ENVIRONMENTAL TOXICOLOGY RESEARCH LABORATORY
NERC - CINCINNATI
INTERIM REPORT
1 Studies on toxicology of catalytic trace metal components
2 Toxicology of automotive emissions with and without
catalytic converters
MARCH 1974
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ENVIRONMENTAL TOXICOLOGY RESEARCH LABORATORY
NERC - CINCINNATI
INTERIM REPORT
1. Studies on toxicology of catalytic trace metal components
2. Toxicology of automotive emissions with and without
catalytic converters
March, 1974
J. F. Stara
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TABLE OF CONTENTS
Identification Number Page
Summary 1-3
A. Toxicological Studies of Catalyst
Metal Components 4-61
A.I. "Metabolic and Kinetic Aspects of Palladium
and Platinum" 4-23
A. 2. "The Acute Toxicity of Palladium Chloride" . . 24-33
A. 3. "Comparative Toxicity of Noble Metal Compounds
on Lactic Acid Dehydrogenase and Glutamic
Qxalaocetate Transaminase in vitro" . . 34-37
A. 4. "Effect of Noble Metal Compounds on Protein
Synthesis in Various Organs of Rats" . . . . 38-41
A. 5. "Dermal Irritancy of Several Pd, Pt/ and Pb
Compounds and of MM?" » . . 42-48
A. 6. "Dermal Absorption of l91Platmm +4 m HC1
Solution" 49-50
A. 7. "Ocular Irritation of Two Palladium Compounds
in Rabbits" 51
A. 8. "Neurophysiology Summary of Pt and Pd" 52-53
A. 9. "A Preliminary Report on the Cardiovascular
Actions of Palladium" 54-61
B. Automobile Exhaust Emission Studies With and Without
Catalytic Converters 62-140
B.I. "Auto Exhaust Facility Modification" . ... 62-70
B.2. "Catalyst Exhaust Exposure Studies" 71-75
B.3. "Design and System Performance for Studies of
Catalytic Emissions" 76-83
B.4. "Exhaust Emissions During Steady Speed Runs
With the Catalytic Converter in the Exhaust
System" 84-90
B.5. "Exhaust Emissions from Catalyst-Equipped
Engines" 91-98
B.6. "Sulfate Emissions from Use of High-Sulfur
Fuel, TAME-K" 99-103
B.7. "Comparison of the Biological Effects of Acute
Exposure to Whole Exhaust Emissions from an
Automobile Engine Equipped with and without
A Noble Metal Catalytic Converter" 104-116
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Identification Number Page
B.8. "Effect of Exhaust Emission from Catalytic
Converter on Aryl Hydrocarbon Hydroxylase". . . 117-123
B.9. "Biochemical Effects of Emissions from an
Automobile Engine with and without Catalytic
Converter" 124-129
B.10. "Exposure of Maternal, Pregnant, and Newborn
Rats to Exhaust from Modern Automobile Engine
with Catalytic Converter and Other Emission
Controls (TAME J)". . 130-134
B.ll. "Gross Morphologic and Functional Damage to
Plant Species by Diluted Exhaust of Automobile
Engines Operated with and without A Catalytic
Emission Control Device" 135-140
11
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Summary
INHALATION TOXICOLOGY OF AUTOMOBILE EXHAUST EMISSIONS AND THEIR
TRACE METAL COMPONENTS ASSOCIATED WITH USE OF CATALYTIC CONVERTERS
J. F. Stara
Introduction
The Division of Health Effects, EPA, instructed the ETRL m July
1973, to reprogram ROAP 21 AFK and develop a new program for toxicologic
assessment of emission products from oxidizing catalysts.
Members of ETRL staff had compiled early in FY 73 a monograph
entitled, "Toxicology of Atmospheric Sulfur Dioxide Decay Products."
In July 73, immediate emphasis was placed on the metabolism, kinetics,
and biological effects of noble metals, palladium and platinum,
because of the extremely limited data found in the literature on this
subject.
The definitive toxicological investigations conducted m this
laboratory utilize animal exposures, placing emphasis on those routes
of exposure that are of major environmental significance. Other
routes, such as intravenous, are used to obtain comparative toxicological
data. For this purpose, ETRL developed a toxicologic matrix which was
geared to investigate the biological effects resulting from inhalation
of the individual catalytic components and the whole exhaust emissions
after passage through the catalytic converter. The protocol used for
the toxicologic investigations is outlined in Figure 1. Whether tms
matrix is followed in part or in full depends on the theoretical
prediction of toxicity and the amount of knowledge presently avail-
able on the pollutant m question.
1 U>50 LC50 ETC
f PHYSIOLOGICAL
SCREEN
PULMONARY FUNCTION
NEUROPHYSIOLOGIC
RESPONSE ETC
» PATHOLOGY
GROSS » LIGHT
4 SENSITIVITY
DERMAL OCULAR ETC
I BIOCHEMICAL SCREEN
GENERATION OF
EXPOSURE
» AUTOMOTIVE
t SINGLE
POLLUTANT
~
EXPERIMENTAL »NIMAL
EXPOSURE
INHALATION
INGESTION ETC
1
—
CHARACTERIZATION
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EMiSjlOM AEROSOL
CHURACTEP STICS ETC
!
UBACUTE EXPOSURE
1 PHYSIOLOGY
PULMONARY
1 CHRONIC EXPOSURE 1
SELECTIVE TESTS FROM
WHICH EVER OF THE
BEHAVIORAL NEUflO FOLLOWING IS OEEMEO
. PHYSIOLOGICAL MOST PRODUCTIVE
REPRODUCTIVE ,
t BIOCHEMISTRY
6H7YMES
METABOLITES
3 PATHOLOGY
J
3
4
LIGHT AND EM j
4 METABOLISM
BIOCHEMICAL
PHYSIOLOGICAL
PATHOLOGICAL
ELEMENTAL
ANALYSIS
OTHER AS
APPROPRIATE
KINETICS BODY S"
BURDEN ETC
1
TEST REPORTS
(OPEN LITERATURE
INTERNAL)
/
/
^
S
TMC APPROACH FOR ANY GIVEN POLLUTANT(S) WILL VARY SOMEWHAT FROM
THIS GENERAL SCHEME DEPENDING ON WHAT IS CUPnruHV KNOWN ABOUT
THE POLLUTANT AND THE TYPE OF INFORMATION NlfDEO TO 'l» THE CAPS
Figure 1
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Toxicological Studies of Catalyst Metal Components
These studies were undertaken to ascertain the relative toxicity
of catalyst metal components such as platinum and palladium. The
acute and subacute studies are required in any comprehensive evaluation
of the noble metals since data of this type is largely unavilable in
the literature.
Intratrachael administration of platinum or palladium resulted in
greater body retention than oral dosage (A.I). Furthermore/ the
differences among the intravenous, intraperitoneal, and oral toxic
dosages of palladium indicated a difference m distribution of palladium
depending on the route of administration (A 2). Biochemical studies
have shown that the relative toxicity of platinum or palladium on
glutamic oxalaoacetate transammase and lactate dehydrogenase was
dependent upon the chemical form of the metal (A.3) In addition,
palladium elicited an increase in ^ C-leucine incorporated into heart
and blood serum Platinum produced a dose-response change in 14C-
leucine incorporation in the lung (A. 4). Dermal irritancy, dermal
absorption, and ocular irritation tests are being utilized as screen-
ing techniques to determine the relative toxicity of various types of
platinum and palladium compounds (A 5, A.6, A.7). Preliminary
neurophysiological screening tests have shown that palladium elicited
short-term changes at higher dosages; these changes may represent
indirect effects produced by changes in other tissues such as the
cardiovascular system (A. 8). Preliminary cardiovascular studies
have indicated that palladium acted as a non-specific cardiac
irritant as well as a peripheral vasoconstrictor (A. 9).
In summary, the preliminary results have indicated that platinum
and palladium salts are toxic in high concentrations. However, the
levels tested in studies reported here will probably not be found in
the ambient atmosphere. Further work is underway to determine the
atmospheric concentrations of these noble metals that may pose an
environmental hazard.
Auto Exhaust Fmission Studies
EPA has available at ETRL an auto exhaust generating system for the
production of irradiated and nonirradiated gasoline engine exhaust-air
mixtures. This system has been utilized in a series of acute and sub-
acute studies which determined the biological effects of exposure of
various experimental animal species to whole automotive exhaust emissions
with fuel additives and/or with or without a catalytic converter (B.I).
Furthermore, the system was utilized to determine whether new
pollutants may be emitted through the use of these converters. These
new emissions may occur under three conditions- (1) conversion of
organic sulfur compounds present in gasoline into sulfuric acid mist
and sulfates, (2) platinum and palladium emission due to catalyst
degradation, and (3) alteration of the emissions with the production
of different quantities and/or new species (B.2). A report on the
specific design and system performance is included (B.3).
Emission studies have indicated that the oxidation-type catalyst
in the exhaust system has resulted in the following changes in exhaust
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emissions. (1) large reduction of carbon monoxide, total hydrocarbons,
and various organic compounds such as acetylene, (2) a nearly total
elimination of aldehydes, (3) less photochemical reactions of hydro-
carbons, (4) the presence of sulfuric acid as a major component in
the particulate (B.4, B 5, B.6), and (5) trace emission of Pt and Pd
particulates most of which are in the respirable range.
The bio-effect studies were undertaken to determine the general
toxicologic effects of an acute exposure to exhaust from engines equipped
with catalytic converters (catalytic exhaust) vs exposure to exhaust from
engines without catalytic converters (non-catalytic exhaust) The results
indicate that aruirals exposed to non-catalytic exhaust exhibited profound
changes in weight of lactating female and infant rats, and in the survival
rate of infant rats. There were no apparent effects on the rats exposed
to catalytic exhaust with the possible exception of minute weight loss in
lactating female rats (B.7).
The clinical data indicates that the only statistical exposure effect
in the catalytic exhaust was an increase in total serum proteins. In the
non-catalytic exhaust exposure, there were significant effects on total
protein, platelet count, red blood cell and white blood cell count, white
cell differential, alkaline phosphatase, hemoglobin, hematocrit, partial
thrombo plastin tissue, serum glutamic oxalaoacetate transaminase, and
serum glutamic pyruvate transaminase levels Lung pathology studies
showed a greater incidence of pathological conditions after non-catalytic
exposure than after catalytic exposure (B.7). In general, these data
indicate a far greater potential hazard to several organ systems with
prolonged exposure to non-catalyst exhaust.
The effect of catalytic and non-catalytic exhaust was tested on
several biochemical systems. Lung aryl hydrocarbon hydroxylase (an
enzyme which is responsible for the biotransformation of various
carcinogens) activity is depressed with exposure to non-catalytic exhaust
However, the catalytic converter significantly reduced the lung aryl hydro-
carbon hydroxylase depression (B.8). Furthermore, serum lactate dehydro-
genase was greatly elevated with non-catalyst exposure but was not
significantly effected with exposure to catalyst exhaust (B.9).
Plant damage occurred in both catalytic and non-catalytic exhaust;
it appeared to be somewhat less m the catalytic exposure. Since plants
were damaged m both types of exposure, it appears that plants are
inherently more susceptible to exhaust damage than animals and that the
effect threshold was exceeded by emissions (B.ll).
In summary, the acute whole emission studies have demonstrated that
acute exposures to automotive exhaust without a catalytic converter
elicited a profound effects on physiological and biochemical function
and produced histo-pathological lesions. In contrast, exposure to exhaust
from a system with a catalytic converter did not result in such demonstrable
physiological or biochemical dysfunction. One may conclude that the intro-
duction of the catalytic converter has reduced the levels of certain
exhaust constituents. This has resulted in a decreased biological effect of
exhaust emissions. However, it should be noted that catalyst-modified
emissions produced changes in plants In addition, it is now known that
catalyst-modified emissions are highly acidic. As a result, longer term
studies are needed and are planned for the future (April, May) These
studies will test the effect of long-term exposure to the more acidic
catalyst exhaust.
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PALLADIUM AND PLATINUM STUDIES
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A.I. METABOLIC AND KINETIC ASPECTS OF PALLADIUM AND PIATINUM
W. Moore, D. Hysell, W Crocker and J. Stara
Automotive manufacturers have indicated that palladium will be used in
exjunction with platinum in automotive catalytic converters. These converters
are designed to reduce the concentrations of carbon monoxide (CO) and hydrocarbons
(HC) m the exhaust stream by oxidizing them into carbon dioxide and water The
control of the concentrations of CO and HC in automotive emissions is necessary
for light duty vehicles to comply with the CO and HC emission standards set
forth in the Clean Air Act - 1970. With the use of palladium and platinum
in automotive catalytic converters, there is the possibility that some of the
material will be emitted to the atmosphere or enter into other segments of
the environment following degradation or disposal of worn-out converters.
1. Palladium
Information on the biological effects of palladium is extremely limited.
Meek et al. (1943) have summarized the toxicological findings and presented
additional data on the toxicology following subcutaneous and intravenous
injections in rabbits. Intravenous infections of PdC^ were extremely toxic,
whereas subcutaneous injections were nontoxic due to the formation of an in-
absorbable complex. When given in small amounts intravenously, the median
lethal dose in rabbits was 18.6 rcg/kg, with a survival period of 12 days
(Orestano, 1933).
The effects of palladium, 5 ppm in drinking water, on the growth and
life span of mice were observed from weaning until natural death (Schroeder
et^ al. 1971). No significant differences were found in the body weights of the
females, however, body weights of the exposed male mice were significantly less
than those of the controls The life span of the exposed male mice was signifi-
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cantly longer than that of the control male iru.ce. The incidence of malignancy
and amyloidosis was higher in the exposed animals
Information regarding human exposure to palladium is also extremely sparse.
In older medicine, palladium has been given orally for the treatment of tubercu-
losis without success, and it has been injected into abdominal fat for the
purpose of reducing obesity (Meek et al. 1943). Exposure of forearm skin to a
1 mg/ml buffered PdCL, solution for 24 hr. did not result in skin irritation
(Meek et al. 1943). Men working with platinum salts occasionally developed
an asthmatic response to platinum exposure These same men had no recurrence
when transferred to jobs involving the handling of palladium (Hunter et al. 1945)
Palladium has been shown to be highly active in some enzyme systems, and
it is possible that traces of palladium may influence, but not necessarily
unfavorably, enzyme systems in plants and animals including man (Christensen,
1971-72).
It is apparent from very limited information that palladium is toxic
when it is absorbed and that more information is sorely needed in view of the
possibility of environmental exposure due to catalytic converters The
purpose of this study was to provide information on the retention, tissue
distribution, excretion and placental transfer of Pd following different routes
of administration.
Animals and Treatments
The outbred albino rats (Charles River CD-I strain) used in this study
were maintained on a commercial diet (Purina Lab Chow) and tap water ad libitum
except where otherwise noted The various treatment groups consisted of
1. Intratracheal Administration
Ten fasted male rats, 180-200 g, were anesthetized with pentobarbital
sodium and placed in dorsal recumbency. The trachea was isolated through a
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ventral midline cervical incision and blunt dissection of the overlying
roasculature. 103PdCl2 (25 yCi m 0.1 ml saline) was injected intratracheally
vath a 1 cc tuberculin syringe and 5/8 in., 25 ga needle. Following closure
of the incision, the animals were maintained in hanging wire cages for 104
days to determine whole body retention of the 103PdCl2/
2. Oral Administration
twenty fasted male rats, 180-200 g, were lightly anesthetized with
ether and given 25 yCi of -^SpdClj m 0.2 ml saline by stomach tube. Ten
rats were placed m metabolism cages for collection of 24 hr urine and
fecal samples to determine routes of excretion They were maintained 104
days for determination of whole body retention of the PdCl2. The other ten
rats were sacrificed 24 hrs. following dosage to establish organ distribution
of the PdCl2. Fifteen non-fasted suckling rats, 30 g, \iere given a sirgle
dose of l^pcic]^ (25 yCi in 0.2 ml saline) by stomach tube These animals
were maintained for comparison with retention of Pd in the adult rats.
3. Intravenous Administration
Twenty male rats, 180-200 g were given 25 yCi PdCl2 in 0.1 ml saline
intravenously (iv) in a tail vein with a 1 cc tuberculin syringe and 5/8 m.,
25 g needle. Ten rats were sacrificed 24 hrs. later for organ distribution,
ten rats were placed in metabolism cages for collection of 24 hr samples of
urine and feces and for whole body counting to determine retention. Thirteen
female rats (16 days pregnant) were given 25 yCi -^PdC^ iv and maintained
in metabolism cages for collection of feces and urine. They were sacrificed
24 hrs after dosage for determination of organ distribution and placental
transfer of the •'-^PdC^. An additional group of 8 female rats were given
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25 pCi PdCl2 iv within 24 hrs post-parturition. The mothers and litters
were maintained 25 days to determine if the ^^Pd was transferred to the
young via the mother's milk.
Sacrifice and Tissue Sampling
Ml rats were euthanatized with an overdose of chloroform anesthesia.
Samples collected routinely were blood, heart, lung, liver, kidney, adrenal,
pancreas, abdominal fat, spleen, skeletal muscle, bone, brain and testicle
from males, ovary from females. In the pregnant females, 4 placentas, 4
fetuses and a pooled sample of fetal livers were also saved. In the young
rats from the milk transfer study, lung, liver, kidney, bone and spleen were
saved. Tissue samples were placed in pre-weighed glass vials for counting.
Radioactive Determinations
103p
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8
significantly affected whole body retention. The percent of 103Pd retained
with time in the rat following three different routes of administration is
presented in Figure 1. Following oral dosing, the retention curve declined
very rapidly during the first 3 days to about 0.4% of the initial dose. The
initial rapid clearance is attributed to passage of the non-absorbed T>d
through the gastrointestinal tract. Extrapolation of the second component
of the retention curve to the intercept indicated that absorption was less than
0.5% of the initial dose. Retention of •L03Pd by the suckling rats following
oral administration was similar to the adults, however, the amount absorbed
and retained with time was significantly higher.
The amount of l"3pd retained following intratracheal dosing was
significantly higher than for oral dosing and also significantly less than
for iv dosing. The greatest amount of Pd retained with tune occurred
following iv administration.
Excretion
Radioactive counts of 24 hr urine and feces samples from the rats
receiving the 103Pd orally showed that almost all of the Pd was eliminated
in the feces and only a trace amount was excreted in the urine (Figure 2).
With iv administration, Pd was eliminated both in the urine and feces in
similar quantities. Toward the end of the study, urinary excretion exceeded
fecal excretion.
Tissue Distribution
The distribution and concentration of ^^pd was determined for
different tissues following oral and iv dosing Twenty~four hours after
oral dosing, detectable quantities of 103p
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Suckling Rat
I Oral
'• Percent of Initial
l' \ Retained
103
Pd
Intratracheally
8 12 16 20 24 28 32
Days After Dosing
Fig. 1. Whole body retention of Pd in adult rats
following oral, iv, and mtratracheal admin-
istration Also shown is whole body retention
of 103pd in suckling rats following oral
administration.
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Excretion of 103Pd following iv and oral
administration.
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11
liver, the concentration in the kidney was much greater than in the
liver. Twenty-four hours after iv dosing, -^Sp^ was found in all the tissues
analyzed with the highest concentrations occurring m the kidney, spleen,
liver, adrenal, lung and bone, respectively.
The rats used in the whole body retention study were sacrificed
104 days post-exposure and the tissues counted. No significant amounts of
103p3 Were found in any of the tissues from the group receiving the oral
dose. In the iv dosed rats, the highest concentrations of 'T'd were found
in the spleen, kidney, liver, lung and bone. For the mtratracheally-dosed
rats, the lung contained the most Pd followed by kidney, spleen, bone and
liver.
Maternal/Fetal Uptake
Thirteen pregnant rats (16th day gestation) were given 25 yCi -^^pd iv
and sacrificed 24 hrs later. During the 24 hr period, the pregnant rats
excreted 44.2% of the initial iv dose. The amount excreted by the pregnant rats
was higher than the amount excreted by the fasted adult male rats during
the first 24 hr. period. The magnitude of the difference in ^^pd concentration
among the maternal organs and also between maternal tissues and the fetuses is
best shown by the counts per g tissue and these values are given in Table 1.
