970, Las Vegas, Nevada.
152
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5. Gage, M., Yang, Y.Y., Coehn, A.L. and Stara, J.F.
Alterations of Wheel Running Behavior of Mice by
Automotive Fuel Emissions. Paper presented at
American Psychological Association Meeting, September
1-8, 1972, Honolulu, Hawaii.
6. Hammer, D.I., Sandifer, S.H., Keil, J.E., Prieste, I.E.,
and Stara, J.F. Cadmium Exposure and Human Health
Effects. Presented at the 5th Annual Conf. on Trace
Substances in Environ. Health, Columbia, Mo., June, 1971
7. Hysell, O.K. Care and Management of Poisonous Reptiles.
Presented at the S.E. Ohio AALAS Meeting, April, 1972.
8. Hysell, O.K. Management of Animal Colony for Toxi-
cological Research. Presented at Symposium Interact,
University of Cincinnati, December 1972.
9. Iltis, R., Burkart, J.K. and Morris, C.H. Constant
Ratio Carbon Monoxide Controller for Fuel Emission
Toxicity Studies. In-house Report.
10. Lee, S.D., Danner, R.M., Butler, K., Miller, R.6.,
Yang, Y.Y. and Stara, J.F. Effects of Pollutants on
Respiratory Carbon-14 Output. Presented at 65th
Annual Meeting of Air Pollution Control Association,
June 18-23, 1972.
11 Lee, S.D. Early Biochemical Effects of Ct^HgCl in
Rats. Paper presented at the ICES Invitational
Symposium, Chapel Hill, North Carolina, September
26-27, 1972.
12. Lewkowski, James P. Long Duration Heterosegmental
Spinal Inhibition. Paper presented at Membrane Physio-
logy Conference at Cumberland, Kentucky on June 19-22,
1972.
13. Miller, R.G., George, E. L., Barkley, N.P. and Richards,
M.K. Determination of Cadmium, Zinc and Manganese in
Biological Samples. In-house report.
14. Miller, R.G., Lee, S.D. and Danner, D.M. The Effect
of Length of Fasting on 14C02 Excretion Following
Injection of U-14C-G1ucose. Presented at the 156th
National Meeting of American Chemical Society, Atlantic
City, New Jersey, Sept. 1968.
153
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15. Moore, W. and Stara, J. F. Review of Toxicologic
Methods for Environmental Pollution. Presented at
Annual Meeting of Veterinary Colleges, Atlanta, Georgia,
May, 1972.
16. Moore, W., Jr. and Ajayi, M.M. A Modified Method for
Growth of Rinderpest Tissue Culture Vaccine. Presented
at Nigeria Veterinary Council, January, 1970.
17. Stara, F.J. and Engel, R.E. Radiologic Contamination
of the Biosphere: Its Implication to Environmental
Health. Presented at the 107th Annual AVMA Meeting,
June, 1970, Las Vegas, Nevada.
18. Stara, J.F. Biological Effects of Automobile Exhaust
and Other Pollutants in Beagles. Presented at the
AALAS Annual Meeting, May, 1971, Houston, Texas.
19. Stara, J.F. Biological Effects of Air Pollution with
Particular Emphasis on Trace Metals. Presented at the
Air Pollution Symposium, Lakeland College, Lakeland,
Ohio, April, 1972.
20. Stara, J.F. Toxicology of Fuel and Fuel Additives.
Presented at Symposium Interact, University of Cincinnati,
Cincinnati, Ohio, December 1972.
21. Stara, J.F., Kimmel, C., Moore, W. and Hammer, D.
Toxicology of Manganese with Particular Reference to
Manganese Fuel Additives: A Review. Submitted for
clearance (internal report).
22. Wiester, M.J., Bonventre, P.F. and Grupp, G. An
Estimate of Heart Damage Induced By Diphtheria Toxin
vs. Studies in an Isolated Heart Preparation. Paper
presented at Ohio Valley Section Society for Experi-
mental Biology and Medicine, Ohio State College of
Medicine, Columbus, Ohio, November 19, 1971.
23. Wiester, M.J. and Iltis, R. Systolic and Diastolic
Blood Pressure Measurement in Monkeys: Non-Invasive.
Paper. Presented at the Association for the
Advancement of Medical Instrumentation, Washington, D.C.,
March 21, 1973.
