atomic energy of canada limited the effects of chronic ... · atomic energy of canada limited the...
TRANSCRIPT
Atomic Energy of Canada Limited
THE EFFECTS OF CHRONIC INGESTION OF ORGANIC
REACTOR COOLANT (HB-40) ON MICE (Mus musculus)
PART ONE
by
STUART L. IVERSON and BRIAN N. TURNER
Whiteshell Nuclear Research Establishment
Pincwa, Manitoba
June 1973
AECL-4220
THE EFFECTS OF CHRONIC INGESTION OF ORGANICREACTOR COOLANT (HB-40) ON MICE {Mus musculus)
PART ONE
by
Stuart L. Iverson and Brian N. Turner
Whiteshell Nuclear Research Establishment
Pinawa, Manitoba, ROE 1L0
June, 197 3
AECL-4220
THE EFFECTS OF CHRONIC INGESTION OF ORGANICREACTOR COOLANT (HB-40) ON MICE {Mus museulus)
PART ONE
by
Stuart L. Iverson and Brian N. Turner
ABSTRACT
Groups of out-bred house mice {Mus musculus) were
chronically exposed to three different concentrations
(approximately 1.0, 0.1, and 0 mg/SL) of equilibrium organic
reactor coolant (HB-40) in their drinking water for three
generations. Coolant concentrations of this order of magnitude
could be detected by the mice but did not cause a reduction
in their fluid intake. The dose rates to adult mice were
approximately 0.25 mg/kg/day, while those to young were
higher. No treatment effects were seen in littar size, number
of litters produced, weight of young, sex ratio, or weight
gain to weaning. X-ray LD5Q ,, 's were higher in animals
exposed to coolant bat not significantly so. When the mice
were given consecutively more concentrated NaCl solutions to
drink, there were no differences in death rate or weight loss
among treatments, indicating that functional kidney damage
had not occurred. No effects were seen in intraspecific
aggressive responses or voluntary activity. An effect of
coolant ingest ion was observed in an open-field test, but the
effect could not be demonstrated when the open-field experiment
was repeated. Evidence to date indicates that chronic
consumption of water containing as much as 1.0 mg/JL organic
coolant has no deleterious effect on this species.
Whiteshell Nuclear Research Establishment
Pinawa, Manitoba, ROE 1L0
June, 1973AECL-4220
Effets d'une ingestion chronique de caloporteurorganique (HB-40) chez les souris (Mus musoulus)
Première partie
par
Stuart L. Iverson et Brian N. Turner
Résumé
Trois générations de souris domestiques(Mus musaulus) élevées par rafraîchissement du sang ontété exposées chroniquement a trois concentrationsdifférentes (approximativement 1.0, 0.1 et 0 mg/£)decaloporteur organique équilibré (HB-40), dans leureau potable. Les concentrations de caloporteur de cetordre de grandeur pouvaient être détectées par lessouris mais elles n'ont pas réduit leur absorption dufluide. Les débits de dose des souris adultes étaientapproximativement de 0.25 mg/kg/jour, tandis que ceuxdes souriceaux étaient plus élevés. Aucun effet dutraitement n'a été constaté dans les portées, dans lepoids des souriceaux, dans la répartition des sexesou dans le gain de poids au sevrage. Les rayons X^50/30 ^ta*-ent plus élevés chez les animaux exposésau caloporteur mais pas de façon significative.Lorsqu'on a donné aux souris, consécutivement, dessolutions de NaCl plus concentrées à boire, il n'yavait pas de différence dans le taux mortel ni dans laperte de poids par suite des traitements, ce qui indiquequ'aucun dommage fonctionnel de rein ne s'est produit.Aucun effet n'a été perçu dans les réponses agressivesintraspécifiques ou dans l'activité volontaire. Uneffet de l'ingestion du caloporteur a été observé lorsd'un essai effectué en champ ouvert mais cet effet n'apas pu être démontre lorsque l'expérience en champouvert a été répétée. Les données disponibles à ce jourprouvent que la consommation chronique d'eau contenantjusqu'à 1.0 mg/t de caloporteur organique n'a pas d'effetdélétère sur cette espace.