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12
Table 1. Pd in Maternal Organs and Fetuses
TISSUE
Kidney
Liver
Ovary
Lung
Bone
Blood
Placenta
Fetal Liver
Fetus
MEAN COUNTS/g
588,479
319,153
29,625
29,211
18,351
3,654
58,321
1,429
757
The pattern of distribution and concentration of p(j in maternal organs
was similar to that previously found in the whole body iv experiment. Most of
the fetuses (35) contained a small amount of 103pd, and the mean value for these
fetuses is given in Table 1. However, radioactive counts for 17 fetuses from
5 litters was not significantly higher than background. The same pattern of
results was obtained for the fetal livers. The amount of 103Pd found in the
fetuses indicated that Pd does not readily move across the placental barrier
in the rat.
Within 24 hrs. following the birth of their young, a group of female
rats were given 25 pCi ^^Pd and the litters counted to see if ^^Pd was
transferred to the young via the milk The retention of -^pcl by the dams
and litters with time following a single iv exposure is shown in Figure 3.
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13
-C3
o
oa
O)
o
CO
o
10
10
10
o
Adult Female Rats
Suckling Offspring
8 12 16 20 24
Days After I V Dosing
28
Fig. 3. Whole body retention of Pd in nursing feirale
rats folia /ing i v. administration and uptake of
1n^~'1 in suckling young via the nulk.
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14
It is evident that a small amount of the 103Pd was passed to the young via
the nalk Twenty-five days after dosing of the dams, the suckling rats were
sacrificed and lung, liver, kidney, bone and spleen taken for analysis A
very small amount of 103Pd (10-50 counts/gram tissue) was found m the
tissues. The bone had the highest level of activity followed by the kidney,
spleen, lung and liver.
2. Platinum
In discussing the toxicology of platinum, it should be emphasized that
much of the information available in the literature deals with the effects of
complex platinum salts The effects of the complex Pt salts appear to differ
somewhat from the effects of platinum alone.
Sodium chloroplatinate, a complex platinum salt frequently encountered
in industry, has been tested in both rats and guinea pigs (Hofmeister, 1945,
Saundelle, 1969). Immediately following the intravenous injection of 20 mgAg
sodium chloroplatinate into a guinea pig, there occurred an intense attack
of asthma resulting m death within 3 minutes. Autopsy revealed the lungs
to be pale and inflated as in anaphylactic shock. The injection of 1-2 mg/kg
resulted in bronchospasm comparable to that caused by 3 ygAg of histamine.
Repeated injections of histamine resulted in reproducible changes in bronchial
motility, whereas sodium chloroplatinate exhibited tachyphylaxis upon repeated
injection Aerosol exposure of guinea pigs to sodium chloroplatinate also
resulted in an intense asthma attack Administration of the antihistamine,
pysilnnine, completely blocked the action of sodium chloroplatinate.
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15
Compared to histanune, sodium chloroplatinate has a long latent period
(45 seconds following iv infection and 15 seconds following application to
guinea pig ileum). This long latent period, along with the observed tachy-
phylaxis, suggests that platinum salts do not act by themselves, but instead
are involved in the release of a substance from the tissues. Evidence that
chloroplatinate may be causing the release of histamme is seen from the fact
that 10 minutes after the intracardiac injection of 40 mg/kg of sodium
chloroplatinate into rats, the plasma histamme level rose to 1500 yg/L
from a norm of 150 yg/L«
Platinosis is a skin or respiratory reaction or disorder resulting
from exposure to soluble complex platinum salts. There have also been reports
of dermatitis resulting from exposure to platinum oxides and chlorides
(Schwarte, 1947) and also to platinum alloys (Scheard, 1955). Skin lesions
from platinum exposure have been described as a dry, scaling rash associated
with cracking and occasional bleeding Instances of skm reaction have
been reported from workers in platinum refineries (Roberts, 1951, Milne, 1970,
Parrot, 1969) and by workers in photographic studios, who handled a paper
containing potassium chloroplatinite (Karasek, 1911).
Scratch tests on human subjects have provided information suggesting
that sensitivity to platinum salts is an acquired reaction precipitated by
previous exposure. Prior to employment in a platinum refinery, none of the
24 subjects were sensitive to the test solution above a 1 10^ dilution.
Following employment, workers who showed no signs of platinosis remained
2
insensitive to dilutions above 1 10 . However, subjects with definite signs
of platinosis has positive responses to dilutions of 1 10^ to 1 10^. The
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16
degree of sensitivity being more or less dependent on the severity of the
subject's syrrptoTis (Roberts, 1951).
Respiratory problems resulting from exposure to complex platinum
salts are initiated with irritation of the nose and respiratory tract,
coughing, sneezing and running of the eyes Continued periods of exposure
nay result in an asthma-like condition associated with wheezing, tightness
of the chest and shortness of breath The complex platinum salts frequently
responsible for platinosis are sodium, potassium, or ammonium chloroplatinate
(Levene, 1971).
Symptoms of both dermatologic and respirato>y toxicity have been
reported for a substantial number of workers exposed to complex platinum
salts (Parrot, 1969, Roberts, 1951, Hunter, 1945). It has also been
reported that 70% of the exposed staff of a platinum refinery had observable
cases of platinosis (Herbert, 1966). Hunter et a^. 1945, found that 52 of
91 employees of London platinum refineries had respiratory disturbances.
Roberts has stated the opinion that all workers exposed to platinum salts
have some degree of platinosis, 60% of which are symptomatic and 40% of which
have no obvious symptoms, but who reveal evidence of involvement such as
irritated conjunctivae and hypertrophy of the respiratory lymphatics (Roberts,
1951).
With the possibility that platinum may be emitted to the atmosphere and
\
the proven toxicity of certain platinum compounds, it is important to determine
the biological fate of platinum
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17
Animals and Treatments
The same procedures were vised in this study as in the palladium study
except the rats were given 25 yCi of 19-lpt.
Radioactive Determinations
The radioactive solution was composed of carrier- free 191,193pt -m
0.5 M HCL.
The solution contained at least 50% Pt and only the ^^-Pt gamma
was counted. All values were corrected for decay rPt has a half- life of
3 days) and the same dilutions and counting procedures were followed as m
the 103Pd studies.
Whole Body Retention
The whole body retention of 191pt following a single exposure was
significantly affected by the route of administration The percent of -^Ipt
retained with time in rats following three different routes of administration
is presented in Figure 4. Following oral dosing, the total net gastro-
intestinal excretion was extremely high resulting in a rapid decline of the
retention curve to less than 1 percent at the end of three days. The data
indicated that the rapid clearance was due to passage of non-absorbed -^Pt
through the gastrointestinal tract. Extrapolation of the second component of
the retention curve to the intercept indicated that less than 1 percent of
the initial dose was absorbed
The whole body retention of 191Pt following intratracheal dosing was
significantly higher than for oral dosing The excretion of approximately
50 percent of the initial dose during the first 24 hours is attributed to
mucociliary and alveolar clearance Whole body retention of l°lpt was the
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80
18
Percent of Initial
Retained
191
Pt
60
40
c
O
V
v.
O
Q.
20
Adult
Intratracheally
Oral
j_
j-Q—O-
-Q I
8 12 16 20 24 28
Days After Dosing
32
191
Fig 4 Whole body retention of Pt in adult rats
following oral, iv, and intratrachael admin-
istration
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19
highest following iv dosing, the short half-life precluded an accurate
determination of the biological half-life.
Excretion
Radioactive counts of 24 hour urine and feces sanples from rats
receiving ^Ip^ orally indicated that almost all of the ^TPt was eliminated
in the feces and only a small amount excreted in the urine (Figure 5). These
values support the whole body data which showed that total net gastrointestinal
absorption was low. Following iv administration, 19lPt was excreted in both
the urine and feces. The urine contained a greater quantity of the 19lPt
Tissue Distribution
The distribution and concentration of -^Pt in tissues was determined
following oral ard iv dosing After single oral dose, the kidney and liver
contained the highest concentrations of °lpt. The amount of radioactivity
found in the other organs was not significantly higher than background The
amount of -^Ip^ found in selected tissues following iv dosing is presented on
Table 2. Most of the tissues did not contain levels of l^lpt appreciably
higher than that found in blood. However, the fraction of l^lpt in the plasma
that was m an "available" form for movement into the various tissues was not
determined The large amount of 191pt found in the kidney suggests that this
organ accumulates this element Concentrations higher than the blood values
»
were also found in the liver, spleen and adrenal. The lower count for the
brain suggested that either 19lPt can be transferred through the blood -bram
barrier only to a limited extent or else much of the circulating l^lpt 1S
complexed to large molecules which do not cross the blood-brain barrier.
-------
20
io6
c IO5
.2
X
LU
lio4
_C
g- 3
X
t)
10
«••••
I 0
- \
- \
- \
- \
- \
= \
:SN\
- H\
E <> t°'-D^N;Dx
_ \ \ \ ,-'D^^ ™ i
\ \ V°" \, 1V
=" \ \ Feces ^^ ;
• V°--
\ \
\ \ Feces
1 \ Y °
I V Oral
\
r \
i Urine °
i i i t i i i i i
2 4 6 8 10 12 14 16 18
Days After Dosing
191
Fig. 5. Excretion of Pt following iv and oral
administration
-------
Table 2. Counts/g Wet Tissue
1 day
2 days
3 days
7 days
14 days
Blood
Heart
Lung
Liver
Kidney
Spleen
Pancreas
Bone
Brain
Fat
lestis
Mrenal
Muscle
Duodenal Segment
22,147
11,819
18,432
36,848
162,227
41,085
22,208
13,146
1150
4487
4186
45,439
4798
12,725
19,732
12,201
16,139
31,274
160,656
45,840
19,487
12,800
2485
4501
6540
42,363
4671
6044
21,671
15,508
17,638
23,528
162.374
44,033
22,618
13,184
s.
1027
4576
4545
58,596
3930
6045
12,774
8805
11,180
25,732
138,101
55,764
14,802
8932
595
3201
3873
26,667
' 3441
4031
7921
4593
5770
4733
30,195
20,973
,3973
5440
265
429
1431
6190
2146
1410
-------
22
Maternal/Fetal Uptake
191
Fifteen pregnant rats (18th day gestation) were given 25 yCi Pt
intravenously and sacrificed 24 hours later. During the 24 hour period, the
pregnant rats excreted 18 8 percent of the initial dose The amount excreted
by the pregnant rats was approximately the sane as the amount (19.3 percent)
excreted by the adult male rats during the first 24 hour period. The con-
191
centration of Pt per gram for different maternal tissues and fetuses is
given in Table 3.
Table 3 -"pt in Maternal Organs and Fetuses
TISSUE
Kidney
Liver
Lung
Ovary
Blood
Bone
Brain
Placenta
Fetal Liver
Fetus
MEAN COUNTS/g
127,064
43,375
17,981
14,639
10,568
9,193
792
27,750
1,421
432
191
The data indicated that there was some transplacental passage of Pt,
however, there appeared to be placental binding or accumulation "^Pt was
present in all the fetuses (60) counted The hemochorial placental barrier of
rats is more easily traversed than the more complex placental barriers found m
other species of experimental animals
-------
23
1. Christensen, G.M. Effects of Meta/ Cations and Other Chemicals Upon
the in vitro Activity of Two enzymes in the Blood Plasma of the
White Sucker, Catostomus commersoni. Chem. Biol. Interactions £, 351-
361, 1971-1972.
2. Herbert, R. Affections provoquees par les composes du platine
Arch Mai Prof. 27_ 877-886, 1966.
3. Hofmeister, F Ueber die physiologische Wirkung der Platmbasen
Naunyn-Schmiedebergs. Arch, exper Path. Pharmak. 16, 393, 1882
4. Hunter, D , Milton, R. and Perry, K.M A. Asthma Caused by the
Complex Salts of Platinum. Brit. J. Industr. Med. 2, 92-98, 1945.
5. Karasek, S R and Karasek, M. The Use of Platinum Paper, Report
of Illinois State Commission of Occupational Disease, p 97, 1911.
6. Levene, G. M Platinum Sensitivity, Brit. J Derm. 85_ 590-593, 1971
7. Meek, F F , Harrold, C C and McCord, C P The Physiological
Properties of Palladium. Indust Med Surg. 12_ 447, 1943
8. Milne, J. E H A Case Of Platinosis. Med. J Australia 2_ 1194-
1195, 1970
9. Parrot, J L , Herbert R , Saindelle, A , Ruff, F. Platinum and
Platinosis Arch. Environ. Health 19_ 685-691, 1969.
10. Orestano, G. The Pharmacologic Action of Palladium Chloride.
Boll See Ital. Biol Sper. 8_ 1154-1156, 1933.
11. Roberts, A. E. Platinosis. Arch Industr Hyg. 4_ 549-559, 1951
12. Saindelle, A., Ruff, F. Histamine Release by Sodium Chloroplatinate.
Brit J Pharmacol 35 313-321, 1969
13. Scheard, C Contact Dermatitis from Platinum and Related Metals.
Arch. Derm. Syph 71 357-360, 1955
14 Schroeder, H A. and Mitchener, M Scandium, Chromium (VI), Gallium,
Yttium, Rhodium, Palladium, Indium m Mice Effects on Growth and
Life Span. J. Nutr 101 1431-1438, 1971.
15. Schwarte, L , Tulipan, L., Peck, S A Text-Book of Occupational
Diseases of the Skm Lea and Fibiger, Philadelphia, 1947.
-------
24
A. 2. THE ACUTE TOXICITY OF PALLADIUM CHLORIDE
L. Hall, J. Adams, I. Washington, K. Campbell,
W. Crocker, D. Hysell, W. Moore and J. Stara
Introduction
As part of the corrprehensive evaluation of the inhalation toxicity
of catalyst emission compounds, acute toxicity studies with palladium
chloride were begun as a rapidly obtained, reliable and inexpensive
first estimate of toxicity in order to quantify the upper limits in terms
of dose and obtain observations for assessing the biological or pharma-
cological effects of these cotpounds.
Pesults
a. Single dose studies
Using the method of Deichman and LeBlanc ^ ' , the approximate lethal
dose (AID) was determined in 200-300 gm rats, (Charles River COBS) using
intravenous, intraperitoneal, and oral modes of administration. In addition,
the intravenous AID was determined in the rabbit and the mtratracheal minimum
lethal dose was determined in the rat. The results are shown in Table 1.
Table 1. Acute Lethal Toxicity of
Species
Rat
Rat
Rat
Rabbit
Rat
Approx. LDjQ
5 mgAg slope 'vl.S
70 mgAg slope -vl.5
>200 mgAg
5 mgAg
6 mgAg*
Route
IV
IP
Oral
IV
ITR
* Minimum lethal dose
-------
25
Marked differences are noted among the different routes of administration,
ranging from 5 mg/kg for IV to greater than 200 mgAg for oral.
Using the more precise method of Litchfield and Wilcoxon^' , the intra-
venous and intraperitoneal H^Q (14 days) was determined. Figure 1 shows
the log probit plot of the intravenous data. The LDso (14 days) was calcu-
lated to be 3.0 mg PdCl2Ag with 95 per cent confidence limits of 2.57-3.49.
The slope was found to be 1.43 with 95 per cent confidence limit of 1 15-1.77.
The (CHI)2 test indicated that the data are not significantly heterogenous.
Following intraperitoneal administration, the I£>50 was calculated to be
123.0 (91.1-166.1) mg PdC^Ag with a slope of 1.84 (1.04-3.27), no signifi-
cant heterogenety was roted (Figure 2).
A limited number of rats from the intravenous and intraperitoneal studies
were housed in metabolism cages and several toxicometric parameters were
measured during the 14 days observation period. Survivors of an acutely
toxic intravenous dose of PdCl2 exhibited a 25 per cent decrease in water
intake and urine excretion. Following intraperitoneal dosing a 7 per cent
reduction in body weight was observed with up to 80 per cent reduction in
food intake. Water intake was reduced markedly initially and then returned
to control levels or above. In one rat a 28 per cent increase in urine
volume was noted at 14 days after dosing, with a constant decreased specific
gravity of 1.030. Proteinuria was noted in all animals following both
i
routes of administration. Elevated urinary ketone bodies were observed in
some animals following both routes of dosing.
Following intraperitoneal infection, necropsy findings indicated a
chemical type "bum" of the visrora in animals dying within 24 hours.
-------
26
Gross pathologic examination of intraperitoneally dosed survivors
at 14 days showed prominent peritonitis with numerous adhesions involving
the liver, intestine, pancreas, and spleen. Involvement did not appear
dose related. The kidneys showed a yellowish-tan appearing renal parenchyma
with cloudy capsule. The liver in 30 day survivors showed reduced liver
mass, and yellowish discoloration with thickened, opaque capsules.
Tissue palladium concentrations were determined by atomic absorption
spectroscopy m rats which died following intraperitoneal injection. The
concentration for four doses in brain, lung, cardiac muscle, liver, spleen,
kidney and testes are shown in Table 2. A dose effect was suggested m
some tissue but the sample size was too limited for absolute confirmation.
However, the tissue levels in brain, heart, and lung showed that palladium
was mobilized from the peritoneal cavity.
In vitro protein binding studies were performed with palladium and
platinum chlorides (PdCl2 and PtCl^, using the Toribara ultracentrifugation
technique^ ' at concentrations up to 200 yg compound/ml using whole plasma
or plasma equivalent albumin. Protein binding was greater than 99 per cent
at all concentrations. Temperature and pH were found not to affect binding.
Following acutely toxic intravenous doses of palladium chloride, death
occurred very rapidly, with a sharp threshold such that if exitus did not
occur withm five minutes, the animals (both rats and rabbits) survived
the 14 day experimental period Rapid death may be due to respiratory
arrest, since breathing ceased while a heart beat was still palpable for
some time. However, cyanosis was not noted; the animals' oral mucosa and
eyes were pink in color. Clonic and tonic convulsions were noted m rabbits
and rats.
-------
70
65
60
55
I 50
CJS
45
40
35
30 -
10
i i i i i i
10
100
DOSE
(mg/kg)
95
90
85
80
70
60 ^
C3
50 i
UJ
CJ
40 £
30
20
15
10
NJ
FIGURE 1, INTRAVENOUS Pd2 MORTALITY DATA PLOTTED BY METHOD OF LITCHFIELD AND WILCOXON (19^9)
-------
70
65
60
55
CO
50
45
40
35
30
10
100
1000
DOSE
(mg/kg)
98
95
90
85
80
70
60 £
£
50 g
oe
LU
40 °-
30
20
15
10
to
00
FIGURE 2, INTRAPERITONEAL P
-------
29
TABLE 2
TISSUE PALLADIUM CONCENTRATIONS IN ACUTELY POISONED RATS FOLLOWING
J15TRAPERITONEAL M2>1INISTFATION (ug/gm Dry Vvt.)
Total
Dose
30mg
24mg
18mg
12mg
Mean
Day/Died
24hr/l
48hr/5
72hr/0
96hr/0
24hr/0
48)ir/2
72hr/2
96hr/0
24hr/0
48hr/l
72hr/4
96hr/2
24hr/0
48hr/0
72hr/0
96hr/l
Brain
< .04
5 42
5 77
4.18
1 26
2.18
4.77
6.95
Lung
-
86 84
74 46
78.52
41 03
30 60
22 86
3.45
Heart
27.32
37 93
26 62
40 12
17 57
11 15
17 42
0
Liver
185 91
302 68
201 54
277 39
67.79
115 60
51 36
37.24
Spleen
330 56
418 65
-
333 02
272.23
317.18
200 04
196 80
192 09
Kidney
843 39
476 82
478 00
590 86
583 94
202 01
177.83
*
Testes
60 91
67 58
42 34
67.44
14.31
33 56
31 56
12.70
-------
30
The sharp threshold for mortality following intravenous administration
suggested some conpartmental (blood) saturation phenomenon. Therefore, ten
rabbits which had survived the first intravenous dose of PdCl? were re-
£*
injected six hours after the first dose with a dose less than or equal to
the original dose. All but one died, and the survivor was the largest
animal used and had received initially the lowest dose.
b. Multiple dose studies
Toxicity of PdCl2 following daily intravenous dosing
Thirty-three rats weighing 250 g were used in this experiment.