154
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ETRL
Administrative Staff
Jerry F. Stara
Wellington Moore
Delno Balser
Di rector
Deputy Director
Administrative Officer
Clerical
Lucille Light
Deborah K. Dean
Carol Haynes
Secretary
Clerk-Steno
Clerk-Steno
Geraldine Vaughn
Natalie Wai trip
Clerk-Typi st
Clerk-Typist
Scientific and Technical Staff
Joan Adams
Helen Ball
Naomi Barkley
Kenneth Bridbord
Joseph Burkart
Kathleen Butler
Kirby Campbell
David Cmehil
Arnold Cohen
Gilbert Contner
Waldon Crocker
Robert Danner
Michael Gage
Lucille Garner
Thomas Garner
Emma Lou George
Larry Hall
Robert Hinners
James Morton
David Hysell
Rumult Iltis
Rose Kremer
Si Duk Lee
James Lewis
James Lewkowski
Deborah Long
Reba Lucas
Myron Malanchuk
Lofton McMillan
Robert Miller
Susan Neiheisel
Charles Morris
Gerald Radigan
Marta Richards
John Rounds
Chemi st
Biologist
Biologist
Program Element Manager
Res. Mech. Engineer
Biological Aid
Veterinarian - Toxicologist
Biological Lab. Tech.
Physical Science Tech.
Research Chemist
Biologist
Chemi st
Research Psychologist
Medical Technologist
Engineering Tech.
Medical Technologist
Pharmacologist
Research Mechanical Engineer
Electronic Tech.
Veterinarian - Pathologist
Electronic Engineer
Biological Aid
Research Chemist
Biological Aid
Physiologist
Biological Aid
Statistical Clerk
Chemical Engineer
Biologist
Chemist
Biological Aid
Electronic Tech
Facilities Mech
Chemi st
Biological Aid
- Automotive
155
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Alvin Schwarberg Mechanical Engineer
Robert Slater Research Chemist
Ruth Stevens Biol. Lab. Tech.
Elizabeth Thall Biological Aid
Isaac Washington Engineering Tech.
Thomas Wessendarp Biologist
Jean Wiester Res. Physiologist
Julius Williams Biological Aid
You-Yen Yang Math. Statistician
Consultants
John W. Clayton, Ph.D., Director,
Center for Environmental Toxicology,
University of Wisconsin, Madison, Wisconsin
Carole A. Kimmel, Ph.D., Instructor,
Harvard Medical School, Boston, Massachusetts
Herbert Stokinger, Ph.D., Chief,
Toxicology Branch, NIOSH and Chairman,
Committee on Toxicology, National Science
Foundation, Cincinnati, Ohio
Raymond R. Suskind, M.D., Chairman,
Department of Environmental Health
Director, Kettering Laboratories,
University of Cincinnati, College of Medicine
Cinci nnati, Ohio
Theodore R. Torkelson, Ph.D., Toxicology Specialist,
The Dow Chemical Company, Midland, Michigan
Neil! K. Weaver, M.D., Associate Medical Director,
Humble Oil & Refining Company, Houston, Texas
156
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ACKNOWLEDGMENT
We extend our thanks to Lucille Light, Carol Haynes,
Geraldine Vaughn, Deborah Dean, and Natalie Waltrip for
their tireless assistance in typing and organizing this
report; Robert Danner, Emma Lou George, Kathleen Butler,
James Horton, and Arnold Cohen for expertly illustrating
it; Delno Balser and Rumult Iltis for helping with many
details related to it, especially the reproducing and
binding; and James Lewkowski for proofreading and critical
evaluati on.
We want to give special thanks to our associates in
animal care, auto repair, electronics, and data analysis
for their excellent effort in giving the needed support
to the research program of the laboratory.
Finally, we wish to acknowledge gratefully the leader-
ship and support given to the Environmental Toxicology
Research Laboratory by Dr. Andrew Breidenbach, Director,
Frank Middleton, Deputy Director of the National Environ-
mental Research Center - Cincinnati; by Dr. Herbert Wiser,
Director, and Dr. Kenneth Bridbord, Program Element Manager,
of the Division of Processes and Effects, U.S. Environ-
mental Protection Agency, headquarters; and by Louis Lefke,
Office of Program Coordination, especially the helpful
efforts of George Shultz.
The Authors
tfU.S.Government Printing Office: 1973 — 757-574/5303 Region 5-11
157
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