L'Energie Atomique du Canada, LimitéeEtablissement de Recherches Nucléaires de Whiteshell
Pinawa, Manitoba, ROE 1L0
Juin 1973
AECL-4220
TABLE Or CONTENTS
1. GENERAL INTRODUCTION 1
2. GENERAL METHODS 3
3. PALATABILITY OF COOLANT SUSPENSIONS . Q
3.1 METHODS.... 8
3.2 RESULTS 10
3.3 DISCUSSION 12
3.4 CONCLUSIONS 12
4. ESTIMATION OF COOLANT INGESTION RATE 13
4.1 METHODS 13
4.2 RESULTS 14
4.3 DISCUSSION 14
4.4 CONCLUSIONS 15
5. INDIVIDUAL WEIGHT FROM BIRTH TO WEANING: THEEFFECTS OF NUMBER OF SIBLINGS 17
5.1 METHODS 17
5.2 RESULTS 18
5.3 DISCUSSION 23
5.4 CONCLUSIONS 26
6. LITTER SIZE, NUMBER OF LITTERS, WEIGHT OFYOUNG AND SEX RATIO 26
6 .1 METHODS 27
6.2 RESULTS 27
6.3 DISCUSSION 32
7. SURVIVAL FOLLOWING X-IRRADIATION 32
7.1 METHODS - 33
7.2 RESULTS 33
7.3 DISCUSSION 35
7.4 CONCLUSIONS 35
8. KIDNEY FUNCTION 37
8.1 METHODS 38
8.2 RESULTS 38
8.3 DISCUSSION 43
8.4 CONCLUSIONS 44
9. BEHAVIOURAL EFFECTS 44
9.1 METHODS 45
9.2 RESULTS 47
9.2.1 ACTIVITY 47
9.2.2 OPEN-FIELD 49
9.2.3 AGGRESSION 51
9.3 DISCUSSION 51
10. CONCLUSIONS 55
11 . ACKNOWLEDGEMENTS 56
12. LITERATURE CITED 57
1 -
1 . GENERAL INTRODUCTION
The organic cooled reactor (OCR) is one of a group
of reactor concepts recently evaluated to determine the
optimal type of power reactor to develop in the near future.
As a part of this evaluation, the effects of its coolant,
HB-40*, on various organisms are being examined. These studies
are centered around the aquatic environment for several reasons.
A power reactor would be located near, and be cooled by, water
from a lake or river. Accidental spills of liquids usually find
their way to lakes or streams, and once there are difficult to
control, and spread quickly by flow or circulation. Aquatic
organisms appear to be sensitive to compounds similar to HB-40
in the water (Guthrie and Acres, 1968).
A major release of HB-40 to the environment
from a power reactor must be considered extremely unlikely
because of built-in safety features. Chronic releases
through the heat exchangers are also unlikely, since leaks
from the primary (organic) to the secondary (steam), and from
the secondary to the cooling circuit would have to occur at
the same time and any loss of coolant would be against a
pressure gradient. Although conclusions reached by this study
apply to the above cases, the reasons for doing the research
are based on a different rationale.
Recent experience seems to indicate that any material
manufactured by man finds its way into the environment in some
quantities at some time. One aim of research should be to
determine how toxic a material is and how much effort and
money should be expended to slow its dispersal into the
environment.
* Tradename of the Monsanto Chemical Co., St. Louis, Mo., fora mixture of partially hydrogenated terphenyls.
- 2 -
Crude oil is a good example of a material that is
not purposely introduced into the environment but finds its
way there in large quantities. Oil is not particularly toxic
but causes damage through its physical presence. Polychlorinated
biphenyls (PCB), also accidentally released into the
environment, are quite toxic, not particularly biodegradable
and are concentrated by organisms. A spill of PCB into a lake
or river would be considerably more serious than a spill of an
equal volume of oil. Essentially, the purpose of this study is
to locate the position of irradiated HB-40 on a scale of
comparative toxicity to the environment, and by so doing define
the amount of effort that should be expended to prevent it from
reaching the environment.