Each rat was given 0.5 mg Pd in .1 ml (2 rng Pd/k9), daily in the tail vein
for five days. Deaths occurred almost ainmediately following injection. The
mortality during the five day period is given in Table 3.
Table 3. Cumulative Intravenous Toxicity of PdCl2
Date
11/26/73
11/27/73
11/28/73
11/29/73
11/30/73
Amount
Injected IV
.5 mg
.5 mg
.5 mg
.5 mg
.5 mg
Total Accum.
Dose mg/kg
2.0
4.0
6.0
8.0
10.0
1
No. of
Acute Deaths
0
2
2
4
3
Accum
Deaths
0/27
2/27
4/27
8/27
11/27
An additional group of six rats was given 0.5 mg Pd on the first day and 1 mg
on the second, five of the six annuals died almost immediately.
-------
31
Discussion
While the goals of this task are to determine the inhalation toxicity
of catalytic components, other routes were examined for a more comprehensive
evaluation. Intiavenous studies were initiated because of their ease, in-
expensiveness, and rapid information return regarding systemic and comparative
intoxication. Intraperitoneal studies were also initiated for the above
reasons, and also because of the similarity of mobilization between intra-
peritoneal and pulmonary depots Oral administration was studied smoe
material deposited m the lung is cleared in part to the gastrointestinal
tract. Intratracheal dosing is a reasonable model for inhalation studies.
Palladium chloride was selected for initial study because palladium
compounds are principal contenders for use as catalysts in automotive exhaust
control devices and, therefore, the toxicity and biological effects of
palladium ion are of prime interest as a form of the metal which might distribute
within the organism.
The large difference between the intravenous, mtraperitoneal and oral
acutely toxic doses suggest a difference in distribution of the palladium ion
depending on the initial depot. Ihis is supported by the tenacious macro-
molecular binding (> 99 per cent protein bound), the extensive peritoneal
pathology after IP administration, and the sharp threshold for acute intravenous
toxicity.
-------
32
The signs accompanying intravenous toxicity were suggestive of acute
central respiratory depression, respiration ceased before cardiac arrest
occurred. Convulsions were noted in some animals which could possibly
have been other than agonal Further studies are needed to elucidate
the mechanism of acute toxicity. However, the tenacious protein binding,
coupled with the sharp threshold and the repeat dosing experiment with
rabbits and rats, suggest saturation of some binding sites before distribution
to the ultimate target site(s).
Two additional effects were observed that merit comment. Respiratory
arrest was not accompanied by frank cyanosis. This would suggest some effect
of palladium on metalloporphyrin proteins Secondly, a pilot study was
performed to determine the effect of palladium chloride on whole blood in
vitro. The addition of this salt to whole heparinized blood caused clotting
in the sample. Further studies are necessary to ascertain the significance of
these findings.
Mortality after intraperitoneal administration appears to involve a
chemical peritonitis, although other direct toxicity may occur, as in the
kidney, since mobilization of the palladium occurred as shown by the tissue
analysis.
Some cormient is necessary regarding the discrepancy between the intra-
peritoneal ALD5Q as determined by the method of Diechman and LeBlanc^'
and the 1^50 as determined by the procedure of Litchfield and Wilcoxon^).
Assuming the LDso to be the most precise value (123 mg/kg), the value of the
-------
33
(70 mg/kg) represents a 43 per cent underestimation. The principal
reason for this discrepancy, although the difference is not particularly
alarming, is thought to be due to the chemistry of PdCLj. The ALDgQ was
determined using aged solutions which have been shown to be more toxic than
the fresh solutions. Whereas the LDcQ was performed with freshly prepared
solution of Pdd-2. In fact, before we were aware of this anomolous
chemical behavior of palladium chloride in solution, an LD^Q determination
was performed according to the method of Litchfield and Wilcoxon'^' with
aged solution. This procedure produced a value for the LDso m good agree-
ment with the ALD5Q method of Diechman and LeBlancd) .
These studies suggest that if palladium ion is formed after either
inhalation or ingestion, a great potential for intoxication exists because
of the enormous affinity of this metal ion for biological ^acromolecules .
References
1. Diechman and LeBlanc. J A I H.A 25 415, 1943
2. Litchfield and Wilcoxon. J. Pharm. Therap. 9£ 99, 1949
3. Toribara. J. Clin. Invest. 36 738, 1957
-------
34
A.3. COMPARATIVE TOXICITY OF NOBLE METAL COMPOUNDS ON
LACTIC ACID DEHYDROGENASE AND GLUTAMIC OXALAOCETATE TRANSAMINASE
IN VITRO
S. D. Lee and R. M Danner
An in vitro study was conducted to study the relative toxicity of
palladium and platinum compounds on two enzymes glutamic oxalaoacetate
transanunase (GOT) and lactate dehydrogenase (LDH) The concepts of
correlation between metal-ion toxicity, enzyme inhibition ard serum
enzyme levels as related to cellular damage in certain organs represent
the hypothesis for this study
t In vitro enzyme test system was as follows heparinized rabbit blood
plasma was pooled and 5 ml portions were sealed and stored at -20°C until
use. The test solutions were prepared from stock solutions which contained
one milligram of metal compounds, which was dissolved in dilute hydro-
chloric acid and made up to a final volume of 10 ml with sodium acetate
^
buffer (pH 7.4, l.OM). The test system used in this experiment was similar
to that of Christensen (1971/1972) with minor modifications The concentrations
of stock solutions was 1.0 mg/ml. Concentrations of 1 x 10~5 through 1 x 10" ^
yg/ml were prepared and tested.
Various reaction combinations were prepared
Control = 0 3 mg plasma, 0.1 ml acetate buffer (pH 7.4, l.OM, 100 pi),
test sample = 0.3 ml plasma, 0.1 ml test solution containing
noble metal compounds, reagent blank = 0.3 ml acetate buffer and
100 pi test solution, enzyme blank = 0.3 ml plasma and 4N HC1
(100 pi), standard = Versatol E (0.3 ml) and 0.1 ml acetate buffer
-------
35
The test solutions were incubated for 30 mm at room tennperature (25°C)
Following incubation, the enzyme activities were determined using DADE reagent sefe
All analyses at each concentration of test solution were performed in duplicate
and average values determined. The enzyme activities were expressed in
International Units/ml for IDH and Reitman-Frankel Units/ml for GOT following
correction for enzyme and reagent blanks
Figure 1 shows the inhibitory effects of palladium chloride (PdCl2)f
dutier of propionyl palladium chloride (C^H^-PdCl) 2 and potassium chloro-
palladate (K2PdCl4 on serum IDH PdCl2 exhibited a "stair-case" type
inhibition pattern and was the most toxic among the three Pd compounds tested,
it reached 80% inhibition at 250 mg/1 (C3H5PdCl)2 was less toxic than PdCl2/
it leveled at a plateau of 35% inhibition between 100 mg/1 and 250 mg/1
K2PdCl4 was the least toxic of the three compounds, it showed a mere 5%
inhibition up to 150 mg/1 and reached 20% inhibition at 250 mg/1.
Figure 2 shows a comparative inhibitory effect of ^H^PdCl^/ PdCl2/
K2PdCl4, PtCl4 and HgCl2. HgCl2 was used as a reference compound since its
toxic effect is well known. The relative toxicity of the five compounds
tested was ranked as follows
HgCl2 >(C3H5PdCl)2 > PdCl2 > PtCl2 > K2PdCl4
These tests were performed to ascertain what type of effects one might
expect if sufficient quantity of Pt and Pd compounds were found to be emitted
\
into the ambient air. The in vitro data on inhibition of IDH and GOT may
serve as a useful information as additional data are determined relative to
the emissions of these compounds
-------
36
lOOr
90
80
70
60
«- 50
o
g 40
~ 30
* 20
10
50
100
(mg/L
150
200
250
Figure 1. Comparative in vitro inhibition of Pd-oxpounds on Serum IDH
-------
37
t_
o
CD
uo
M—
o
a
o
«&••
•rS
c
_
,
100
90
80
70
60
50
40
30
20
10
0
(CoHrPdCI)
/
HgCI2 /'
- / /
/ /
/ ,'—/-"" -PtlCI2
X x' /
X x^ / p^pj
y s* / .•••**'** r lUl^
i X .•/'""
- 1 ,''..'•""'/
• 1 ^^<.«* / ^^^**i7 n J f*
"/ ^'*''*' /' ^^*^^ lydk
-* /x '* ^ ^^^^"^
- I - y
/ • ' ^
• ''!• ^
' '' /-'
""""" / t t » 1 1
,50 100 150 200 250
mg/L
Figure 2. Comparative in vitro inhibition of SCOT
-------
38
A.4. EFFECT OF NOBLE METAL COMPOUNDS ON PROTEIN SYNTHESIS
IN VARIOUS OPJGANS OF PATS
S D. Lee and R M. Banner
Experiments were conducted to detect early biochemical effects of intra-
gastric administration of noble metal compounds (PdC^ and Pt[304]2) on protein
synthesis in various organs as determined by the rate of incorporation of
C-leucme. Experimental animals (rats) were given PdC^ (1 mg/kg body weight)
24 hrs before sacrifice. Control animals were given saline solution One
hundred forty yCiAg body weight of -^C-leucine was injected through the tail
vein of all rats (control and treatment groups) and allowed to metabolize for
one hour before sacrifice, The -^C content of purified protein m liver, kidney,
lung, heart and blood serum were examined.
Each of the excised organ samples was homogenized with 0 25 M sucrose
3il v/w. An aliquot of the homogenate was used to precipitate protein
with 10% trichloroacetic acid v/v The precipitate was washed twice with
5% trichloroacetic acid v/v and then twice with 95% ethanol The concen-
tration of protein was determined by the Biuret method. Radioactivity levels
were measured in a Packard Liquid Scintillation Spectrometer The observed
values were expressed in terms of dpm/mg protein and per cent alteration with
reference to control
As can be seen in Table I, no change was observed in kidney ard lung, and
only a slight decrease in liver. However, there was a marked increase in ^C-
leucine incorporation into the heart and blood serum protein. The increases
were 137% ard 49% in heart and blood serum, respectively.
A different pattern of C-1-leucine in rats given 0 5, 1.0 and 5.0 mg
per kg body weight of Pt(504)2 was observed.
-------
39
As depicted in Table II, Pt(SO.)2 caused a different response in 14C-1-
leucine incorporation into protein of five organs examined The most
pronounced change was observed in the lung where a definite dose-response
was observed with increasing concentration incorporation of ^C-l-
leucine rose 10.3% for 0.5 mg/kg/ 22.7% for 1.0 mgAg and 109 4% for 5 rag/kg
body weight, respectively.
The incorporation of C-1-leucine in kidney showed a reverse trend
+16.9% for 0 5 mg, 62% for 1 0 mg/kg and no change for 5 mg/kg body weight,
respectively. The changes in the brain showed similar pattern as the kidney
There was a 30% increase at 0.5 mg/kg body weight for liver and no other
apparent changes were indicated. Treatment with 5.0 rogAs body weight of
Pt(SO^^ resulte<3 m a 9% decrease About 30% and 26% increase for 0.5 mg
and 1 o mgAg levels were observed, respectively Apparently, Pt(SC>4)2 at
the concentrations used m this study did cause a significant disruption m
protein synthesis in organs tested
-------
40
Table I. Effect OF PdCJ-2 on -^C-Leucine Incorporation Into Protein
Control
Experimental
% Change
dpm/mg Protein
Liver
1,484 (4)*
1,361 8 (4)
- 8.0
Kidney
1,708.0 (4)
1,728.2 (5)
+ 1.2
Lung
1,566.5 (4)
1,540 6 (5)
- 17
Heart
955 (4)
2,265 (4)
+ 137 2
Blood
Serum
1,877.3 (3)
2,796 5 (4)
+ 49.0
( ) * Denotes number of animals
-------
41
Table II Effect of Pt(S04)2 on 14C-l-Leucine Incorporation
Into Protein
nxjAg B w' ^"^\
0
0.5
% Change
1.0
% Change
5.0
% Change
cpm/mg Protein Corrected for Organ Weight
Liver
1056 (6)*
1374 (4)
+ 30 1
1088 (4)
+ 3.0
1110 (4)
+ 5.0
Kidney
2332 (6)
2807 (5)
+ 16.9
2477 (4)
+ 6.2
2331 (4)
0
Lung
1732 (6)
1911 (5)
+ 10.3
2239 (4)
+ 22.7
3627 (4)
+109.4
Heart
1355 (6)
1356 (5)
0
1326 (4)
- 2.1
1233 (4)
- 9.0
Brain
882 (6)
1142 (5)
+ 29.5
1111 (4)
+ 26.0
949 (4)
+ 7.0
( )* = number of animals used.
-------
42
A 5. DEKMAL IRPJTANCY OF SEVERAL Pd, Pt and Pb COMPOUNDS AND OF MMT
K.I. Campbell, E L. George, L L. Hall and J.F. Stara
A necessary aspect of general toxicologic characterization of
potential environmental pollutant substances is the evaluation of dermal
irritancy. A series of such tests were performed on several palladium
and platinum compounds, for their relevancy to catalytic automotive emission
control devices, and on two lead compounds and the gasoline antiknock
additive, 2-methyl cyclopentadienyl manganese tricarbonyl (MMT).
The test procedure used was essentially that in standard use by
the National Institute of Occupational Safety and Health, ' ' a modi-
fication of the official Food and Drug Administration procedure ' ' In
each test six (6) healthy, male albino rabbits weighirg 2 to 3 kg tTere
used Up to seven (7) pairs of sites 2 x 2 on were used on the closely
clipped dorsolateral aspects of the trunk of each animal, the sites on
the right side being abraded and those on the left remaining intact. Test
materials in the solid (powder) state were applied in 0 1 gm quantity per
site, mixed with about 0 1 ml deionized water and spread over the site,
liquid materials were applied directly in 0.1 ml quantity* Each application
was covered immediately with a gauze patch and further secured with tape
(and overwrap in one test) and use of a leather restraining harness.
After 24 hours, harnesses and coverings were removed, and test sites were
washed with mild soap, rinsed, and dried. The skin reactions were then
evaluated and scored, and again scored 48 hours later. Skin reactions
were evaluated and scored using a grading system summarized in Table 1
-------
43
The assigned rating was calculated as the average of means of the 24 and
72 hour scores for the test group, rated separately for intact and
abraded skin, as illustrated in Table 2. Ratings were interpreted
according to the scheme summarized in Table 3.
The materials tested, the dermal irritancy (intact skm) and
cellular toxicity (abraded skin) responses observed, and the corresponding
interpretations are shown in Table 4. Results were interpreted
conservatively, i.e. based on the test in which the most severe responses
were observed Many of the test materials showed a delayed healing of
the abrasion lines themselves, in addition to or regardless of the
standard response criteria.
Severity of response to some of the compounds tested more than
once was quite variable Skin character and hair gra-rth patterns among
rabbits in the specified weight range were somewhat variable, and
these could be factors in irritancy responses and evaluations. We
recommend selection of rabbits for uniformity on these additional criteria.
Close but gentle (atraumatic) clipping in preference to shaving, and over-
wrapping in preference to taping for security of patches, are also
recommended. In addition to tests for dermal irritancy, tests for
sensitization should also be performed. Sensitizations may be far more
serious or chronic than direct irritation, they may develop at lower and
more common levels of exposure, and opportunity for development may be
greater by virtue of extended or repeated exposure by ingestion and
inhalation as well as cutaneously
-------
44
~ J
Table 1
Evaluation of Skin^ Reaction to ^est !laterial_
Grac'e Valve ard
Reaction
nH't}icJra (regardless of decree)
and ederra confined to test area
)
Intact £
0
(Non -irritant)
1
(liild imtcuit;
2
('irritant)
and cdeira extending be^ord test area 3
(Strong imtart)
(deep reaction involving denras)
(Corrosive)
bradcxl in
(Kild cclliLJeir tc
(Cellular t
(Strorg cel2u]r^
-------
45
Table ?
Exarrple Calculation of
\
\
Test Rating
Intact Skin Peacticn
(Dermal lTitarc\)
nm?l Fiirccr
1
2
3
4
5
C
" K=6
* \
?4 Hr
1
0
2
1
3
JO
72 Hr
1
1
1
0
2
J.
Total
2
1
3
1
3
JL
-
rcan
1.0
0.5
1.5
0.5
1,5
0.5
5,5
traded Skoji Reaction
(Cellular Tc^ic^t-/)
24 Hr.
2
i
\
2
2
2
Jk
72JIr__
»
3
2
2
1
2
J.
Total >3an
*
5 2.5
3 1.5
4 2.0
3 1.5
4 2.0
J2 3.0
10.5
Derrral irritation rct^rq for
intact son - 5 5/6 = 0.9.
In this a^rplG, a ron-
iiiitant.
imtation rating for
abxac'ed ckn, = 10 5/6 - 1.8. —
In Uiis e>arple, a raid cellulaM
-------
46
TALI C 3
Interpret?J^on of Skin Jest Ratings
Intact Skin
Abraded Skin
Rating
0-09
1 - 1.9
2-- 4
0 -0 9
1 - 1.9
2 - 4
Interpretation
Non-irritant, probaoly safe
for intact human skin contact
Mild irritant, may be safe for
use, but appiopriate proactive
measures are recommended during
contact
Too irritant for human Skin
contact, avoid contact
Non-toxic to cellular conoononts
of abraded skin, probably
for human skin contact
safe
Mild cellular toxins, may be
safe for abraded skin contact
provided protective measures
are employed
Cqllular IDA ins too imiant
for abraded skin contact,
avoidance of contact is ?Q«nsed
MIXED REACTIONS
Intact Sinn
0-09
1 - 1.9
2-4
Abraded SVi n
0-09
1-19
2 - 4
1 -'1.9
2-4
2 - 4
Safe for human skin contact
Safe for intact human skin
contact, may be safe for
skin contact when protection is
maintained ,^_
Safe for intact human skinj con-
tact with abraded skin should
be avoided
May be safe for intact and
abraded skin contact \'hen pro-
tection is maintained
May be safe for intact hunan
skin contact ./hen protection
is maintained, but contact with
abraded si in is to be avoided
Unsafe for intact and abraded
human shn contact, avoid contact
-------
Table 4 - Rated Responses to and Interpretation of Direct
Dermal Irntancy and Cellular Toxicity Tests in Rabbits
Material
Tested
Seventy Rating*
Intact Skin Abraded Skin
(Irntancy) (Cellular Toxicity)
Interpretation
Dei om zed water
(negative control )
Glacial Acetic Acid
(ethanoic) acid
(Positive control)
Potassium chloropalladite,
Potassium chloropalladite,
K2[PdCl6]
Palladium chloride,
PdCl
Ally palladium chloride
dimer, (CH
Dichlorodiammine palladium
(II)Trans,
Ammonium chloropalladite,
(NH4)2PdCl4
Ammonium chloropalladat
(NH4)2PdCl6
Palladium monoxide, PdO
Platinum (II) dichloride,
PtCU
0
2.6
0
(0)
0
(0)
0
(0 1)
0 8
0
(0)
1 5
(3 1)
2 8
(4)
0 2
Platinum (IV)tetrachlonde, 1 8
PtCl4 {2.7)
Platinum(IV)dioxide, 0
Pto2
Lead chloride, 0
Pd C12
Lead monoxide 0
Pb 0
2 - Methyl cyclopentadienyl 0 1
manganese tricarbonyl (HIT)
0 1) Safe for human skin con-
tact
3.2 2) Unsafe for human skin
contact
1.6 3) Safe for intact human
(1.9) skin may be safe for abrad-
ed skin when protection is
maintained
1.6 4) Safe for intact human
(2) skin abraded skin contact
should be avoided
06 5) Safe for intact human
(1) skin may be safe for abrad-
ed skin when protection is
maintained
1.8 6) Unsafe for human skin
contact
02 7) Safe for human skin con-
(0 5) tact
2.5 8) Unsafe for human skin
(3 7) contact
3.2 9) Unsafe for human skin
(4) contact
0 10) Safe for human skin con-
tact
06 11) Safe for human skin con-
tact
2 6 12) Unsafe for human skin
(3.8) contact
0 13) Safe for human skin con-
tact.
0 14) Safe for human skin con-
tact.
0.1 15) Safe for human skin con-
tact.
08 16) Safe for human skin con-
tact
*Rating in parentheses indicates the most severe test result where tested more than
once, those without indicate the single test rating or average of 2 or 3 test
ratings
HBased on most severe or single test result
-------
48
References
1. Course Manual "Toxicologic Investigative Techniques" Occupational
Health Research and Training Facility, Division of Occupational
Health, U.S Department of Health, Education and Welfare, 1964
2. Johnson, G T , Perone, V B , Busch, K.A , Lewis, T R and Wagner, W D
Protocols for Toxicity Determinations, Unit 1, Acute Projects
Toxicology Branch, NIOSH, Cincinnati, Ohio, 1973 (Draft)
3. Perone, V.B. Personal communication, 1973
4. Code of Federal Regulations, Chapter 1, Paragraph 191.11
-------
49
A. 6. DERMAL ABSORPTION OF 191PLATINUM+4 IN HC1 SOLUTION
K. Campbell, E George, W Moore, W. Crocker, F. Truman
In conjunction with tests of dermal imtancy of platinum compounds
an experiment to assess transcutaneous absorption of ionic platinum was
performed In each of 5 rabbits, 10 yl of a solution containing 191Pt+4
in 0.5M HC1 was spread over a closely clipped 1 on square area of dorsal
skin in the scapular region The nuclide dose was 8 36 yCi, a Packard
gamma scintillation spectrometer (Model 5375) was used for counting
Samples of blood before application and at 4, 24, 48 and 72 hours post-
application, and 72-hour terminal samples of skin (incorporating the
site of application), liver and kidney were counted. Counts were
corrected for background and decay, expressed as counts per minute (CPM)
per gm of sample (except for the skm specimen for which only total count
was pertinent), and the fraction of the original applied dose was calculated
Results showed that at the 72 hour terminal period, on the average,
53.41% of the original dose was in or on the skin at the site of application
and that very small fractions appeared in the blood or in the tissues. Of
the sequential blood samples, the earliest (at 4 hours post-application)
contained by far the greatest fraction of the applied dose (0 0074%), sub-
sequent samples contained less than 1/10 as much Among the tissues at
sacrifice, the concentration of activity in kidney was about 2.7 times that
in liver and 14 3 times that in blood. The results are summarized in Table 1.
Data of this experiment do not permit conclusions as to total amounts absorbed
vs lost from the s'-in, the fractional distribution to other tissues, and the
amounts excreted, they do suggest early minor transcutaneous absorption, with
distribution to blood, liver and kidney There was no visible sign of
dermal irritation at the site of application.
-------
Table 1. Tissue Levels of ^ Pt Activity Following Dermal
Application of 191piatmum+4 ^ HCl Solution
Specimen
Blood
Pretreatxnent
4 Hr. post- treatment
24 hr. " "
48 Hr. "
72 Hr. " " (terminal)
Skin, terminal
Liver, terminal
Kidney, terminal
CPM/gm
mean
0
92.4
1.4
6.8
2.1
594,493
Total*
11.35
30.3
Proportion of Dose Applied, Dec. Fract.xlO"
Mean Range
C
74.0
1.24
6.60
1 82
534,100
10.25
27.0
0
0
0
0
0
274,000
3
6
- 0
- 240
- 3.2
- 30
- 8
- 732,200
- 16
- 48
Remark
n=5
"
"
"
"
Ave. 53.41% of
orig dose
n=4
n=5
*Based on entire skin sample.
-------
51
A 7. OCULAR IRRITATION OF TWO PALLADIUM COMPOUNDS IN RABBITS
D Hysell, S Neiheisel and D Cmehil
The test was performed as outlined in the Code of Federal Regulations,
Title 21, part 191 12, revised as of April 1, 1973 Six albino rabbits,
having no known ocular abnormalities, were restrained and 100 mg of the
test material was deposited on the surface of the right eye The left
eye was maintained as a control. The animals were examined for ocular
inflammation 24, 48 and 72 hours following application of the material
In the case of PdO (Table 1) no reaction was noted in any of the six
rabbits In one animal, the test material was still present in the
corgunctival sac at the end of 72 hours, but was completely covered with
a thick rrucous material
All six animals receiving PdCl showed a severe corrosive type lesion
of the conjunctiva with severe inflammation of the cornea and anterior
chairber of the eye (Table 1) This was noted at 24 hours and persisted
throughout the test period.
These test results would indicate that at the dosage levels used,
PdCl was a severe irritant, PdO was not
Table 1
Compound
PdO
PdCl
Fraction of animals shaving reactions at specific test intervals^
24 hours
0/6
6/6
48 hours
0/6
6/6
72 hours
0/6
6/6
-------
52
A.8. NEUROPHYSIOLOGY SUMMARY OF Pt AND Pd
J LewkovvSki, T Wessendarp,
W Moore and J Stara
The visual evoked potential in the rat is being utilized as a
screening technique to test the relative short-term effects of various
toxic agents Over 120 anesthetized rats have been exposed via intra-
venous iT3ection in the past year The resultant changes in the visual
evoked potential has been analyzed using various methods including computer
averaging techniques
The results have indicated that this screening technique may be
important in assessing the significant acute effects of various pollutants
on central nervous system function The table below indicates the mean
threshold dose of a particular cation which elicited a reproducible change
in the visual evoked potential in 50% of the animals studied.
VISUAL EVOKED POTEOTIAL SCREEN
Mean Reproducible Dose-Effect Threshold
Ion (mg/kgj
Co 0 010
Cd 0 10
Pd 0 40
Cr 0.80
Ba 20
Mn 20
Pt Minimal effect
Therefore, the relative short-term effect of the intravenous adminis-
tration of these cations on the rat visual evoked potential may be ranked
as follows
Co > Cd, Pd, Cr > Ba, Mn > Pt
-------
53
Assuming a similar blood level of Pd and Pt, it would appear that Pd
has a greater effect on the central nervous systeir. function under the
experimental conditions of this particular screening technique It should
be noted that this effect may not necessarily be a direct effect on central
nervous system function. Further experimentation is underway to determine
whether these effects are direct or are due to indirect factors, such as
changes in blood pressure or some other physiological changes
In addition, more quantitative and less subjective methods to determine
these thresholds are currently being utilized in an attempt to more pre-
cisely determine the relative central nervous system toxicity of these metals
-------
54
A. 9. A PRELIMINARY REPORT ON THE CARDIOV2\SCULAR ACTIONS OF PALLADIUM
M. J. Wiester
Introduction
Palladium chloride has been shown to be extremely toxic when given
intravenously (I.V.). Rabbits, rapidly injected with 0.6 mg/Kg quickly
die with damage chiefly to the heart. The nature of the heart damage
was not further defined and there is very little other information in
the literature addressing this subject. The purpose of this study was
neasure the effects of different palladium chloride solutions on heart
rate, ECG pattern, blood pressure, cardiac contractility (dp/dt) and
breathing for one hour following intravenous injection.
Methods
Sprague Dawley (f rats (300 + 50g) were surgically prepared one day
prior to use. Surgery consisted of catherization of the abdominal aorta
with tubing (#50 P.E.) for measurement of blood pressure and tubing
(#10 P.E.) v/as inserted into the femoral vein to acoomodate I.V. injections
Both catheters were guided through the subcutaneous tissue to the bacK region
and via a puncture wound through the skin to the outside. Six small silver
electrodes, fitted with micro-strip connector pins, were inserted under the
skin and sutured. Electrodes were arranged laterally so that four were
near the lutfos to record the BOG and two were on the lateral surface of
the rib cage for respiratory measurements. After surgery, the rats were
returned to their cages and given food and water ad libitum (Purina Lab
Chow and tap water).
For testing, an unanesthetized animal was placed m a plastic tubular
holder for the duration of the experurent and sensor leads fed to a recorder
(Figure 1). The measurement system is diagrammed in Figure 2. After a
-------
55
FIGURE 1. PLASTIC RAT HOLDER. HOLDER WITH RAT AND POLYGRAPH LEADS
-------
56
/x
PRESSURE
TWWSbUCLR
\/
^
t
rz
f
sr
DIFFCRCtmATOR
dp/clt
V
\
f
\1
'
V
IMPEDANCE
PHeiMOGRflPH
^7
1
f
\7
'
^
ECG-
PREflMPUFIER
^
\
^
7"^
^
RECORDER
FIGURE 2 Block Diagram of Recording System
Pressure Transducer
Differentiator
Impedance Pneumograph
ECG Preamplifier
Polygraph
For measurement of arterial blood
pressure - calibrated with a Hg
manometer (Miller Insttument)
For a recoid of maxinum rate of
change of aortic piessute - time
constant of 1 msc Calibrated
with an osciloscope
Monitors rate and relative depth of
respiration (Narco Bio-Systems)
(Grass) Lead 2 was recorded
(Grass 7C)
-------
A COHTROU rc
,t
B_tO &6COMD5 AFTFn DOSS
,-U'
\ / !
I ' I
C 60 MIM /"=ieR DCSS
01
Fig 3 Pat i?4 A Polygraph recording 25 minutes into the control period Aortic blood pressure is 165/i25
Electronic differentation of tne blood pressure signal is displayed as dp/dt The uptsard deflection shov.s maximum rate
ofpressure development, wmch for this pressure pulse is 3,000 trmHg/sec dp/dt reflects the contractile state of cardiac
rusc'^ The respiration record sho,vs the rate and relative depth of breatmng The ECG is derived from lead 2 For
this lead a proiiinant P wave and R wave can be defined The Q, S, and T \a/es are soirc\ hat less specific However, the
pattern is dependable ana remains unchanged throughout control periods heart Rate = 460 beats/mm
B This section she /s the Measurements inr^diatoly following tne I V injection of 2 04 mg/kq PdCl2 Gross abnormalities
can be seen in the ECG PVC's are not frequent enough to cause a detrimental fall in blood pressure Breathing was not
altered Similar irregularities continued for approximately 3 minutes The animal survived
C One hour following the injection blood pressure haa increased to 185/140 irarllg, dp/dt = 3660 imHg/sec , tne ECG showed
no gross abnormalities, heart rate = 408 beats/irin and bieathing was uncranged
-------
58
thirty minute stabilization or control period the palladium solution
was injected and washed in with saline. Hie total volume of the dose
and wash solution Wcis 1 ml. and total infusion time was one minute.
Effects of the injection were then observed for 60 minutes. Control
animals were injected with 1 ml. of saline and treated the same.
Results and Contrente>
Palladium chloride exerted an immediate cardiovascular effect in
the unanesthetized rat. The most pronounced effect was seen on the
electrical integrity of the heart. A total of ten animals were dosed
in amounts ranging laetween 1.14 - 5.9 mg PdCl2/Kg and in each instance,
premature ventricuLir contractions (PVC) were noted within one minute
after dose initiation. PVCs were never seen during the 30 minute control
periods or in control animal experiments (5 rats). Doses between 1.14 -
1.75 ng/Kg resulted in mild episodes of PVCs following the injection
with no consequential fall in blood pressure. These arrhythmias
continued for 3-4 minutes then the BCG stabilized. This stability,
however, was dependant on the quiet state of the animal. If the rat
moved or showed signs of distress, PVCs> reappeared. Rats that received
doses between 1.75 - 5.9 mg/Kg experienced gross alterations in the BCG
pattern following injection. If the cardiac arrhythmias were inter-
mittent or of sucn a nature as to allow adequate filling and pumping,
then the animal survived (Figure 3). These surviving animals were able
to maintain sufficient blood pressure levels during the critical 3-4
minute period following injection.
These surviving rats also reestablished a stable ECG during the one
hour observation period, and like the low dose animals, were susceptible
to arrhythmias, if they became agitated. Rats that succumbed after
-------
59
receiving PdC]^ intravenously suffcred gross alterations in the EGG
acxsompanied by a precipitous fall in blood pressure. After the aortic
pressure fell, breathing became erratic and the EGG continued to
deteriorate (Figure 4). Death usually occurred within four minutes
after infection. Additional EGG abnormalities, other than PVCs, that
were seen after injection of Pdd^ were extra p waves, large S waves
and various degrees of A-V block.
Rats surviving a PdCl9 injection developed elevated blood pressures
t*
which persisted throughout the one hour observation period. Systolic
pressure increased 20-50 ranHg and diastolic 10-20. Heart rates
correspondingly decreased, and dp/dt changed very little.
Intravenous PdCl^ appeared to have no initial effect on respiration.
Changes in breathing were seen, however, the changes followed gross cardiac
arrhythmias and falling blood pressures. If the rat reestablisned a
steady and productive heartbeat, thus, survived the injection, breathing
returned to control values and remained stable.
Results from preliminary experiments described above indicate that
PdCl^f when injected I.V., acts as a non-specific cardiac muscle irritant
as well as a peripheral vasoconstrictor. Since the chloride salt strongly
dissociates in solution (PdC^ % Pd** + 2 C1-) the palladium ion itself
nay be the irritant. Effects seen might be due to the release of
i
catecholamines or to stimulation of adrenergic receptors located in the
cardiovascular system by the metal ion.
-------
'_ 'A1
A COWTROI. PERIOD
/v/
Bieoo PRE4SURE
"HIM i/nMvii'iMiH < ' ,.,, ;,,
f 17. rf,• r,/1cfnn,ri tfr
-------
61
1. Orestano, G. The Phannacologic Actions of Palladium Chloride,
Boll. Soc. Ital. Biol. Sper. 8 1154-1156, 1933
-------
WHOLE EXHAUST EMISSION STUDIES
-------
62
B.I. AUTO EXHAUST FACILITY MODIFICATICN
R. G Hinners and J. K Burkart
Introduction
The auto exhaust generating system has recently been modified at this
(1 2)
laboratory and the following will update other papers ' describing the
earlier facilities for the production of irradiated and nonirradiated
gasoline engine exhaust-air mixtures It is also intended as a reference
for biologically oriented papers discussing health effects of completed
studies The Toxicity Assessment of tobile Emissions (TAME) project
represents a series of acute and subacute bio-effect studies which test
exposures of experimental anurals to whole automobile exhaust emissions
with fuel additives ard/or with or without a catalytic converter Briefly,
the exhaust gases are generated by an engine-dynamometer unit and mixed
with clean conditicned air in a dilution system to produce the desired
concentration The exhaust gas mixture is divided with one part flowing
direct to animal exposure chambers, and the remainder flowing through
irradiation chambers to other animal chambers. The changes include an
air dilution tube for the immediate mixing of the entire raw exhaust
emissions with conditioned air and a large mixing chamber after the dilution
tube. Information is also provided on air supply, engine cycle, fuel supply
and other minor changes that have been made.
Dilution Tube
The effluent from the engine exhaust system is passed into an air
dilution tube, through flexible stainless steel tubing, connected to the
muffler The dilution tube is 23 in in diameter and made from 10 ga SS
-------
63
plate, rolled and welded. Dilution air enters the tube through a 90° elbow
from a remote supply source. Located between the flanges of these two tube
sections is a mixing baffle plate, with a 7-1/4 in. diameter hold bored in
the center Ihe incoming dilution air under pressure, is forced through
this hole to mix with the raw exhaust The tailpipe exhaust inlet elbow
enters 90° to the tube axis and is bent 90° again, so that the flow axis
of the exhaust outlet coincides with the center line axis of the dilution
tube The exit end of the 2-in diameter SS exhaust elbow is in the same
plane as the baffle Located on top and outside the dilution tube, at
the baffle plate, are two quick-disconnect couplings One allows the
end of the flexible 2-in. I D. exhaust pipe from the muffler to connect
with the dilution tube and the other connects to the outside atmosphere.
A blank plug is installed in the disconnect to the dilution tube when the
exhaust is vented outdoors System back pressure at this point is 4 in.
water This feature provides the capability of varying the modes of engine
operation for aerometry and allows interruption of animal exposures.
By closing a damper m the air supply line, the dilution ratio can be
controlled. To retain the particulate matter in suspension and prevent
condensation, it is necessary to dilute the whole exhaust with at least 8
parts of air to 1 part of exhaust For each pound of fuel burned, approxi-
mately a pound of water is formed and some condensation occurs if the exhaust
\
is not immediately diluted with dry air Also to prevent condensation, the
outside of the dilution tube is insulated since engine room temperature often
exceeds 90°F and dilution air temperature averages 50°T and 67% relative
humidity The main portion of the dilution tube consists of two 7 ft. long
flanged sections before reducing through a transition to a 6-in. diameter and
entering the mixing chamber
-------
64
Mixing Chamber
The diluted auto exhaust enters the mixing chamber, formerly used as
an irradiation chanter, through a 6-in diameter SS pipe opening in the
side wall An elbow discharges the exhaust in front of and parallel
with a tube-axial ian, controlled at a low rpm by a Zero Max unit, to
mix the entering auto exhaust with the chamber atmosphere
The chamber is, 23-1/2 ft long, by 4 ft wide, and 8 ft high, with a
volume of 683 ft^ The sides consist of a framework of aluminum structural
members holding metal panels to replace the plastic windows The aluminum
sheet metal panels are clamped and sealed by means of pressure screws and
gasketed channels. Previous studies with a reference fuel, to which had
been added methylQclopentadieny1 manganese tricarbonyl ^M?) as an
antiknock additive required darkress, due to the light sensitivity of the
MMT. The top, bottom, and ends of the chamber are formed of 1/4 m thick
aluminum plate welded on both sides at all seams to prevent leakage
At the end of the chamber opposite the entry port is a 6-in diameter
line with a motori2ed damper control vented to the atmosphere. Another
6-in. diameter outlet pipe from the chamber supplies the exhaust either to
irradiation chambers or to raw exhaust animal exposure chambers A pressure
sensor, which is ad} us table and located downstream of the chamber exit line,
controls the motorized damper in the vent line to maintain 2-m of positive
water pressure in the chamber
Irradiation Chambers
The photochemical reactions that result from the exposure of the diluted
raw exhaust to artificial sunlight take place in five irradiation chambers
-------
65
Fluorescent lighting panels composed of blue lamps, black lamps and sun
lamps outside the chamber pass intense ultraviolet radiation through
windows of Teflon FTP fluorocarbon film. One irradiation chamber is
needed to provide the atmosphere for each animal exposure chamber Normal
flow through the irradiation chambers is 11 cfm, which results in 15 air
changes per hour in the animal exposure chambers In some instances,
however, the flow has been reduced by one-half that of normal, which, of
course doubles the irradiation time One of the original irradiation
chambers used in previous exhaust studies has been converted into a mixing
chamber which is described separately
At a volume of 683 ft3 and 11 cfm flow, 43 minutes is needed to
achieve 50% of inlet concentration when "building up" from zero Approxi-
mately five times 43 minutes (3-1/2 hours) are needed to reach equilibrium
at the inlet concentration, decay tune is also 3-1/2 hours
Air Supply
The air purifier unit provides, at maximum, 550 cfm of CBR (chemical,
biological, radiological) filtered and conditioned air Inside building air
is passed through a cooling coil to lower the temperature to 40°F (saturated
at coil outlet), there is no reheating or humidification. Therefore, if the
relative humidity of the outside air drops below 36 grains of moisture per
pound of dry air, the relative humidity in the final exposure chamber will
also vary Most of the tune, there is no problem maintaining constant
relative humidity, but occasionally on very dry days, there is a change
-------
66
The humidifiei is turned off, because of the constant reed for cool
dry air to mix with hot wet raw exhaust. Exposure chambers on control air
are supplied from a separate CBR filtered source, with controls set to
maintain 72° + 2°F and 55 + 5% relative humidity in the animal chambers.
The same air is also ducted to the air filter inlet of the engine being
used for the study smce a change in humidity effects the NC^ emissions
from the engine
Engine Cycle
The dynamometer driving schedule for the Chevrolet engines consists
of a repetitive series of idle, acceleration, cruise, and deceleration
modes of fixed tune1 sequences and rates The following Table I and
Figure 1 is the modified "California Cycle" used m the fuel emission studies.