The effects of ingested HB-4 0 in mammals appear to be
primarily on the liver and kidney. Ott and Pirrwitz (1970)
examined the effect of non-irradiated HB-4 0 on liver function,
as shown by the sulfobromophthalen (BSP) retention test, and
found no effect after a dose of 1.5g/kg in the rat. Irradiated
coolant is, however, of much different composition (R.B. Stewart,
pers. conun.) . It is more toxic since its LD50 to mice was found
to be 6,000 mg/kg as opposed to 12,500 mg/kg for unirradiated
HB-4 0 (Adamson and Weeks, in press). The same authors report
irreversible nephritis after 16 weeks ingestion of 600 mg/kg
irradiated coolant per day as well as changes in f.ie ultra-
structure of the liver at doses above 250 mg/kg coolant per
day. Irradiated HB-40 contains both biphenyl and meta- and
para-terphenyl, which causes increases in BSP retention times
(Ott and Pirrwitz, 1970). The terphenyl isomers, particularly
meta-terphenyl, are known to cause liver and kidney damage and
a decrease in growth rate in rats (Cornish, Balor and Ryan,
1962) .
This study was designed to examine the effect of
suspensions of equilibrium reactor coolant (irradiated HB-40)
on various parameters in laboratory mice (Mus musaulus). The
- 3 -
parameters estimated are functional ones and were selected
primarily from an ecological point of view. The objective
was to obtain an indication of the ability of an exposed,
population to maintain itself in the environment. Food chain
effects were, however, not studied.
2. GENERAL METHODS
The mice (Mus musaulus) used in this study were
obtained from the University of Manitoba, Department of Animal
Science, and are an outbred strainrecently derived from the
wild type. All animals were held in Carworth disposable poly-
propylene mouse cages with Carworth chrome-plated wire bar
covers. Cages were bedded with spruce planer shavings and all
animals were provided with Purina lab chow, and water or
coolant suspension, ad lib. Temperature in the animal room
was 20-22C and relative humidity was 10-40 percent. The
photoperiod was 16L8D (on at 0600 hr CST).
Coolant suspensions were produced in a continuous
flow contactor (Figure 1) which was constructed and operated
primarily for studies of chronic toxicity to fish. The
composition of equilibrium coolant added to this device is
shown in Rafale 1. Infrared and .ultra-violet spectrophotometry
and mass spectrometry show that the coolant suspended in water
was not demonstrably different in composition from bulk
coolant (no selective suspension or solution could be
demonstrated) (J.E. Guthrie, pers. comm.). The coolant
suspension made by this method is similar in settling times
and composition to that made in a device (Acres and Guthrie,
1968) v/hich simulates a pinhole leak in a reactor heat
exchanger (J.E. Guthrie, pers. comm.).
Coolant and river water were drawn from the device
into 4.55 i- glass jugs as required. One-tenth coolant was
composed of 1 part coolant suspension and 9 parts river
- 4 -
HEAD TANK15 I
u v.STERILIZER
40 WATT
AOUAFINE
SP-36-S
STRAINER
WATER SUPPLY
DIRECT FROMRIVER
HEATING ANOAERATION
TANK115 I
Q
FILTER
30 MICRON
GLASS FIBER
STIRRERHAYWOODGORDON
PO-2
STORAGE
TANK
50 I
FILTER
30 MICRON
GLASS FIBER
TO FISHTANKS
1-2 l/m
SOURCE OF
"RIVER WATER"SOURCE OF
" COOLANT"
Figure 1. Flow diagram of contactor for providing coolantsuspension. The contactor tank was emptiedweekly and 4.55 i of fresh equilibrium coolantadded .
- 5 -
water. Samples were taken from each jug and analyzed for
coolant content by the ultra-violet absorbance methods
(W.A. Boivin, pers. comra.). Concentration of coolant in
the full strength drinking solutions, based on 183 samples
taken between October 14, 1971 and November 7, 1972,averaged
0.97 mg/A. with a standard error of 0.10 mg/X..
TABLE 1
Composition (weight percent ± standard deviation) of bulkirradiated coolant (R.B. Stewart, pers. comm.) used asfeed stock for contactor. Coolant was taken directly
from the A circuit of WR1 reactor as required.
Volatiles
Biphenyl
Methylated Biphenyls
Hydrogenated Terphenyls
Meta-Terphenyl
Para-Terphenyl
Less Volatile thanP-Terphenyl
0.8
5.7
10.0
19.0
10.0
4.5
51.0
± 0.1
± 0.5
± 0.1
± 2.0
± 1.0
± 0.4
± 5.n
Drinking solutions were made available to the mice
in standard glass water bottles with rubber stoppers and
glass tubes. All materials in contact with coolant were
equilibrated before each use, to saturate absorbing surfaces.
The derivation of the groups of experimental
animals is shown in Figure 2. Fifteen pairs of mice were
selected from laboratory stock, and from their first litters
90 (45 males and 4 5 females) were randomly selected and
assigned to the three treatments. These mice, the C^
- 6 -
GENERATIONS
LABORATORY STOCK
(15 PAIR)
TIME
SEPT-PAIRED
C, GENERATION
PAIRED(15 PAIR)
REPLICATE I(22 PAIR)
vC 2
REPLICATE(22 PAIR)
PAIRED(15 PAIR)
REPLICATE I(22 PAIR)
REPLICATE 2(22 PAIR)
Vc3
kJ
PAIRED(15 PAIR)
C 2
REPLICATE I(22 PAIR)
VCZ
REPLICATE 2(22 PAIR)
SEPT.-BORN
OCT.-WEANED
EXPOSEDOCT.- TO
TREATMENT
NOV.-PAIRED
DECrBORN
.JAN -PAIRED
JAN.-BORN
•FEB.-PAIRED
F i g u r e 2 . D e r i v a t i o n o f t h e e x p e r i m e n t a l s t o c k .
- 7 -
generation, were held in unisex groups of 4 per cage, and
were exposed to the treatment for 30 days. They were then
randomly paired within treatments. From the young produced
in the first litter, 44 mice were randomly selected at
weaning and paired in each treatment, forming Replicate 1 of
the C- generation. From the second litter produced by the
C, generation, the same procedure was followed to produce
Replicate 2 of the C,, generation. The C, animals were then
discarded.
The C2 generation forms the life span and reproduction
part of the experiment. The life span, and total number and
biomass of young produced by these animals will be measured.
The birth date, number in the litter and total weight of the
litter are recorded at birth. At that time the weights of
the adults are also recorded. When the young are weaned at
21 days of age, number in the litter, percent males and
total weight are recorded.
Some of the CU mice were utilized for short-term
experiments. A weekly schedule of the number of animals
required was set up, and this number plus 10 percent was
selected randomly from the number to be weaned in a given
week. The selected animals were housed individually after
weaning, toe-clipped for identification, and held until
approximately 50 days of age. At that time the exact number
required was randomly selected, and the experiment began.
Only males were used in short-term experiments. Specific
methods utilized in short-term experiments are described in
conjunction with the results of the experiments.
In summary, the experimental design provides 132
pairs of mice (the C2 generation) exposed to experimental
conditions (coolant) since before conception, to yield data
on reproduction and life span. It also provides a large
supply of animals exposed to coolant for one generation longer
(the C3 generation) which are used for short-term experiments.
- 8 -
3. PALATABILITY OF COOLANT SUSPENSIONS
Since it was planned to administer irradiated HB-40
to mice in their drinking water it was necessary to determine
if such suspensions would be acceptable to them. The purpose
of this experiment therefore was to determine if: 1. house
mice can detect coolant suspensions in the concentrations used;
2. they avoid or prefer them when given a choice; 3. enforced
consumption of coolant causes a change in fluid intake.
3.1 METHODS
This experiment was carried out before the continuous
contactor was completed. Coolant suspensions were made by a
batch-contacting process (O.E. Acres, pers. comm.), and coolant
concentrations in the three treatments were 5.0 mg/£, 0.5 mg/x,
and 0 mg/jl (control) . Thirty male and 3 0 female Mus musaulus
weighing 20-30 g were selected for the experiment and held
individually in disposable cages.
To acclimate the animals to experimental conditions,
each was provided with two water bottles placed side by side
in the cage top for 10 days. Throughout the experiment all
water bottles were weighed to 0.1 g daily at 1000 hr, and the
amount of solution drunk from each bottle determined by
subtraction. Bottles were re-filled only when less than half
full. The experimental design is shown in Table 2. At the
beginning of phase I, drinking solution bottles were assigned
to the left or right position randomly, and left in that
position for the first 10 days of phase I. During the second
10 days of phase I, and all of phase III, the two drinking
solution bottles in the cage were switched in position, or
not switched, depending on the flip of a coin. In phase II
each animal had only a single bottle.