Table I
Mode
Idle
Accelerat lor
Cruise
Decelerat-ion
Cruise
Acceleration
Peak
Decelerat-ion
Speed, M P H
0
0 to 30
30
30 to 15
15
15 to 49
49 to 50
50 to 0
Tame, seconds
20
14
15
11
15
29
1.5
31.5
Total 137 sec.
-------
67
TIME (SECONDS)
FIGURE 1 CYCLE SPEED TRACE
(137 SECONDS)
-------
68
Replacement oi the California Cycle with the IA-4 cycle controller
was considered at one time. However, after consultation with other
experts in the field, it was decided to continue with the California Cycle
because the exhaust is being further diluted to prescribed levels and both
cycles are very similar sauce they reflect transient as well as cruise
operation. The key to this research is comparative toxicity either
cycle is satisfactory to achieve this goal A simple repetitive cycle
that is easily cont_rolled over long periods of time (weeks) is of prime
importance to toxicologic investigations
Fuel Selection
The gasoline selected for use in the Chevrolet engines as a standard
reference, baseline* fuel for evaluation of engine, fuel and additive
variables was American Oil Co. Unleaded 91 Octane Test Fuel, Intermediate
Grade Indolene Clear As a reference it was important that it be of quite
precise and reproducible composition and character, including absence
of lead and other additives (except as specifically noted), and similar to
high-volume regular market gasoline This gasoline has been used for such
purposes m research and development by industry and other agencies The
lubrication oil se Lected was Texaco Havoline 30W, API service specification
SE. Table II shown below, represents a comparison and product analysis of
the two gasoline deliveries used for exhaust emission studies during 1973.
-------
69
Table I. Fuel Properties
Shipment No.
Date Delivered
Quantity, gallons
Octane No., research
Octane No., motor
Lead Atm. Abs., gir/gal.
Phosphorus, gm/gal.
Sulfur, wt. %
Aromatics, Vol %
Olefins, Vol. %
Gum, Existent, mg/100 cc
Gravity/ °API
Oxidation Stability, minutes
Ried Vapor Pressure, Ibs
#1
3/30/73
2,000
91.4
82.9
0.01
0.002
0 04
25.4
11.8
0.8
61.4
600+
9.1
#2
10/29/73
1,500
91.3
82.5
0.01
0.00
0.05
23.5
9.9
1.0
61.5
600+
9.0
Note. Shipment #1 used for studies G, H, I and J. Shipment #2
used for study K with Thiophene added to produce 0.10%
by weight sulfur
Fuel Storage and Handling
Local fire and safety regulations require flammable liquids to be
stored outside the building, so two underground fuel storage tanks were
installed on the property near a blacktop surface driveway. To promote
chemical stability of the fuel during storage, the tanks are maintained
under slight positive pressure with nitrogen supplied from cylinders and
-------
70
controlled by a pressure regulator. A double acting pressure and vacuum
relief valve on the vent outlet compensates for changes due to fuel being
pumped out or temperature increase, which would alter the pressure of the
nitrogen gas cover Each tank is of 2000 gallon capacity and equipped with
an electric fuel pvitp rated at 15 gallons per minute
Outside the bvalding wall and next to the engine room is an 18-gallon
marine fuel tank setting on a weight scale and connected to a remote electric
fuel gauge located in the instrument panel. Transportation of the test fuel
from the main underground storage supply to the one-day supply tank is
effected by a mobile safety dispenser cart made especially for transporting
flammable liquids The 60-gallon-capacity cart carries the Underwriters
Laboratories' approval as a portable flammable liquid tank and is equipped
with transfer pump and grounding reel. The cart also has a drain, and
removing the pump gives access to a 4" handhole for reaching and cleaning
the tank interior between fuel changes. Similarly, the 18-gallon marine
tank can easily be inverted for cleaning when required.
An alteration in the composition of the reference fuel for a study is
made by the addition of the required amount of chemical to a full cart
batch. Thus studies requiring the testing of fuel additives such as
methylcyclopentadienyl manganese tricarbonyl (MMT) or thiophene to increase
the sulfur content can be conducted by mixing only the amount of fuel necessary.
References
1. Hinners, R. G Laboratory Produced Automobile Exhaust Facility.
Biomed. Sci. Instrum. _! 53, 1963
2 Hinners, R G , Burkart, J K and Centner, G L Arimal Exposure
Chambers in Air Pollution Studies. Arch Environ Health. 13_ 609-615,
(Nov ) 1966.
-------
\
1
71
B.2. CATALYST EXHAUST EXPOSURE STUDIES
R G. Hinners and J.K Burkart
During 1973, animal exposure studies were conducted in ETRL, NERC-
CincinnatL, to assess the relative health hazard of automobile exhaust
emitted from engines equipped with and without catalytic converters using
similar engine settings. Automotive exhaust catalysts were developed to
lower exhaust emissions of carbon monoxide, hydrocarbons by oxidation, and
oxides of nitrogen by reduction. They are the three pollutants specifically
listed in the Federal Clean Air Act of 1970. The regulations also require
that no pollution control device shall emit "noxious or toxic" substances
Three possible conditions could result m such emissions 1) As the hot
catalysts promote the oxidation of carbon monoxide and hydrocarbons in
automotive exhaust, converting them to carbon dioxide and water, it may
simultaneously convert the organic sulfur compounds present in all gasoline
into sulfuric acid mist and eventually sulfates 2) The metals used in
the converter, such as platinum and palladium, may be emitted under conditions
of catalyst degradation from the exhaust pipe in fine particles and be
suspended in the air. 3) The total emissions may be altered and may produce
different quantities or new species.
In order to perform the assigned tasks, this laboratory recently
acquired and installed two new engines equipped with catalytic converters
from General Motors Company* and the Ford Motor Company * (See Table 1)
-------
72
Table 1
FORD - 400 C.I D (1975 Prototype) R-6 Engine with R-14 Calibration and
the Following Controls
(1) EGF (exhaust gas recirculation)
(2) Air Puitp
(3) Fli,.idic spark delay valve
(4) Various tenperature sensing triggers
(5) Catalytic converter of monolith, noble metal
oxadation type. IWo converters of thjs type
are required, one for each bank of cylinders.
(Cc'talyst by Matthey-Bishop Co )
GENERAL MOTORS - 350 C I.D. (1973 Production Engine)
(1) EGR
(2) Art Purp
(3) Caialytic converter, pelletized type, noble metal
ox.dation catalyst. One converter after Y pipe
(Catalyst by Engelhard Co.)
-------
73
The schematic plan view of the engine-dynamometer unit and dilution tube
is presented in Figure 1.
During TAME studies H, I, J and K, the 1973 Chevrolet engine was
operated continuously for 7 days using the California Cycle Comprehensive
data for comparison of study engine operating conditions is given m Table 2
TAME 'K' was designed to test emissions and bioeffects of a high-sulfur
gasoline free of other undesirable substances such as lead. Thiophene was
added to produce a sulfur content of 1000 ppm m the control fuel, Indolene
Sulfur compounds present in gasoline are mainly in the form of polysulfides
and thiophene compounds with an insignificant amount of hydrogen sulfide
It was reported and confirmed by our testing procedures that the addition
of oxidation catalysts to the automotive exhaust system causes an increase in
the emitted particulate material (consisting mainly of hydrated sulfuric
acid droplets) as a result of the oxidation of organic sulfur compounds in
gasoline. Recent national averages of the sulfur content are between 210
and 260 ppm for premium gasoline, and between 390 and 440 ppm for regular
gasoline The "Indolene" motor fuel used at the ETRL facility had a sulfur
content 0.04% by weight or 400 ppm.
-------
DOUBLE ENGINE-DYNAMOMETER UNIT
(PLAN VIEW)
-a R
1 Vibration Isolating Stand
2 1973 Chev V-8 350 C I D
3 Turbohyd romatic Transmission
4 Dynamometer Absorption Unit
5 Flywheel
6 Catalytic Converter
7 Muffler
8 Dilution Tube
9 Baffle Plate
10 Dilution Air Supply
11 1975 Ford V-8 400 C I D
12 Catalytic Converters
FIGURE 1
-------
TAME H
TAME I
TAME J
TAME K
Dates
Fuel
Engine
Engine Hrs.
Study Hrs.
Eng. Miles
Cuirm Catalyst
Hrs.
Catalyst Miles
Total Fuel
(Ibs)
Fuel, tb/Hr.
Exh. Oxygen (%)
Air/Fuel Ratio
Oil Consunption, qts.
Dilution Ratio
Dilution Air Flow
Average, SCFM
Dilution Tube Temp.
Average, °F
9/10 - 17
Ref . Only
' 73 Chev.
w/catalyst
62-230
168
4600
244
4880
1533
9.10
4.9
—
1-1/8
8.0/1
318
106
10/10 - 17
Ref. Only
'73chev.
No catalyst
255-425
170
8500
244
4880
1545
9.08
N.A
14 4 cycling
12 4 idle
1/2
9.6/1
305
101
10/24 - 31
Ref. Only
'73Chev.
w/catalyst
444-615
171
12,300
465
9300
1601
9.40
4.2
—
1/4
8.7/1
310
114
11/14 - 21
Ref. + Sulfur
1 7 3 Chev.
w/catalyst
675-841
166
16,820
632
12,640
1495
9.02
4.7
—
1/4
9.5/1
324
101
Ul
Table 2. Comparison of Study Engine Operating Conditions
-------
76
B.3. DESIGN AND SYSTEM PERFORMANCE FOR STUDIES OF CATALYTIC EMISSIONS
J. Burkart and R Hinners
All of the catalyst studies m this report were performed with the 1973
Chevrolet engine, new headpipe, standard muffler and fabricated stainless
steel tailpipe New road load data supplied by the EPA Motor Vehicle
Emissions Lab were used, they are equivalent to an increase of inertial
weight from 3400 Ibs (used on 1972 Chev) to 4000 Ibs. No attempt was
made to adjust idle mixture, as in earlier TAME A through G studies and
carburetor "limiters" remained in place. In TAME "H1 the engine was run
"as received" except for setting idle speed, dwell and timing. Maintenance
performed before T'AME I, J and K consisted of oil and filter change, new
points, condenser, spark plugs and setting hot idle speed, dwell and
tuning In addition, before TAME 'K' new spark plug wires were installed
For each of t±e continuous one week studies shown in Table 2 of the
article B-2, approximately 3400 miles were accumulated on the California cycle
Separate cumulative ergine miles and catalyst miles are reported since
the catalyst was removed in TAME 'I1 and additional steady speed runs
(without animal exposures) were made to characterize emissions The dilution
ratio is determined by the ratio of average tailpipe C02 "to dilute CO^
Because the variability of tailpipe C02 throughout the cycle is small,
the problem of obtaining a proportional sample is negligible. Samples for
CO- detection flow at a constant 1 liter per minute thru a refrigerated
cooler, dessicant dryer and paper filter to the Beckman 1R Model 315. This
instrument, converted for OX^, is calibrated 15% CO- full scale and zeroed
-------
77
on dilution air, however, because of the cooler some C02 loss ln
condensate was unavoidable
For all studies a continuous trace at constant sample flow on two-
Mosley (2 pen) recorders was made of the following emissions
(1) tailpipe CO
(2) dilute CO
(3) tailpipe THC
(4) tailpipe C02
The above, along with "spot" checks of tailpipe oxygen and dilute C02,
monitored engine and dilution system operation.
TAME schematic Figure 1 shows sampling points throughout the system
starting with engine (E), catalytic converter (C) and standard muffler (M).
The numbers will be referred to for aerometry sample identification except
when exposure chambers are sampled, the chamber number and treatment (I, NI,
clean air) is used Average total particulate losses on a percentage basis-
are also shown starting with 100% at point 5 in the dilution tube An
overall loss of 39% occurs by the tune the NI chamber is reached. The
diagram shows only part of the exposure chambers receiving autoexhaust and
there are control chambers which receive filtered air from a separate supply
During the studies, the entire tailpipe volume was mixed with the
quantities of air given in Figure 2 and resulted in the dilution tube
temperatures shown The dilution air temperature for all studies ranged
from 48° to 55°F. Figure 3 depicts tailpipe conditions of exhaust oxygen
content and average catalyst temperature (on center line of tailpipe one-
inch from catalyst oftlet) At the tailpipe, the seven day trend during
the catalyst studies (H, J, K) was oxygen decrease, C02 increase and catalyst
temperature increase.
-------
78
Table 1 shows the GM catalyst efficiency when TAME 'I1 (w/o catalyst)
average emissions are used as the basis for comparison It is noted that
the catalyst is mere efficient in terms of CO than for HC under the hot
cycling condition.
Some initial loss of efficiency may be due to the higher oil consunption
during TAME 'H1, also by the end of that study #3 plug had "fouled."
Table 1 GM CATALYTIC CONVERTER EFFICIENCY
Average Tailpipe Concentrations* (PPM)
S. I £ £
Carbon Monoxide 56 5376 400 380
Hydrocarbons (as methane) 96 1056 191 171
Total Per cent Reduction Below TAME I
Carbon Monoxide 99% X 93% 93%
Hydrocarbons 91% X 82% 84%
*Calculated from dilute concentrations multiplied by
dilution ratio.
-------
79
Basic specifications for the 1973 Chevrolet engine are shown below
in Table 2.
Table 2 Chevrolet Engine System
1973
Displacement 350 C.I.D.
Compression Ratio 8.5/1
Carburetor Type Roch. 2GV 1-1/2
Carburetor No. #7043114
Distributor* # 1112168
Mech. Adv. Unit C 4815
Vacuum Adv. Unit C6020(46914)
Dwell 30°
Initial Timing 8oBTC
Maximum Vacuum Advance 14°
Emission Control Equipment Air Pump
Rich tune (A/F M.4.5/1)
Tuned port Vac. Adv.
EGR 11633 (IF 7040437)
Data for exposure chamber temperature and relative humidity are presented
in Figure 4 The temperature profiles appear favorable, however, the
relative humidity for chambers receivug exhaust were consistently above
60 per cent relative humidity while the reverse was true for control air
chambers
-------
/ /
VENT
®76
DILUTION AIR
IRRADIATION CHAMBERS
.
--O
V,
EXPOSURE CHAMBERS
CO
o
MIXING CHAMBER DILUTION TUBE
FIGURE 1. TAME SCHEMATIC SHOWING SAMPLING POINTS -.-O
-------
330
E
%*•
V
320--
Oi
<
z
o
310--
300
DR=8/1
012345678
TAME H
(CATALYST)
u. 120°
o
O.
LU
I—
UJ HO°J
Z
O
100°--
Q
O
< 90°
DR=9 6/1
DR=8 7/1
TAME I
(NO CATALYST]
TAME J
(CATALYST)
DR=9 5/1
TAME K
(CATALYST W/
HIGH SULFUR FUEL)
012345678 012345678 012345678 012345678
DAYS DAYS DAYS DAYS
FIGURE 2-DILUTION AIR FLOW AND DILUTION TUBE TEMPERATURE
oo
-------
O
d.
UJ
»—
I—
>-
—i
<
t—
(J
-O
950<
900(
f\ ^ XV f\
oou •
800'
CATALYST
/
z
UJ
O
x
O
UJ
Q_
O
>
<
12345678
DAYS
TAME H
12345678
DAYS
CATALYST
TAME J
DMA
4
3
2
1
0
CATALYST
WITH
HIGH
SULFUR
FUEL
TAME K
CO
to
FIGURE 3 TAILPIPE CONDITIONS
-------
83
70% 80°FH
60% 75°F-
50% 70°F
IRRADIATED EXHAUST
i ( I i i I 1
1234567
i i i i i i i
1234567
ji r^ i i i i
1234567
570% 80°F-]
NON IRRADIATED EXHAUST
1U
360% 75°F-
0£
efi
u.
o
UJ
ce
50% 70°F
i i i r i i i
1234567
I I I T I I I
1234567
I I I I I I I
1 234567
70% 80°F_|
CLEAN CONTROL AIR
UJ
60% 75°F-
50% 70°F
1234567 1234567 1234567
DAYS DAYS DAYS
TAME I TAME J TAME K
• • SOLID LINE TEMPERATURE
*--•• DASH LINE RELATIVE HUMIDITY
FIGURE 4 EXPOSURE CHAMBER TEMPERATURE
& RELATIVE HUMIDITY
-------
84
B.4. EXHAUST EMISSIONS DURING STEADY SPEED RUNS WITH THE
CATALYTIC CONVERTER IN THE EXHAUST SYSTEM
M. Malanchuk, N Berkley, G Centner,
M. Richards and R Slater
Introduction
In preparation for studies on the exposure of animals to the exhaust
emissions from catalytic-equipped systems, preliminary runs were made with
the 350 C I.D. Che\Tolet engine operating at constant speeds. Information
was sought that would indicate the levels of constitutents different from
those of previous i~uns made under different engine operating conditions
The data were needed particularly with reference to sulfur compounds and
acidity of the emissions Preparations were made to test for sulfates,
sulfur dioxide, suJfuric acid and nitrate components Sample procedures
were adapted with £>uch changes as were considered expedient to get
quantitative result.
Experimental Procedure
The main effort was directed to the sampling of particulate in order to
establish the natuie of the anticipated changed character of the particulate.
Since membranes-type filters used to collect aerosol from the catalytic
converter system deteriorated from the corrosive action of the sample, quartz
fiber material was used and found favorable for such samplings. Not only
did the material resist breakdown, but also in the aqueous extraction medium
it did not show any resulting changes in the nature of the solution (e.g pH)
upon standing for as long as 20-30 hours.
The sample fiLters were handled m two different ways Every filter was
weighed immediately after sampling Some were permitted to stand overnight.to
equilibrate in the room atmosphere (70-75°F, 40-60% R.H.) until the weight
-------
85
had stabilized. These final weights were used to calculate the particulate
concentrations in the sairpled atmospheres Other filters, immediately after
they were weighed following the sampling, were placed in a measured volume of
distilled, deionized water. Conductance and pH measurements were then
made to determine ion concentrations mainly, the acidity of dissolved
samples. The aqueous extracts were also used for analysis of particular
ion radicals like the sulfate and nitrate groups
Analytical procedures included the barium chloranilate method^ ' and
(2)
also nephelometry for sulfate, the phenol-hypochlorite reaction and
ion-specific electrode for amroma and ammonium compounds, and the
(3)
hydrazine reaction for nitrate
Bubblers containing distilled water or a weak acid solution were used
to scrub sampled atmospheres for nitrate - and for ammonium-producing
components. A sampling train of bubblers similar to that used in stack
sampling'^ was arranged for separation of 862 from SCU m atmospheres
drawn from the exhaust pipe before and after the catalytic converter and
from the arimal exposure chambers. The first bubbler in the train, con-
taining isopropanol, collected 803 The succeeding two bubblers, containing
hydrogen peroxide, collected the SC>2 ard converted it to the sulfate form.
In some cases, the follow-up bubblers contained tetrachlormercurate instead
(4)
of peroxide to trap the SO- for analysis by the West-Gaeke method
None of the animal exposure chambers from which the atmosphere was
sampled contained any animals. These chambers previously were hosed down
thoroughly with hot water to minimize, if not eliminate, sources of contaminating
deposits.
-------
86
Results
The effects of different engine speeds and of different concentrations
of sulfur in the fuel are seen in the concentratior values of exhaust
emission conponents in Table I.
Values of gaseous conponents are listed first - carbon monoxide (CO),
total hydrocarbons, as methane (THC), nitrogen oxides (NC^) with a break-
down into nitric oxide (NO) and nitrogen dioxide (IC^)/ the aliphatic
hydrocarbons of the 04-^5 grofp examined, olefins of the C2-C^ group, and
acetylene Values of particulate material are listed as total particulate
and the sulfate and nitrate concentrations in that particulate
The first three column groups of concentration values were obtained
from the operation of the engine with the base fuel, Indolene gasoline, and
the use of the catalytic converter unit in the immediate exhaust system. The
last two column groups show the concentrations obtained when the Indolene
gas was "spiked" with an organic sulfur compound to double the concentration
of sulfur in the fvel, in one case, the catalytic converter unit vras retained
in the exhaust system, in the second case, the unit was removed before the
run was started
The samples were mainly collected from the animal exposure chambers
receiving the diluted exhaust emissions that had been exposed to the irradi-
ation lights, I, and those chambers receiving diluted emissions not treated
to the irradiation effects, N-I. Some samples were collected from the
exhaust system in -the immediate range of the engine, viz , the particulate
samples identified as diluted exhaust (Dil'd Exh )
-------
87
In order to relate the values from the higher engine speeds and the
runs with higher gasoline sulfur to the "base" 15 irph with its 7 5/1 dilution,
the actual values of those runs have been adjusted to equivalent values
for a 7.5/1 dilution which are given m brackets. Thus, for total
particulate weight in the diluted exhaust, it is seen that the significantly
different values of 6 10 irg/M3 at 30 mph and of 8.40 mg/ta3 at 50 nph
become nearly the same value as the 5 03 mg/M3 at 15 nph when the dilution
adjustments are made for the 6 2/1 and 4.9/1 at 30 itph and 50 mph, respectively.