TABLE 2
E x p e r i m e n t a l d e s i g n . Each g r o u p w a s c o m p o s e d of 10 m a l e and 10f e m a l e a n i m a l s a s s i g n e d r a n d o m l y at t i m e s m a r k e d R. T h e a n i m a l sw e r e g i v e n the d r i n k i n g s o l u t i o n s s h o w n in the body o f the t a b l e
A\.
I
Group
Group
Group
1
2
3
Acclimation(10 days)
water-water
water-water
water-water
Phase I(20 days)
water-water
water-0.5 coolant
water-5.0 coolant
0
5
Phase II(20 days)
water
. 5 coolant
. 0 coolant
Phase III(10 days)
water-water
water-0.5 coolant
water-5.0 coolant
I
I
- 10 -
3.2 RESULTS
Phase I
The mice given a choice between two bottles containing
water (Group 1, Table 2) did not drink significantly more from
one than from the other (t = 0.62, 19df, p > 0.05). Those
given a choice (Table 3) between 5.0 mg/& coolant or water drank
more water (t = 6.00, 19df, p < 0.01), as did those given a
choice between 0.5 ppm coolant and water (t = 3.30, 19df,
p < 0.01). Randomly moving the drinking bottles during the
last 10 days did not affect this preference.
Phase II
Analysis of variance indicated that there were no
significant differences in liquid consumption (Table 3)
(F = 2.99; 2, 59df; p > 0.05). One male, however, did not
drink any of the 5.0 mg/s, coolant and died on day 6 of the
experiment.
Phase III
When returned to a free choice situation (Table 3) ,
none of the groups drank significantly more water than
coolant (Group 1: t = 1.99, Group 2: t = 0.91, Group 3:
t = 1.43, 19df for each, all p > .05).
- 11 -
TABLE 3
Mean and standard deviation of the grams of liquid consumedper mouse per day. Each mean is based on 20 animals.
Group
1
Group
2
Group
3
4
5
6
Phase
water
.6 ± 2.
water
.2 ± 2
water
.4 ± 2
6
0
7
5
.9
I
water
3.4 ± 2.0
.5 coolant
2.6 ± 2.1
.0 coolant
1.7 ± 1.9
Phase II
water
7.2 ± 1.7
0.5 coolant
7.0 ± 1.9
5.0 coolant
7.0 ± 2.0
4
4
3
Phase
water
.7 ± 2.
water
.5 ± 2.
water
.9 ± 1
2
2
.4
III
water
3.3 ± 2.4
0.5 coolant
3.5 ± 2.0
5.0 coolant
3.4 ± 1.6
- 12 -
3.3 DISCUSSION
The results indicate that under the experimental
conditions used, Mus could detect coolant concentrations as
low as 0.5 mg/s,. When given a choice between water and
coolant, they preferred water, but not to the complete
exclusion of coolant. When forced to drink coolant exclusively,
they did not decrease their liquid intake. After having been
forced to drink coolant, the animals no longer discriminated
against coolant when given a choice between it and water.
Although mice can detect coolant in the concentrations used,
it is apparently only slightly unpalatable.
Therefore it will be possible to study chronic
effects of coolant ingestion by mice, since they accept the
solution readily and do not decrease their water consumption.
No harmful effects of ingestion of these concentrations of
coolant were observed. The one mouse which died refused to
drink and probably died of dehydration.
3.4 CONCLUSIONS
Administration of coolant suspensions in water at
concentrations similar to those used here is an acceptable
procedure since there was no apparent decrease in fluid
intake.
- 13 -
4. ESTIMATION OF COOLANT INGESTION RATE
This phase of the study was designed to estimate
the amount of coolant ingested per kg of body weight per day,
to allow comparison with other toxicological studies. It was
not practical to measure individual water (and thus coolant)
intakes continuously in the experimental animals. Ingestion
estimates were made, instead, by determining the relationship
between fluid intake and weight in lactating and nonlactating
animals.
Under constant environmental conditions, diet and
activity, water intake is dependent upon: 1. body mass, which
is directly related to skin surface, lung surface and metabolic
rate; 2. growth, which necessitates the addition of approximately
0.78 g of water per gram of weight gained; 3. lactation, which
involves the ingestion of large amounts of water for milk
production. The coolant dose to male mice can be estimated
simply from the relationship between body mass and water intake,
since males grow relatively little during adult life, and do
not lactate. The dose to the females, however, must take into
account all three factors, since females show cyclic growth
during pregnancy, and may be lactating. The dose to the
pre-weaning young cannot be estimated, since it is not known
if milk contains coolant.
4.1 METHODS
Water consumption by pairs of C~ mice was measured
by the method of weight difference. On a given day, the male
and female in a cage were weighed individually, any young
present weighed as a group, and the water bottle was weighed.
Twenty-four hours later, the water bottle was re-weighed, and
- 14 -
the difference in weight recorded as water consumption. Over
800 water consumptions were determined during April - August,
1972.
The results were divided into two groups: 1. those
from cages in which there were no young mice; 2. those from
cages in which young mice (2-12 days old) were present.
Regressions were then calculated separately for these groups
to determine the relationships between weight and water
consumption.
4.2 RESULTS
The regression of y (water consumption) on x (weight)
for non-lactating animals (males and females) was y1 = 0.17x + 3.39;
that for lactating animals was: 'y1 = 0.25x +0.83. F tests
indicated that both regressions were significant (non-lactating:
F = 63.8; 1, 294df; p<0.01; lactating: F = 249.1; 1, 309df-
p<0.01). These relationships were used to determine the amounts
of fluid consumed by lactating and non-lactating mice of
various weights. Since a known amount of coolant was present
in each gram of drinking water, the intake in terms of mg
coolant per kg body weight could be calculated. This relation-
ship between weight, lactation and coolant dose is shown in
Figure 3.
4.3 DISCUSSION
The results indicate that the coolant dose to adult
animals varied between approximately 0.22 and 0.27 mg/kg.
Figure 3 shows that larger animals ingested less coolant per
gram body weight than smaller ones, and that a female nursing
a litter received a higher dose than a non-lactating animal
- 15 -
of the same total biomass. The difference associated with
lactation was less than expected, and may indicate a reduced
water loss per individual as a result of the animals huddling,
producing a modified macrohabitat in the nest. These, findings
agree with previous observations, and increase confidence in
the methodology. Consideration of the error bands of the
regressions indicated that a predicted dose rate of 0.22 mg/kg
would not be significantly different from one of 0.27 mg/kg.
We therefore cannot say that any group of adult animals was
exposed to a higher dose rate than any other.
The younger animals were, however, exposed to higher
doses, since they drink considerably more water in relation to
their weight (Figure 3). It appears that each animal goes
through a period of high dose rates, from the time it is
weaned until it weighs 25 - 30 grams, and from then on is
exposed to a relatively invariable dose rate. Age and weight
are therefore important variables controlling coolant dose,
but female mice are not exposed to a significantly higher dose
because of lactation.
4.4 CONCLUSIONS
Young animals go through a period of high coolant
intake per kg body weight during the time they are growing
rapidly and have a high surface to volume ratio. The adult
animals, however, are all exposed to the same intake of
approximately 0.25 mg/kg/day.
V.o>
E
>Q
tELUQ.
DO
SE
l-2
CO
OL
0-35
0-30
0.25
0.20
1 '
\
\
AE \
1 I
1
\
\
\
|
1 1
\
| I
1 ! 1 1 I 1 1
LACTATING FEMALES AND YOUNG
NON-LACTATING INDIVIDUAL?
TD
r — T
B
I 1 1 1 i 1 |
1 1
—
/A
1 I
CTl
I
10 20 30 40WEIGHT (g)
50 60 70
Figure 3. The relationship between coolant dose (mg/kg body weight) and bodyweight for lactating females with young, and for non-lactating miceThe doses are calculated for a concentration of coolant in waterof 1.0 mg/ji,. The letters indicate mean weights of various groupsanimals: A. mean weight of 26 females immediately prepartum. B.females postpartum. C. 26 males the same age as the females. D.of 343 lactating females plus litters. E. 332 animals at 21 daysage. F. 118 males at 44 days of age.
ofsameweightof
- 17 -
5. INDIVIDUAL WEIGHT FROM BIRTH TO WEANING:THE EFFECTS OF NUMBER OF SIBLINGS.
Body weight during the period from birth to weaning
should be a good indicator of possible damage to an organism,
since it shows the combined effects of several physiological
functions of both the mother and offspring. Essentially,
birth weight reflects the ability of the mother to nourish the
fetus, and, considering litter size as a variable, whether
this capability is reduced by the stressor (chronic ingestion
of coolant) at high levels of demand. Under constant environ-
mental conditions, the growth rate of a mouse for its first
two weeks of life depends on the milk supply of its mother.