Since the data applies to only single runs at each of the five different
sets of conditions (except the "base" 15 nph with regular Indolene fuel,
there were two runs for which the average values were calculated), the
information must be considered as tentative Duplicate runs will be made
to establish reproducibility and to confirm the results presented
Nevertheless, large differences in the results between runs were
seen which may be acceptable for what they indicate. The large increase in
the nitrogen oxides at the highest speed of 50 mph was to be expected (see
columns 1-3 of Table 1). There was a concomitant decrease in hydrocarbons.
Particulate levels remained about the same with the changes in engine speed
Apparently particulate sulfate concentrations also renamed the same at
different speeds, however, they seem to make up most of the total bulk of
the particulate Nitrate was present at much loser concentrations.
-------
_»_ i,
88
With the high s.ulfur (2 x) Indolene gas as fuel, the carbon monoxide
and hydrocarbons (total and individual) levels remained the same (column 4).
The big difference was seen in the total particulate and sulfate contents,
about 1-1/2 - 2 times as much.
The engine operating with the high-sulfur fuel but without the
catalytic converter had, as expected, much higher levels of carbon monoxide
and hydrocarbons The nitrogen oxide levels were at similar concentrations
as those for the 15 mph with regular Indolene fuel. The particulate levels,
column 5, were only 1/3 to 1/4 of those for the catalytic converter, column 4.
Sulfate in the partxculate was almost negligible by comparison.
Measurements of acidity of the particulate from the catalytic-equipped
exhaust emissions was, without exception, high enough to allow for all the
sulfate to be consi'3ered present as sulfuric acid That is to say, that the
hydrogen ion concentration as (2H+) that was measured with a pH meter v/as
greater than the sulfate (SO^-) concentration, sometunres by a factor as high
as three times. Such high acidity was found unaccountable on the basis of
the anionic components measured and of the total particulate determined, so
that additional study is needed to resolve that phenomenon. An indication of
relative acidity levels with different operating conditions is shown in Table 2.
The particulate, column 5 of Table 1, in the no converter condition, on the
other hand, was almost completely neutral as measured by pH meter on its
\
aqueous extract.
Measures of sulfur components, SO , in the undiluted exhaust before and
J*L
after the catalytic converter showed 50-90 per cent decrease of sulfur across
the converter That is to say, that there is a considerable hold-up of
sulfate in the catalyst bed itself
-------
TABLE 1. COMPARISON OF EXHAUST EMISSIONS, STEADY SPEED RUNS
Exhaust Dilution Ratio
CO,ppm*
Exp. Ch N-l
THC,ppm
Exp. Ch: N-l
ppm
Exp. Ch: N-l
NO, ppm
N0,
Exp. Ch: N-)
Exp. Ch: N-I
Al Iphatlcs ppm
C4-C5 Exp. Ch: N-l
Olefins ppm
C2-Ci, Exp. Ch: N-l
Acetylene ppm
Exp. Ch: N-l
I
Particulate, mg/M^ -
- Dll'd Exh
Exp Ch N-l
I
Sulfate, mg/M3-Dll'd Exh.
Exp. Ch N-l
I
Nitrate, mg/m3-Dll'd Exh,
Exp. Ch N-l
I
Regular Indolene Gasoline
With Catalytic Converter
High-Sulfur Indolene
w/catalytic w/o catalytic
15 mph
7.5/1
7
7
9
8
20.0
19.4
14.7
13.2
5.3
6.2
0 107
0 108
0 450
0.426
0.025
0.024
5.03
2.24
1.98
4.33
4.80
3.01
0.32
0.01
0.01
30 mph
6 2/1
8
8
9
9
28.4
31.8
21.2
21.8
7 2
10.0
O.I 13
0.113
0.465
0.456
0.018
0 020
6 10
5.00
4 72
6 18
5 00
3.94
50 mph
(7.5/1)
(7)
(7)
(8)
(8)
(23.5)
(26.3)
(17 5)
(18 0)
(6 0)
(8.3)
(0 093)
(0.093)
(0.385)
(0 377)
(0 015)
(0 017)
(5 04)
(4.14)
(3 90)
(5 II)
(4 14)
(3.26)
!:::!
4.9/1
10
10
4
4
102.0
104.8
71.9
63.6
30.1
41.2
0.031
0.026
0.204
0.195
0.012
8.40
6.25
5 71
6.93
4.90
2 72
0.47
0.00
0.31
1(7 5/1)
•*
(7)
(7)
(3)
(3)
(66.6)
(68.5)
(46.9)
(41.6)
(19.7)
(26.9)
(0 020)
(0.017)
(0.133)
(0 127)
(0.008)
(5.49)
(4.08)
(3.73)
(4.53)
(3.20)
(1.78)
(0.31)
(0.00)
(0.20)
15 mph
8.1/1(7.5/1)
7 (8)
7 (8)
8 (9)
8 (9)
0.109(0 1 18)
0.115(0. 124)
0.433(0.467)
0.447(0.483)
0.018(0.020)
0.028(0 030)
6.46 (6.97)
7.63 (8.24)
5.79 (6 25)
13.16 (14.20)
11.86 (12 80)
10.65 (1 1.50)
0.36 (0.39)
0.01 (0.01)
0.01 (0.01)
15 mph
8/0/1(7.5/1)
491 (522)
93 (99)
21.1 (22.5)
20.3 (21.6)
13.3 (14.2)
9.6 (10.2)
7.8 (8.3 )
10.7 (11.4) oo
VD
1.92 (2.05)
2 50 (2.66)
2 33 (2 48)
0 54 (0 58)
0 49 (0 52)
0 49 (0.52,
0 28 (0.30)
0.04 (0.04)
0.17 (0.18)
-------
- i
90
References
1. Kufta, R. J "Stationary Source Testing." Apollo Chemical Corp.
September 1, 19"'2
2. Weatherburn, M W. "Phenol-Hypochlorite Reaction for Determination
of Armenia" Anal. Chem. 39_ 971, July, 1967
3. Hauser, T.R "tiethod for Analysis for Nitrate by Hydrazine Peduction"
Water Research _L_ 1816, 1956
4. West, P.W and Gaeke, G.C. "Fixation of Sulfur Dioxide as Disulfito-
mercurate (11), Subsequent Colorametric Estimation" Anal. Chem. 28.
1816, 1956
-------
91
B.5. EXHAUST EMISSIONS FROM CATALYST-EQUIPPED ENGINES
M Malanchuk, N. Barkley, G. Centner, M Richards,
R. Slater, J. Burkart and Y Yang
Introduction
Some early studies in the automobile industry have indicated that
oxidation-type catalysts in auto exhaust systems generated high levels of
sulfuric acid aerosol, as much as 0.1 gram of the acid per vehicle mile
It was hypothesized that the engine combustion process converted organic
sulfur compounds in the gasoline into sulfur dioxide, and that the dioxide
was oxidized by the catalyst to sulfur trioxide which reacted with water
vapor in the exhaust to produce sulfuric acid droplets.
Therefore, unlike the constant engine speed runs as descirbed in the
previous article, cycling speed runs more nearly simulating automobile
operation in the streets were used for the animal exposure studies
Measurements of exhaust emission components were made to determine the
levels of such toxic components to which the animals in the studies were
exposed The effective changes in exhaust composition were determined
when the catalytic converter unit was added to the exhaust system, and when
high-sulfur fuel was substituted for the reference Indolene gasoline
Emission components present in relatively high concentrations were
monitored in much the same way as in previous runs
Sampling and Analytical Procedures
The instrumentation and methods used for key components are summarized
in Table I Atomic absorption spectrophotometry was used for trace metal
determinations in particulate.
-------
92
Table I. AJZROMETRIC CHARACTERIZATION OF EXHAUST EMISSIONS
Pollutant compone-vt Analytic method Automatic Manual Where determined*
Carbon monoxide (CO)
Total hydrocarbons
(THC) , as CH4
Nitrogen oxides
includes NO ard N02)
QI to 05 hydrocarbons
(several compounds)
Cg to CIQ aromatic
hydrocarbons (several
compounds)
Aldehydes, total
Particulates, total
mass
Particulate size
distribution
Aerodynamic
Photonomeric
Particulate compo-
sition
Ozone, "oxidant"
Nondispersive X
Infra-red spec-
troscopy
Flame lonization X
spectroscopy
Chemiluminescence X X
spec , coloruretry
using Saltzman
reagent
Gas chromatography X
Gas chroroatography X
MBTH according to X
Hauser
Filtration gravimetry X
Stage impaction X
(Anderson)
Photoelectronic X
(Royco)
Infra-red and ultra-
violet spectrophotometry X
Chemiluminescence spec.
EPM, EC
EPM, EC
EPM, EC
EC
EC
EC
EC
EC
EC
EC
*EPM - Exhaust or priirary exhaust air mixture, EC - exposure chamber
-------
93
Particulate saitples were collected on pure quartz fiber filters
after early rneirbrane filters deteriorated from exposure to the high
reactivity of the collected sample of catalyst-treated emissions.
Bubbler and unpmger samples of the atmospheres were used for
collecting ammonia- and sulfur-based gases.
Results
Table II lists the concentrations of various engine exhaust components
measured during the series of studies of the catalytic converter system
Individual hydrocarbons measured by gas chromatography are shown in
Table III, the aromatic compounds were not measured after it was discovered
that those concentrations were so low in the catalyst-equipped system
atmosphere as to be near or below detection level.
TAME J and TAME H (see Hinners report) were run under the same
engine operating conditions, i e they were "duplicate" runs However,
reference to the data of Table I shows considerable differences in values
between the two runs. When TAME H was performed, the engine was probably
not fully broken in and the catalyst was quite new, the piping system for
conducting the emissions to the exposure chambers probably had not as yet
attained equilibrium conditions of surface exposure characteristics (i e.
mainly deposition of particulate and adsorption of organic vapors) for the
new engine system. TAME J, run at a later date, when a more stable system
should have been established, was considered to have the more accurate
atmospheric component values than TAME H. Therefore,the concentration values
from TAME J were used for comparison to those from TAME I for the purpose of
evaluating the effect of the catalytic converter upon the make-up of the
-------
94
auto exhaust emissions reaching the animal exposure chambers. That comparison
is emphasized by the large percentage reduction values of several atmospheric
components due to the use of the Pt-Pd coated, pelleted catalyst converter
(Table IV), and also seen m the greatly reduced concentrations of individual
hydrocarbons, TABLE III
Since the dilution of the raw exhaust with clean air was not as great
in TAME J (8.7/1 as in TAME I (9.6/1), the reduction values listed in the
third column of Table IV were adjusted by a factor appropriate to the
differences in dilution values, about ten per cent of the TAME J values,
to obtain the more accurate "normalized" values listed in the fourth column
A barely detectible concentration of platinum, 0.029 yg/m3, was
measured in the diluted emissions of the animal exposure chamber
A barely de-cectible concentration of platinum, 0 029 yg/rrH, was
measured in the diluted emissions of the animal exposure chamber - this
result, of course, for a system using a catalytic converter unit that was
quite new and that was shown to be adsorbing a large proportion of the
sulfur gases in the exhaust gases
On the basis of an average flow of 1 W^/mm of raw exhaust produced
at a calculated average speed of 22 mph on the engine dynamometer, it was
estimated that the 0 029 yg Pt/m3 represented a loss of nearly 0 62 yg/Pt/mi
If it is estimated that there is 0 04 troy ounce of the noble metal in
the catalytic unit (1 244 gm, i e ) then 0.5 x 10~4 per cent of the
platinum was lost per mile Such a loss over 50,000 miles of operation
would mean a total loss of 2 5 per cent of the platinum originally present.
-------
.. 1.
95
Conclusions
The incorporation of the oxidation-type catalyst m the exhaust system
resulted in drastic changes in the exhaust emissions
a. The effectiveness of the catalyst was revealed in the large
reduction of carbon monoxide, total hydrocarbons and various individual
organic compounds (such as acetylene)
b. An almost total elimination of aldehydes was achieved.
c. In TAME I (without catalyst), the high value of particulate in the
irradiated atmosphere along with the low value of nitric oxide, NO, and
the measurpd presence of ozone indicated that much more photochemical
reaction of hydrocarbons occurred than in TAME J (with catalyst) That
activity was greater m the case of the olefins than in the acetylene, and
negligible for the aliphatics.
d. Gross evidence (color, weight stability) of the particulate in
TAME I, indicated that the nature of the sample was mainly organic The
particulate in TAME J, on the other hand, was strongly acidic, liquid in
nature and lost significant weight upon standing Analysis showed sulfate
to be the primary constituent Such facts suggested the presence of sulfuric
acid as the major component in TAME J particulate.
More detailed reports of sulfate and acid measurements are given in the
related articles, "Sulfate Emissions from Use of High-Sulfur Fuel, TAME-K"
and "Exhaust Emissions During Steady Speed Runs with the Catalytic Converter
in the Exhaust System."
-------
96
TABLE II.
ENGINE EXHAUST EMISSION VALUES FOR CATALYTIC CONVERTER SYSTEM STUDY
Exhaust Dilution Ratio
CO, ppm.
Exp. Ch
THC, ppm
Exp. Ch.
NOV, ppm.
x Exp. Ch.
NO, ppm. Exp. Ch.
NO,, ppm Exp Ch'
£
Aldehydes ppm
Exp. Ch
Methane, ppm
Exp. Ch
AHphatics
ppm C,-C5
Exp. Ch
Oleflns
ppm C2-C4
Exp. Ch.
Acetylene, Exp. Ch
Ozone, ppm
Exp. Ch
3
Particulate, mg/M
011 'd. Exh.
Exp. Ch
N-I
I
N-I
I
N-I
I
N-I
I
N-I
I
N-I
I
*
N-I
I
N-I
I
N-I
I
N-I
1
N-I
T
X
N-I
I
TAME-H
10-16 Sept.
'73
8/1
7
8
12
13
11.0
11.0
8 5
8.0
2.5
3.0
.....
.....
.....
1.86
2.05
TAME-I
10-16 Oct.
'73
9.6/1
651
559
110
95
11.9
5.1
6.7
C0.5
5.2
4.6
10.20
14.62
1.30
1.32
13.24
9.23
3.28
3.06
0.0
OA
«•»
1.08
0.69
3.19
TAHE-J
24-30 Oct.
'73
8.7/1
46
41
22
22
"12.9
12.6
11.1
9.6
1.8
3.0
0.08
0.10
0.61
0.58
0.89
0.79
0.03
0.03
1.02
0.96
1.09
TAME-K
14-21 Nov.
'73
9.5/1
40
38
18
18
12.6
11.2
10.8
9.7
1.8
1.5
0.18
0.11
6.53
6.13
0.44
* 0.39
0.91
0.82
0 04
0.04
5.97
6 53
5.85
-------
97
Table III.
Gas Chroratographic Measurements of Hydrocarbons,ppm
Component
n-Butane
i-Butane
n-Pentane
i-Pentane
Acetylene
Ethylene
Propylene
Butane-1
Isobutylene
1,3-Butadiene
Methane
TAME-I
N-I
0
0
0
0
3
6
1
0
0
0
.61
.08
.20
.41
.28
.85
.81
26
.63
.41
0
0
0
0
3
5
0
0
0
0
I
.61
.08
.23
.40
.06
.10
.71
.08
.20
.08
TAME-J
N-I I
0
0
0
0
0
0
0
.30
.05
.09
.17
.03
.82
.04
Bid
Bid
Bid
0.29
0.05
0 09
0.15
0.03
0.72
0.04
Bid
Bid
Bid
TAME-K
N-I I
0
0
0
0
0
0
0
6
.21
.03
.05
.15
.04
.81
06
-
-
-
53
0.
0.
0.
0.
0.
0.
0.
-
-
-
6.
18
03
05
13
04
74
04
13
Bid - Below level of detection
-------
98
TABLE IV.
COMPARISON OF EXHAUST EMISSIONS. TAME-I AND -J
Exhaust Dilution
CO, ppm
Exp. Ch
THC,PPM
Exp. Ch
NO.ppm _
x Exp. Ch
NO, ppm Exp. Ch
N09,ppm Exp. Ch
C
Aldehydes, ppm
Exp. Ch
Aliphatlcs, ppm'
C4-C,
H ° Exp. Ch
Olefins, ppm
Exp. Ch
Acetylene, ppm
Exp. Ch
Ozone, ppm
Exp. Ch
Parti cul ate, mg/M
DiTd. Exh.
Exp Ch
Ratio
: N-I
I
N-I
I
N-I
I
N*I
I
N-I
I
N-I
I
N-I
I
N-I
I
N-I
I
N-I
I
3
N-I
I
TAME-I
10-16 Oct.
'73
9.6/1
t
551
559
110
95
11.9
5.1
6 7
0.5
5 2
4.6
10.20
14 62
1.30
1.32
13 24
9.23
3.28
3.06
' 0.0
0.4
1 08
Q.69
3.19
TAME-J
24-30 Oct.
'73
8.7/1
46
41
22
22
12.9
12.6
11.1
9.6
1.8
3.0
0 08
0 10
0.61
0.58
0 89
0 79
0 03
0.03
1 02
0.96
1 09
% Conc'n
Reduction
I-vJ
91.7
92.7
80.0
76.9
99.9
99.9
53.1
56.1
93.3
91.4
99.1
99.0
Normalized
% Reduction
Value
92.4
93.3
81.9
79.0
99.9
99 9
57.7
60.0
93 9
92.2
99 2
99 1
-------
99
B.6. SULFATE EMISSIONS FFOM USE OF HIGH-SULFUR FUEL, TAME-K
M. Malanchuk, N. Barkley, G. Contner and M. Richards
Introduction
To supplement the data on exhaust emissions from catalyst-equipped
systems studied in which regular Indolene fuel was used, in study TAME-K
a high-sulfur content gasoline was substituted In that study a
quantity of thiophene was added to the reference Indolene fuel to provide
a sulfur level twice as great, 0 10 percent, as that normally present. A
more detailed analysis of the particulate was made m order to establish
the concentration of sulfate and of the expected high acidity.
Experimental Procedure
The high acidity of the aerosol produced in the exhaust emissions from
oxidative catalytic equipped systems was indicated in preliminary runs of
the 350 C I D Chevrolet engine. Aerosol collected from an exposure chamber
onto an electrostatic precipitator plate was a water-white liquid and proved
to be very acid by pH-paper test. Also, membrane- type filters used to
sample the exposure chamber atmospheres remained an undisoolored white and
deteriorated upon standing several hours, sometimes to the point of breaking
into fragments.
Therefore, quartz fiber filter material (Pallflex type 2500-QAO) was
used to collect aerosol samples at all the sampling points of the piping
system. Every filter was weighed immediately after sampling Some were
weighed again after several hours or overnight standing to allow for
equilibration with the room atmosphere and stabilization of the sample
weight. Others that were used for aerosol acidity measurements were (total
-------
100
or portions of) then placed without delay after the early weighing into a
beaker of a specified quantity of distilled, deionized water At
least 30 minutes was allowed for water extraction of the sample before
the initial measurements of conductance and of pH were made Final
measurements of ion concentration were made 16-40 hours later.