During the third week of life, the young shift their diet from
milk to solid food, resulting in the growth rate becoming more
dependent on the abilities of the young.
In studies examining the effect of a stressor on
pregnancy and subsequent growth of young, it is often impossible
to determine whether the effect is on the mother, offspring,
or both. Other techniques could be developed to answer this
question, but for ecological purposes the pathway of cause and
effect would matter little if growth was retarded by the
stressor during this period of life. In a natural situation,
weaning, dispersion, and establishment of a home range is a
period of high mortality in young mammals. The additional
disadvantage of suboptimal weight during this time would further
increase mortality.
5.1 METHODS
All young born to the C2 mice between 29 February
and 12 May were included in this study. Young were weighed
individually to 0.1 g 24 to 48 hours after birth, and at
- 18 -
approximately 3 day intervals until weaning. The parameter
"litter size" is the number of young in the cage, alive or
dead, at 24 hours of age. In a small number of cases some
young may have been eaten before the litter count was made.
In the one or two cases in which the female died before the
young were weaned the young were excluded from this analysis,
5.2 RESULTS
The results of a multilinear regression of weight
on age and litter size for each treatment are shown in Table 4
and Figure 4. Increase in weight with age can be seen, as
well as a decrease in weight at both birth and weaning with
increasing litter size. The decremental effect of larger
litter size on weight was greater in animals ingesting
coolant (slope of -0.3792 vs -0.2634, Table 4) than in those
drinking water. The coolant animals also had a slight
tendency to gain weight more rapidly (slope of 0.3298 vs
0.3567, Table 4). There is little evidence for a deleterious
effect of coolant, since at nearly every combination of age
and litter size the animals ingesting coolant weighed more
than the controls. However, at litter sizes over 10, and
ages of less than 10 days the coolant animals weighed less.
Multilinear regression was satisfactory as an
initial description of the relationships between the variables,
but comparison of the data with the fitted planes indicated
that the relationship was not completely planar. Weights at
low litter size - low age and at high litter size - high age
were overestimated, while those at the other two extremes
were underestimated.
- 19 -
AGE(days)
WATER
1/10 COOLANT
COOLANT
13 9 7 5LITTER SIZE (number)
Figure 4 . Multilinear regression of weight on ageand litter size for the three treatments
- 20 -
TABLE 4
Multiple regression of weight on age and litter size.
Age slope (b)
Litter size slope (b)
Intercept
N
F
Water
0.3298
-0.2634
4.2995
2616
4032
1/10 Coolant
0.3267
-0.3327
5.0471
2871
6203
Coolant
0.3567
-0.3792
5.4378
3002
4836
The data were therefore partitioned into 3 day age
classes and weight was regressed on litter size for each age
class and each treatment (Figure 5, Table 5). These regressions
show that the inverse relationship between weight and litter
size, although present at birth, increased in magnitude with
age (slope at 0-2 days of -0.06 to -0.11 vs slope at 21-23 days
of -0.49 to -0.69, Table 5).
As in the multilinear regressions, the estimated
weights of mice ingesting coolant were higher than the controls
at most combinations of age and litter size. The exceptions
are at ages from 4 to 10 days in litter sizes over 10 (Figure 5)
Growth rates between 13 and 22 days of age were nonlinear
(Figure 5). Between 13 and 19 days of age, the growth rate
decreased from its previous level, but from 19 to 22 days it
increased to approximately its earlier level. This may reflect
the changeover from complete dependence on milk to dependence
on solid food.
- 21 -
WATER
1/10 COOLANT
COOLANT
Figure 5. Regression of weight on litter size atvarious ages for the three treatments.
- 22 -
TABLE 5
Regressions of weight on litter size,formula, sample size and calculated
The regressionF are shown.
Age
0-2
3-5
6-8
9-11
12-14
15-17
18-20
21-23
9-
y =
/\y =
9 =
9 =
9 =
9 =
9 =
Water
2.46X -
N =
F =
3.94X -
N =
F =
5.76X -
N =
F =
8.12X -
N =
F =
10.06X -
N =
F =
10. 95X -
N =
F =
11. 25X -
N =
F =
13.02X -
N =
F —
0.06
404
80
0.07
327
28
0.11
332
25
0.25
369
119
0.39
290
152
0.44
358
259
0.45
204
101
0.49
332
142
9
9
9
9
9
9
9
9
1/10
= 2.