The aqueous extracts were subsequently used for determination of
sulfate (SO."), of airmonium (NH4+), and of nitrate (NC^") Sulfate was
analyzed by the barium chloranilate method '^ ', ammonium by phenolhypochlorite
reaction,^' and nitrate by hydrazine reduction.^ '
Alternate analytical methods were used in some cases to confirm the
concentrations determined. A nephelometric method was adapted to sulfate
measurement, and ion specific electrode applied to ammonium measurement.
Those methods had lurated use for the present group of samples because of
sensitivity and reproducibility requirements.
Gas samples for nitrate - and for ammonium - producing components in
the atmospheres were collected by absorption into distilled water or into
a weak acid solution For separation of SC>2 from SO-j, the procedure /of
drawing the gas through a bubbler containing isopropanol and then through
hydrogen peroxide, ^0-, solution or through tetrachlormercurate, TCM,
contained in two follow-up bubblers was applied The ammonium and the
nitrate product concentrations were determined by the same methods cited
for the aerosol analysis. The SO^ sulfate in the first bubbler and the
SC>2 sulfate in the peroxide bubblers of the three bubbler-train were analyzed
by the chlorarilate method The SO^ m the TCM absorption liquid was analyzed
(4\
by the West-Gaeke method.v '
-------
- i
101
The animal (population) occupancy of an exposure chairiber was noted by
the number of cages and activity wheels A cage might have three adult
rats, or a litter of recently-born rats with their mother, or a group of
four hamsters An activity wheel was associated with a single mouse. A
record of the animal occupancy was kept for comparison with levels of
ammonium and acidity of the particulate in the atmosphere of the exposure
chamber.
Results
A condensation of the analytical values for several ionic components
is given in Table 1. Gas and particulate values are listed for the
diluted atmosphere sampled immediately after the exhaust pipe and for the
atmospheres in each of several exposure chambers.
Total particulate for TAME-K is shown in the fourth column of values
of Table 1 of the article, "Exhaust Emissions from Catalyst-Equipped Engines."
o
The sulfate value for the diluted exhaust pipe emissions, 46.5 y mol/fa , m
Table 1 of this article represents almost 75 per cent of the weight of the
total particulate, 5.97 mg/M . Considering the very highly acid nature of
the aerosol (particulate), one must assume that the sulfate is most likely,
totally, sulfuric acid. On the basis of an average emissions volume generated
by the engine of 1 M /mm and an average of 22 m.p.h equivalent road
speed for the engine operation (California cycle), the total particulate
value of the diluted exhaust was calculated as approximately 0.16 gm/mile.
Actually, the acidity is so high that at the present tune it is un-
accountable in terms of the amount of aerosol reported Further work is
required to explain this phenomenon The point should be made that those
-------
102
filter samples which were not used in the extraction scheme did lose, after
standing overnight, as much as 50 per cent of their weight sometimes. It
was this final weight upon which the total particulate calculations were
made.
Although values are given for (NH.+) and (NO^") in the gas phase,
Table 1, it is assumed that the analytical procedures are accounting mainly
for ammonia (NH3) and for the contribution of nitrogen dioxide (NO2) to
these ion concentration values. The SO^ in most cases is probably a fine
mist of acid aerosol in the submicron size range of 0 1 micron or less.
The particulate analyses of Table 1 show that the aerosol in the
diluted exhaust pipe emissions (first column) is a highly acid sulfate.
The aerosol m exposure chamber #15 (second column) is non-acidic and
contains ammonium (2^K^+) nearly quantitative to the sulfate (804") measured.
It is not unreasonable to think, therefore, m terms of the acid such as
sulfuric acid H2SC>4 or of the salts such as ammonium sulfate (NE^^SO^
The amount of sulfate measured in the exposure chambers themselves represented
an average of at least 15 per cent of the sulfur present m the fuel.
References
1. Kuffa, R J Stationary Source Testing. Apollo Chemical Corp Sept 1972
2. Weatherburn, M W. Phenol-Hypochlorite Reaction for Determination of
Ammonia Anal Chem 39_ 971, July 1967
\
3. Hauser, T. R Method for Analysis for Nitrate by Hydrazine Reduction.
Water Research !_ 205-216, 1967
4. West, P. W. and Gaeke, G. C Fixation of Sulfur Dioxide as Disulfito-
mercurate (11), Subsequent Colormetric Estimation. Anal Chem 28_ 1816,
1956.
-------
Table 1
K - Atmospheric Component Concentrations,
GAS
PARTICULATE
(NH4+)
(N03-)
(S04=)
(S02)
(2NH4+)
(N03")
(S04=)
(2H+)
Exh.Pipe
Emissions
Diluted
-
1.6
0.2
46.5
170.6
Exp.Ch#15
Irrad.
15-10 cages
0 4
7.3
15 0
2.0
38.6
1.2
32.8
0.2
Exp Ch #18
Non-Irrad.
2-0 cages
0.1
2.4
28 0
0
4.6
0
34.3
122 9
Exp.Ch.#6
Non-Irr.
No Animals
1.1
0
35.0
109.9
Exp.Ch.#22
Irrad.
4-2 cages
+ 6 wheels
0.22
8.70
7.9
0
34.2
54.7
Exp.Ch.#24
Non-Irrad
2 cages
+ 6 vheels
0.33
6.25
21.9
0.2
31.3
-
Exp.Ch.#23
Non-Irrad.
12 cages
41.2
0
25.5
0
Exp.Ch.#17
Irrad.
6 wheels
11.4 S
0.6
31.3
97.0
-------
104
B.7. COMPARISON OF THE BIOLOGICAL EFFECTS OF ACUTE EXPOSURE TO WHOLE
EXHAUST EMISSIONS FROM AN AUTOMOBILE ENGINE EQUIPPED WITH AND
WITHOUT A NOBLE METAL CATALYTIC CONVERTER
D. Hysell, W Moore, L Garner, D. Cmehil, S. Neiheisel,
H. Ball, Y Yang and J Stara
This study was undertaken to compare the biological effects which
might result from an acute exposure to catalyst treated exhaust (TAME J)
vs. nontreated exhaust (TAME I). The exposure facility and emission
chemistry are discussed in detail in other reports (See Hmners, et al.
and Malanchuk et al ). The experimental animals included young adult male
rats and hamsters, lactating female rats and their sud- Img young Biologic
parameters studied included mortality, body weight, hematology and blood
chemistry, and pathology.
Body Weight and Mortality
The animals in this portion of the study were exposed to treatment
atmospheres 24 hrs/day for 7 days There were 7 treatment groups consisting
of clean air (CA), irradiated exhaust (IH), and non-irradiated exhaust (RH) for
both TAME I and J and a carbon monoxide control (CO) with CO levels comparable
to those encountered in the emissions produced in TAME I Each treatment group
consisted of 10 lactating female rats and their 2-week old litters (10 suckling
rats/litter) which were weighed at the beginning and end of the study Each
treatment group was examined several tunes daily for possible mortality
The results indicate that the IH and RH treatment groups in TAME I showed
the most severe changes in weight of lactating female (Fig. 1) and infant
rats (Fig. 2), ard survival rate of infant rats (Fig. 3) There appeared to
be minimal changes in infant survival in the high CO group (Fig 3). There
-------
TAME I (no catalyst]
-CO
350
GO
s
ca
1,250
QQ
200
150
100
j I I I
I I I I
TAME J (catalyst)
RH
01234567
Days of Exposure
01234567
Days of Exposure
FIGURE 1, BODY WEIGHT OF LACTATING FEMALE RATS
-------
55
50
45
E
eg
j^ 35
f 30
^
25
20
15
10
5
o
OQ
TAME I (no catalyst]
TAME J (catalyst]
01234567
Days of Exposure
01234567
Days of Exposure
FIGURE 2, BODY WEIGHT OF INFANT RATS
-------
TAME I (no catalyst)
100
90
80
"r5
I 7°
^ 60
50
40
30
20
10
i i i
IH
....
TAME J (catalyst)
IH&RH
CA
0123456
Days of Exposure
01 234567
Days of Exposure
FIGURE 3, SURVIVAL OF INFANT RATS
-------
108
were no apparent effects in TAME J with the possible exception of weight
loss in IH and EH lactating female rats (Fig 1).
Henatology and Blood Chemistry
For this portion of the study, adult irale rats were maintained m six
treatment atmospheres (CA, RH, IH for both TAME I and J) 24 hrs./day. Five
animals per group were removed on days 1-5, anesthetized and exsanguinated by
abdominal aorta catheterization. The clinical laboratory determinations
included hemoglobin (HB), hematocrit (KCT), red (EEC) and white blood cell
(WBC) counts, platelet counts, reticutocyte counts, white blood cell
differential, partial thromboplastin time (PIT), prothrombin tune (PT),
fibrinogen determination, total serum protein, alkaline phosphatase, SCOT,
SGPT, blood urea nitrogen (BUN), Na, K, Cl, and Ca. Standard laboratory
procedures were used in these determinations The results are presented
in Tables 1 and 2.
Examination of the results indicates that in TAME J, the only statistically
apparent treatment effect was an increase in total serum proteins in exhaust
exposed animals. In TAME I there were statistically significant treatment
effects in both raw and irradiated exhaust exposures on total protein, platelet
count, BBC and WBC counts, white cell differential, alkaline phosphatase,
hemoglobin, hematocrit, partial thromboplastin time, SCOT and SGPT levels
The irradiated exhaust exposure also produced a treatment effect on levels
of BUN fibrmogen. It should be emphasized that while the treatment effects
were statistically significant, the data may not be physiologically significant
They do, however, indicate a potential hazard to certain organ systems with
prolonged exposure. In explaining the effects, the RBC related changes and
alkaline phosphatase levels could relate to the high levels of CO, the WBC
-------
109
TABLE I.
Treatment Mean Values for Selected Heraatologic
Parameters in Male Rats
KBC/otm (x 106) •
WBC/otm (X 103) :
Platelets/arm (x 106)
Lyrnphocyte neutraphil
ratio
HB (gin %) •
ICT (%).
CA*
RHb
IH c
CA
RH
IH
CA
RH
IH
CA
PH
IH
CA
KH
IH
CA
RH
IH
Without
catalyst
7 074
7.616
7.784
9.1
11.9
12.0
0.95
1.07
1.10
5.3
1.7
1.0
14.9
16 5
16.7
41.6
46.6
47.4
With
catalyst
7.141
7.004
7.073
9.3
9.0
8.7
0.93
0.97
0.92
5.3
5.1
5.3
14.6
14.7
14.5
40.8
41.0
40.1
a: clean air control atmosphere
b: Nonirradiated exhaust atmosphere
c: Irradiated exhaust atmosphere
-------
110
TABLE II.
Treatment Mean Values for Selected Blood Chemistry
Parameters in Male Rats
Total protein, (gm %)
Alkaline phosphatase
(Int. t/nits)
SCOT (R-F Units)
SGPT (R-F Units) :
Fabrinogen (mg/dl) .
BUN (mg %) .
PTT (seconds) •
CA a
RHb
CA
RH
IH
CA
RH
IH
CA
RH
IH
CA
RH
IH
CA
RH
IH
CA
RH
IH
Without
catalyst
6.0
6.3
6.8
79.1
54.2
40.8
161.6
185.3
196.7
48.5
60.5
53.7
170
165
220
23.8
21.0
28.3
19.9
21.7
22.4
With
catalyst
5.8
6.1
6.0
80.9
91.4
83.4
169.7
174.4
174.8
40.0
40.7
42.0
175
175
170
22.5
21.8
21.4
19.9
19.3
19.2
a* Clean air control atmosphere
b: Nonirradiated exhaust atmosphere
c: Irradiated exhaust atmosphere
-------
Ill
changes would suggest a rather severe acute uiflanmatory response, the
other changes relate to hepatic and/or renal dysfunction.
Pathology
Tissues from the adult male rats used in the Hamatology and Blood
CHemistry Section of this study were saved m 10% formalin for pathology.
]ji addition, an equal number of adult male hamsters were exposed and
tissues saved for pathology Sections of hematoxylin and eosm stained
]ung, liver and kidney were examined microscopically for abnormalities.
In TAME I, 2 of 5 IH hamsters shewed acute inflammatory pulmonary
changes after one day exposure. The lesion was characterized by an
infiltrate of polymorphonuclear neutraphils (PMN) into alveoli at the
level of the terminal bronchioles After 2 days, the lesions consisted
of prominent macrophage and EMN exudate in alveoli at the level of the
terminal bronchioles plus a patchy acute purulent bronchiolitis and
pneumonia with some ulceration of bronchiolar epithelium. By the end of
5 days, the IH animals had a subacute purulent bronchiolitis and pneumonia
(Fig. 4). The alveoli at the level of the terminal bronchioles had
thickened septae with some crescentic epithelial caps. Many were plugged
with an admixture of fibrin, macrophages and PMN.
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112
-;%&»
C 4) ""•
Figure 4. Subacute purulent bronchiolitis and pneurtoraa in
hamsters exposed to irradiated exhaust in TAME I
for 5 days
In the RH hamsters, the pulmonary changes were not apparent until day 2,
and thoughout the study the changes were confined to the alveoli at the level
of the terminal bronchioles. The lesions were not as severe as in the IH group
and tended to be more proliferative than exudative. By the end of 5 days, there
were some alveoli with thickened septae with an increase in alveolar macro-
phages (Fig. 5).
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113
Figure 5. Pulmonary changes in hamsters e>q?osed
5 days to non-irradiated exhaust in
TAME I.
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114
In both the PH and IH hamsters from TAME I, no treatment related
changes were noted in liver and kidney until 5 days of exposure. In
the IH group, all 5 animals showed vacuolar change (possibly lipidic
degeneration) in hepatic parenchymal cells (Fig. 6). Two FH annuals
showed similar changes.
Figure 6. Cytoplasrnic vacuolar change in livers frcm
hamsters exposed 5 days to exhaust in TAME I.
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115
Three of the IH animals also had sutular vacuolar changes in renal
tubular cells (Fig. 7).
Figure 7. Renal tubule cytoplasmic vacuolar change in
hamsters exposed 5 days to irradiated exhaust
m TAME I.
Pulmonary changes noted in TAKE I rats paralleled those noted m the
hamsters but were not apparent as early in the study and were not as severe.
Extramedullary heiratopoeisis was present m the livers of the IH and RH rats
by day 4 probably as a result of the high CO levels.
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116
Tissues from TAME J have not yet been examined. For the purpose of
coinparison, TAME H (identical to TAME J in design) results are included.
No pulmonary changes were noted in IH and RH rats. The changes noted in
hamster pulmonary tissue were rather mild and mainly present in the IH
group only after 2 days of exposure. The lesion was confined to the
alveoli at the level of the terminal bronchioles and consisted of an
increase in macrophages, thickened septae and some crescentic epithelial
caps. The changes were rather typical of lesions ascribed to exposure
to N02- No changes in liver or kidney were noted.
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117
B.8. EFFECT OF EXHAUST EMISSION FROM CATALYTIC CONVERTER
ON ARYL HYDROCARBON HYDROXYIASE
L. Hall, I. Washington, J. Adams, K. Campbell and Y Yang
Introduction
Chemical carcinogenesis is of considerable concern in relation to
exposure to environmental pollutants. Due to the association of microsomal
metabolism with carcinogenesis the effect of catalytical modified auto
exhaust on aryl hydrocarbon hydroxylase (AHH), one of the mixed function
oxidase responsible for the biotransformation of some known potent
carcinogens was determined
METHODS
Male Syrian hamster retired breeders [>8 mos. old] were exposed
continuously for five days to automobile exhaust using the system described
by Hinners et al (ETRL) These exposures were carried out in the three
studies, TAMEs I, J, K, described elsewhere in the Catalytic Report
In each study after five days continuous exposure to either clean
air (CA), nonirradiated (MI), or irradiated (I) exhaust, the hamsters were
sacrificed with pentobarbital (IP), exsanguinated, and the puncture, and the
lungs were removed in_ toto and quickly immersed in cold saline (4°C) The
lungs from three animals were then trummed of bronchi and connective tissue,
weighed,and placed in cold 0.15 M KC1 for homogenization Aryl hydrocarbon
hydroxylase was assayed as described by Dixon et al * ' using a homogenate
concentration of 25 mg/ml and an incubation period of 60 minutes. The
results are expressed as the fluorescence equivalent to picomoles of
3-hydroxy-benzo pyrene formed/min/mg tissue
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118
RESULTS
Table 1 shows the hamsters ' lung AHH activity following exposure to
either clean air, nonirradiated, or irradiated exhaust in TAMEs I, J and K.
Statistical analysis of the data showed that significant enzyme reduction
occurred as a result of exposure to both nonirradiated and irradiated
atmospheres in the reference study (TAME I - without catalyst) Exposure
to catalyst-modified exhaust (TAKE J) resulted in a depression of mixed
function oxidase (MFC) activity in both experimental groups, but statistically
significant (p = 05) depression was noted only in the group exposed to
irradiated exhaust The use of high sulfur fuel with the catalyst (TAME K)
produced a depression following exposure to irradiated exhaust, although
<
the decrease did not reach statistical significance at the p = / 05 level.
Due to the innate variability in enzyme activity of control animals
berween experiments, the data were normalized to percent of control for
further comparison between studies Following exposure to nonirradiated
exhaust a reduction in AKH activity of 52, 12 and 0 per cent was seen m
TAMEs I, J, and K, respectively Following irradiated exhaust exposure, the
depression in TAME I was 55 percent, 28 percent in J, and 18 percent m K
Duncan's multiple range test revealed that TAMEs J and K effects were
similar and different from TAME I
Table 2 shows the lung weight/body weight ratios for the three studies
Only in TAME I (without catalyst) was a significant increase in the ratio
noted The severity of this exposure was also evident in the mortality of some
of the other experimental animals, which did not occur in the other studies.
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119
Table 1. Aryl Hydrocarbon Hydroxylase in Hamster Lung
Clean Air
Non-irradiated
Exhaust
Irradiated
Exhaust
I
0.01192
0.00579*
0.00529*
J
0.01789
0 01671
0.01352*
K
0.01134
0.01150
0.00974
1 Activity expressed as equivalent to the formation of
3-hydroxy-BP in piconnoles/nan/nig tissue
* Significantly different from control at p'= .05
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120
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121
Table 2. Lung Weight/Body Wsight Ratio in Hamsters Exposed to Auto Exhaust
TAME
I
J
K
n.
12
12
12
CA
.536
.505
.521
Non-Irrad.
.696*
.498
.531
Irrad
.725*
.492
.528
* Significantly different from control at p = .05
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122
DISCUSSION
TAME I, the reference study, shewed profound toxicological and biological
effects as manifested by death, lung weight/body weight ratio changes, and
depression of AHH activity. However, no gross effects were noted in TAMEs J
and K. Ozone (0.4 ppm) and N02 (4.6 ppm) was measured m the exhaust chamber
atmospheres in TAME I. Palmer et al. ^ ^' noted a 33 per cent decrease in
hamster lung AHH activity following a three-hour exposure to 0.75 ppm ozone,
but no effect on bronchial AHH was noted for N02 concentrations up to 50 ppm
for three hours.' ^' Thus ozone probably had significant effect on AHH
activity in TAME I. The contribution by the other exhaust components, many
with known biological effectiveness, is not known. No ozone was detected,
and no significant change m N02 concentration was found in TAME J or K
which would account for the depressed AHH activity. The biological effective-
ness as reflected in AHH activity/ therefore, may reside in the organic
fraction of the atmospheres. Further work is necessary to resolve this problem.
Two additional comments are needed. The lack of statistical significance
in the AHH depression in the NI atmosphere of TAMEs J and K and in the
irradiated exposure in TAME K is thought to be due to the small sample size
(4/treatment) rather than to lack of effect. Depression of AHH activity has
been a consistent finding following exposure to auto exhaust in several
studies (ETKL Annual Report, 1972).
The apparent lack of AHH induction is curious, since Holt and Keast ^'
\
found induction of AHH in the lungs of mice exposed to cigarette smoke,
which contains several components such as N02/ aldehydes and polycyclic hydro-
carbons, also present in the exhaust. The significance of the depression and
lack of induction is not clear, but due to the association of this enzyme
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123
system with carcinogenesis it seems important to determine the impact of
effects on aryl hydrocarbon hydroxylase and its relationships to cancer
initiation and promotion.
In summary, the use of the catalytic device significantly reduced
but did not eliminate the depression of lung aryl hydrocarbon hydroxylase
by exposure to auto exhaust Whether this residuum of biological
activity in catalytic modified exhaust reflects the lower part of the dose
response spectrum, or the formation of potent new chemical species, is not
yet clear Additional research is necessary for insight into this aspect
of catalyst evaluation.
AOOX3WLEDGMENT
We acknowledge with gratitude the sample of 3-hydroxy-benzpyrene
from Dr. H.V. Gelboin, National Cancer Institute, that was used to
standardize our work.
References
1. Dixonetal Cancer Pes. 30_ 1068, 1970
2. PaJjter et al Cancer Res. 31^ 730, 1971
3. Arch, Environ. Health 25_ 489, 1972
4. Holt and Keast, Expenentia 29_ 1004, 1973
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124
B.9. BIOCHEMICAL EFFECTS OF EMISSIONS FROM AN AUTOMOBILE ENGINE
WITH AND WITHOUT CATALYTIC CONVERTER
S. D. Lee, V. N. Finelli, L. McMillian, and R. M. Danner
As a part of the toxioologic studies of automobile engines with and
without catalytic converters, the biochemistry group, in collaboration
with Dr. Finelli of the Department of Environmental Health at the University
of Cincinnati, has studied early biochemical alterations in rats exposed
to auto exhaust emissions.
Materials and Methods
Experimental Animals - Each exposure experiment consisted of thirty
female Sprague-Dawley rats, each group weighing approximately 200 grains,
divided into three groups of ten animals Clean Air (CA), non-irradiated
(N-I), and irradiated (I).
Exposure Conditions - The exposure system has been described by
Hinner et. al., earlier in this report. The concentrations of major
exhaust components in the exposure chambers were also described in a
earlier report by Malanchuck, et. al. Temperature and humidity in the
exposure chambers were kept constant throughout the experiment at 22°C
and 50 percent relative humidity respectively. The exposures were conducted
24 hours a day, for 7 consecutive days. Two animal exposure experiments
were conducted using the exhaust from the same engine with and without
the catalytic converter. In addition, an experiment was performed by
exposing a group of animals to carbon monoxide alone (experiment CO) at
a concentration of 575 mg/m3 (500 ppm) which approximately reflects the
carbon monoxide level observed in exposure chambers when emissions from
the engine without the catalytic converter was tested.
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125
The following parameters were determined: hematocrits, serum
latate dehydrogenase (LDH), serum glutanic oxaloacetate transaminase
(SCOT), and serum lysozyme. Serum IDH and GOT were determined by
using DADE reagent sets (American Hospital Supply Corp., Miami, Florida,
white lysozyme was assayed with Worthington kit (Wbrthington Biochemical
Corp., Freehold, N. J.). Blood samples were obtained from animals by
tail vein puncture.
Results and Discussion
Figure 1 shows the drastic effects of the exposure to emissions from
the engine without the catalytic converters on the hematocrit. At the end
of the 7-day exposure, very high hematocrit levels were observed in the
experimental animals, 62.3 + 1.5 percent for N-I and 66.2 + 0.5 percent
for I, as compared to a normal value of 43.2 + 0.9 percent for the clean
air group. During a recovery period of 3 weeks, the hematocrit values
were obtained weekly and a gradual return to normality was seen in the
animals of both N-I and I groups. The animals exposed to carbon monoxide
showed a average hematocrit of 62.5 + 0.9 which is equal to the value
found for N-I group in the experiment without converter.
The hematocrit in the animals expsoed to emissions from the engine
when equipped with the catalytic converter did not differ from control
values. From the above data it seems that the elevation of the hematocrit
is due to the carbon monoxide concentration in the exposure chambers. The
levels of carbon monoxide in N-I and I groups in the experiment without
the catalytic converter were 551 and 559 ppm, respectively, while for
N-I and I with the catalytic converter the carbon monoxide levels were
reduced to 46 and 41 ppm. The increased hematocrit may be due to poly-
cythemia and/or dehydration. Total serum protein or albumin analyses
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126
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127
were not obtained and therefore the occurence of dehydration cannot be
confirmed; however the data collected from histological examination of
the experimental animals, presented in a report by Hysell et. al.,
revealed the presence of a large numbers of reptured red blood cells
which may indicate a polycythenic response.
In order to assess organ damage in exposed animals, the activity
of LDH, GOT, and lysozyme in serum was assayed. These intracellular
enzymes are characteristic of appropriate organs and an increase of
enzymatic activity in serum would indicate presumably a leakage of
enzymes from injured cells. Serum GOT was not significantly elevated
in any of the exposed animals, this would indicate that neither liver
nor heart were damaged by exposure to various types of emissions and to
carbon monoxide. Serum LDH was elevated in the anmals exposed to
emissions from engine without catalytic converter. Figure 2 shows that,
at the end of the exposure period, the animals from both N-I and I groups
presented approximately 200% increase in serum LDH activity. In the
recovery period, while the N-I group values tended to return to normal,
the I group values presented an unexplained erratic behavior, moreover
we cannot explain the low value obtained in the third week for the CA
group. No significant changes in LDH activity were observed in the
experimental animals when the converter was used and in the animals
exposed to 00. Serum Lysozyme activity was not assayed in the experiment
conducted without catalytic converter, however, in the experiment with
converter, the exposed animals did not show any statistically significant
elevation. From the above preliminary results it appears that the target
organs of the toxic components present in the emissions from engine
without catalytic converter are probably the lungs and/or kidneys.
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128
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129
It can be concluded that the introduction of catalytic converters
into the automobile exhaust system, not only has reduced the levels of
certain exhaust constituents but has effectively decreased or eliminated
biological effects studied.
Reference
1. Stara, J. F. and R. Hinners Toxicology of Atmospheric Pollutants
Associated with the Use of Automobile Catalytic Converters, Annual
Report, Environmental Research in 1973, U S. Environmental Protection
Agency, National Environmental Research Center, Cincinnati, Ohio
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130
B.10. EXPOSURE OF MATERNAL, PREGNANT, AND NEWBORN RATS TO EXHAUST FROM
MODERN ADTCMBIIE ENGINE WITH CATALYTIC CONVERTER AND
OTHER EMISSION CONTROLS (TAME J)
K. Canpbell, E. George, L. Hall and J. Stara
Toxicity to newborn and pregnant rats of diluted exhaust from a proto-
type automotive engine system including oxidative catalytic converter was
evaluated by two experiments in the TAME J study. Details as to test atmo-
sphere generation and characterization are provided in preceding reports
(See Hinners et al., Malanchuk et al.). Similar studies were performed in
TAME K (high sulfur fuel used) but data are not sufficiently complete to
permit a comparative evaluation of the sulfur effect.
Experiment A
Ten litters of day-old Charles River COBS suckling rats and their
dams were exposed for six days in each of three atmospheres purified air
(controls), nonirradiated (NI) exhaust, and irradiated (I) exhaust.
Dams and litters were weighed and pups were counted when placed in exposure
(Day 0), when removed to clean air (Day 6), and subsequently while housed
in purified air (on Days 11, 15 and 19). On Day 19 the young were 20
days of age and this was designated as "weaning date." Maternal body weight
and infant survival and growth of treated groups were compared to control
values.
Results, which are summarized in Table 1, are preliminary in the sense
that the data has not as yet been subjected to statistical analysis Neverthe-
less, the largest contrasts between treated and control means are 7 per cent
for maternal weight gam deficit (for Day 6), 3 per cent for infant growth
deficit, and 8 per cent for infant survival to weaning, all in NI. Without
-------
Table 1. Maternal Body Vfeight and Infant Growth and Survival in Rats Exposed to
Catalytic Converter-Treated Automotive Emssions m TAME J, Experiment A.
Treatment
Bioef feet Criterion (Clean Air (Control)
Maternal Lactating Rats:
Pre- treatment body weight,
grrs (mean)
Mean fraction of pre-treat-
nent. body weight Day 0
Day 6 (End of Expos.)
Day 11
Day 15
Cay 19 (Weanling)
Infant Growth:
Pre- treatment body weight,
gms (mean)
Fean fraction of above
Eiy 0
Lay 6
Day 11
Day 15
Day 19
Infant mortality & Survival:
Original No.
No lost, % survival to age 7 Days
No lost, % survival to weaning
275.1
1.00
1.163
1.270
1.245
1.283
8.76
1.00
2.139
3.337
4.257
5.708
100
0, 100
1, 99
Non- Irradiated
292.0
1.00
1.085
1.252
1.172
1.262
9.09
1.00
2.138
3.285
4.112
5.666
99
1, 99
8, 91.9
Irradiated
310.0
1.00
1.105
1.208
1.212
1.226
8.74
1.00
2.191
3.310
4.252
5,707
98
0, 100
0, 100
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132
additional data by which a consistent pattern might be discerned as a
basis for interpretation and validation, these relatively snail differences
are suspected as being neither statistically* nor toxicologically significant
Most infant deaths in NI occurred between 16 and 20 days of age. The
cause of these was not clear, and they are not as yet considered as clearly
treatment-related It would thus appear, at least tentatively, that despite
the low exhaust dilution ratio, the test atmospheres from this engine and its
emission controls were not sufficiently harmful to be detected by the
methods used It should be noted that in the previous study (TAME I)
in which the catalyst was not used, there were obvious body weight deficits
and morbidity in exposed anomals, suggesting a oeneficial effect of the
catalytic converter m regard to the latter criteria.
Experiment B
Twelve Charles River COBS female rats pregnant 15 days were exposed
for 6 days to each of 3 atmospheres purified control air, nonirradiated
(NI) exhaust, and irradiated (I) exhaust, as with Experiment A After
exposure, all were transferred to brood cages and housed in clean room air.
Body weight of the maternal animals were recorded when they were placed
in to exposure (Day 0), when removed from exposure to room air (Day 6), and
subsequently on days 11, 15, 19, 25 and 29. Offspring were counted at
parturition following exposure (Day 8), and weighed and counted on days
11, 15, 19, 25 and 29. 'On Day 29 the offspring were 20 days of age, this
date was designated as the weaning date Maternal body weights and infant
survival and growth in exhaust-exposed groups were compared to those in the
control group.
*Subsequent statistical analyses indicated that the infant survival on NI was
significantly less than in CA and IR, however, this suspicious pattern was not
observed in a subsequent experiment (TAME K) The weight-data analyses suggested
no significant treatment effects
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133
The resulting data, which are summarized in Table 2, and which have
not yet been statistically analyzed, are tentatively interpreted as in-
dicating no significant effect by either treatment (NI or I) on maternal
body weight, litter size, or infant survival or growth. The largest
contrast noted was a mean 9% smaller litter size in NI, but litter size
varied considerably in all groups, and the mean I litter size was 3.5%
larger than control. The largest single contrast m maternal body weights
was -4.5% in I, and in infant body weight was +8.2% in NI. Without
additional data to establish a clear pattern it is doubtful that the
pattern and magnitudes of mean differences observed in this experiment are
statistically or toxicologically meaningful. Since this experiment was
not performed in TAME I, a comparative evaluation of converter-no converter
emission toxicity en these criteria is not yet possible, but it is speculated
from other observations in TAME I that effects might well have been demonstr-
able with this system.
+ Subsequently completed statistical analyses indicated no significant
treatment effects on maternal or infant body weights or on infant
survival in this experiment
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134
Table 2. Maternal Bcdy Vcaght and Infant Ocwth and Survival
in Rats After Jn Dtoro Exposure to Catalytic Converter-
Treated /utcnotive Emissions in TAMC 3, Experiment B
fioeffect Criterion
Maternal rats — Body VJsight
lilln body weight (gir) and
faction of initial (pre-
e>pos) weight. N=12
^ay 0 (Pre-e>cposuLe)
Day 6 (Post-exposure)
•ay 11 (3 day after partur)
Day 15
fay 19
Day 25
•ay 29 (v;eanmg)
Lo^-ng orrspring —
I'ftn body \ eigit (gm) and
fraction of 1st weight
(age 3 days)
%ay 11 (3 days of age)
Cy 15
y 19
l)ay 25
Day 29 (weaning, age 20 days)
•
IrBant mortality and survival
Sginal No. and mean litter size
lost and % survival 1st 3 days
I'M lost and % survival to v^eanmg
Treatment
lean Air (Control)
227.5 1.00
303.3 1.333
278.6 1.225
301.5 1.325
318.9 1.402
331.2 1.456
321.8 1.415
-
9.08 1.00
16.25 1.790
24.26 2.672
36.78 4.051
46.63 5.135
115 9.58
2 98.3
3 97.4
Non-irradiated
4(
221.0 1.00
299.4 1.355
262.8 1.189
285.9 1.294
308.9 1.398
327.0 1.480
310.9 1.407
8.71 1.00
16.03 1 840
24.24 2.783
37.96 4.358
48.38 5.555
105 8.75
1 99.0
2 98.1
Irradiated
234.6 1.00
297.4 1.268
280.1 1.194
297.0 1.266
322.5 1.375
326.7 1.392
316.9 1.351
8.46 1.00
15.68 1.853
23.70 2.801
36.37 4.299
46.29 5. 472
119 9.92
0 100.0
1 99.2
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135
B.ll. GROSS MORPHOLOGIC AND FUNCTIONAL DAMAGE TO PLANT SPECIES BY DILUTED
EXHAUST OF AUTOMOTIVE ENGINES OPERATED WITH AND WITHOUT A
CATALYTIC EMISSION CONTROL DEVICE
K. Campbell, R. Miller and J Enright
Effects on vegetation of automotive exhaust from engines with and
without an oxidative catalytic converter, and operated on low-sulfur Indolene
fuel, were assessed by exposing multiple plant species to clean air (control),
irradiated and nonirradiated atmospheres in each of the two sequential
"TAME" experiments 'I1 (without converter) and 'J1 (with converter) Details
concerning the experimental atmosphere generation and characterization are
provided in preceding reports (see hinners et al. and Malanchuk et al ). In
TAME 'K1, in which the atmospheres were produced by the same engine fitted
with converter, but usung high-sulfur Indolene, the time-related aspects of
vegative damage by exhaust to a single plant specie were investigated These
studies were conducted collaboratively with faculty and student representatives
of the Department of Biology (Botany), University of Cincinnati Results
reported are based on preliminary data evaluation.
Experiment A Damage to vegetation by automotive exhaust from an engine
operated on low-sulfur fuel with (TAME J) and without (TAME I) an oxidative
catalytic converter.
Phytotoxicity in exposed plants was evaluated on the basis of
\
morphologic alterations (gross visible alterations of leaf and stem character)
m all species and functional changes (C^-determined photosynthetic activity)
in some. Post-exposure recovery was also observed Effects of irradiated (I)
and nonirradiated (NI) atmospheres were compared in each experiment and
-------
136
between experiments Nine species in varying young stages of development
were used in duplicate in each experiment. Of these, five were the same
in both and four were different, and developmental stages were comparable
between exposures. The species used in each experiment are shown in Table 1.
Duration of exposure was 127 hours for TAME 'I1, and 151 hours for TAME 'J'
Table 1. Plant Species Used in TAME 'I1 and 'J'
TAME 'I* - Without Catalytic Converter
Taxas cuspidata Japanese yew
Picea abies Norway spruce
Ilex hetzi Japanese hollyberry
Cotoneaster lofast Cotoneaster
Euonymus coloratus
Alianthus altissntia Tree of heaven
Episcia cupreata (Silver Sheen) Episcia
Pinus strobus VJhite pine
Easilico
TAME 'J1 - With Catalytic Converter
Dancus carota Red-cored chantenay carrot
Phaseolus mungo Bean
Oxalis corniculata Oxalis
Coleus blumei Coleus
Cotoneaster lofast Cotoneaster
Euonymus coloratus
Episcia cupreata (Silver Sheen) Episcia
Alianthus altissima Tree of heaven
Basilico
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137
In both experiments, exposure to both NI and I atmospheres damaged
all species with varying degrees of severity. Although modes of damage
were also variable, the most generally common were wilting, bleaching and
loss of leaves. In some species, younger leaves were damaged first and in
others the older leaves showed earliest damage Generally, even though
exposure duration was a little longer in TAME 'J1 than in 'I1, damage was
somewhat less in TAME 'J' (with converter) than m TAME 'I1 (without converter).
However, this difference m severity was less remarkable than the difference in
gross toxic severity shown by animals, suggesting that the plants were
fundamentally more susceptible subjects than animals and that their damage
"thresholds" were greatly exceeded in both studies Three of the five species
used in both studies showed less damage in the converter-treated emissions than
in the non-converter emissions TAME 'I' atmospheres killed Cotoneaster,
Allanthus and Episcia with no post-exposure recovery, in TAME 'J' atmo-
spheres Cotoneaster showed slightly less damage even though it was wilted
badly, and Alianthus and Episcia showed post-exposure recovery, i.e., formed
new buds after apparent death with loss of original leaves. Basilico
seedlings, however, recovered after the 'I1 exposure but died from the 'J1
and Euonymus damage was similar in both exposures.
In TAME 'I' damage produced by the NI atmospheres was generally more
severe than that by I Five of nine species showed greater visible damage
by NI than by I while the reaminder were about equally damaged by NI and
I. There was a drastic depression of C -measured photosynthetic rate and,
in Euonymus, photosynthesis was depressed more by NI than by I, while the
reverse was true for Picea. In three species visible damage was delayed
-------
138
until about one week post-exposure, at which tine needle burn was observed
in Pinus and Picea, and bleach m Taxus. In Ilex there was direct damage
to petioles.
In TAME J, also, all nine species were damaged by both NI and I
but the visible damage severity by NI and I was generally not as distin-
guishable as in TAME I. Photosynthetic depression by I was greater than by
NI in Euonymus, while the reverse was true for Cotoneaster The species
showing delayed effects in I were not used in J
Within the context of the vegetation damage from the severe exposure
conditions in TAME studies I and J, it is tentatively concluded that,
in general, there is a detectable but minor advantage to the use of the
catalytic emission control system.
Experiment B The tune course of severe damage to Episcea Cupreata by
diluted automotive exhaust in TAME K
Since plant species exposed for several days to the TAME I and
J atmospheres exhibited severe to fatal damage, specimens of one of the
most susceptible species were exposed for varying durations in this experi-
ment in order to better understand the temporal "dose-response" relationships
of the damage at similar pollutant levels In each of the control (clean air),
nonirradiated (NI) and irradiated (I) atmosphere chambers duplicate
Episcia Cupreata (Silver Sheen) plants were exposed for periods of
t
six days, one day, sax hours and one hour. The six-day plants were examined
daily. Degree of visible damage was subjectively rated on a scale of 0-12,
Degree of visible damage was subjectively rated on a scale of 0-12, essentially
as described previously (Campbell, 1973) After exposure, photosynthetic
rate and recovery were also assessed
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139
As noted in previous studies, foliar damage was distinctly visible
as early as six hours of exposure, and progressively became severe as
exposure continued. In this study, generally, the I plants were damaged
more than the NI plants. There was a visually distinguishable difference
in the pattern of damage shown in the two types of atmosphere, as well,
in terms of spacial pattern of discoloration and of degree of wilt and
turgor. There appeared to be a somewhat steeper slope to the degree of
damage x duration of exposure curve than in the previous studies (TAME I
and J). Preliminary results are illustrated in Figure 1.
A subsequent study of duration - and concentration - related effects,
using the new Ford engine {with catalyst), shorter exposure periods and
multiple lower exposure levels (greater dilution), was conducted recently.
Preliminary data evaluation suggested grossly visible effects at 3 hours
of exposure or less in the high-level atmosphere (corresponding to those
reported above), and duration and concentration relationships in both gross
and functional effects. CO and total HC levels were substantially lower,
and NCjx levels higher, than in TAME K
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12 -
TOTAL INJURY
Without converter/
low sulfur (TAME I)
With converter,
low sulfur (TAME J)
With converter,
high sulfur (TAME^K)
Non-
irradiated
O
Irradiated
*».
o
2345
DURATION OF EXPOSURE, DAYS
6
FIGURE 1, D/WAGE TO FOLIAGE OF EPISCIA PLANTS EXPOSED TO AUTOMOTIVE EXHAUST
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