= 4.
= 7.
= 8.
= 10.
= 11.
= 12
= 14.
Coolant
55X -
N =
F =
64X -
N =
F =
58X -
N =
F =
90X -
N =
F =
42X -
N =
F =
05X -
N =
F =
.49X -
N =
F =
41X -
N =
F =•
0.07
440
44
0.14
421
62
0,34
315
263
0.36
388
232
0.44
362
266
0.45
371
198
0.52
255
177
0.61
318
113
y
9
9
9
9
9
9
9
Coolant
= 3.
= 5.
= 7.
= 10.
= 11.
= 11
= 12
= 15
07X -
N =
F =
27X -
N =
F =
41X -
N =
F =
05X -
N =
F =
24X -
N =
F =
49X -
N =
F =
.67X -
N =
F =
. 61X -
N =
F =
0.11
410
90
0.20
412
140
0.28
410
159
0.45
404
244
0.47
352
206
0.46
395
173
0.51
255
160
0.69
363
.192
- 23 -
To further examine the relationships between the
variables, the data were partitioned by litter size, and
weight was regressed on age (Figure 6, Table 6). More
variability is shown by this method of analysis, possibly
because some of the sample sizes were smaller (Table 6) .
However, the same relationships were present, with growth being
more rapid in animals from smaller litters. As was seen in
the other analyses, at most combinations of age and litter
size, animals exposed to coolant weighed more than the controls.
This analysis showed a greater difference between coolant and
control animals, but the difference was concentrated in litters
of 3-4. In this range of litter sizes the animals exposed to
coolant showed a faster growth rate (slope 0.56 and 0.55 vs
0.48 and 0.41) than the controls.
5.3 DISCUSSION
Considering the results of all three analyses
together, there appears to be no evidence that exposure to
coolant decreases the weight of animals at birth or at any
point up to weaning. The only non-homogeneity of any
magnitude is the more rapid growth of animals in small litters
exposed to coolant. This led to underestimation of the
weights of young mice from large litters in the multilinear
regression (Figure 4). It may also have contributed to the
underestimation of weight in medium-aged mice from large
litters in the regression of weight on litter size (Figure 5).
No biological explanation is proposed for the rapid weight
gain of coolant-exposed mice from small litters. Above a
litter size of 4, the variation of the coolant group is
similar to that in the other 2 groups, and no trends other
than a gradual decrease of weight with increasing litter size
can be seen. It is probable that the extremely rapid weight
gain of small litter size groups is a chance fluctuation
associated with the small sample size. Thus we did not
- 24 -
22
AGE(doys) 10
WATERI/IO COOLANT
COOLANT
LITTER SIZE (number)
Figure 6. Regression of weight on age at variouslitter sizes for the three treatments.
- 25 -
TABLE 6
Regressions of weight on age. The regressionformula, sample size and calculated F are shown.
Littersize
1
2
3
4
5
6
7
8
9
10
11
12
13
9
9
9
9
9
y
9
9
9
Water
= 1.77X +N =F =
= 4.14X +N =F =
= 2.41X +N =F =
= 2.60X +N =F =
= 2.46X +N =F =
= 1.68X +N -F =
= 2.34X +N =F =
= 2.28X +N =F =
= 2.38X +N =F =
' = 1.67X +N =F =
v = 2.25X +N =F =
k = 1.88X +N =F =
' = 1,78X +N =F =
0.491223
0.29189.4
0.48105458
0.41121275
0.402751392
0.422461024
0.31182299
0.343691560
0.295692024
0.261921887
0.24308522
0.2584901
0.33105433
1/10
9= 1.
'y* = 2.
9= 2.
9 = 2.
-y = 2
"y = 2
"y = 1
'y- = 1
•y = 1
•y^ = 1
-y = 1
9 = i
Coolant
19X +N =F =
24X +N =F =
-
97X +N =F =
21X +N =F =
85X +N =F =
53X +N =F =
.95X +N =F =
.76X +N =F =
• 97X +N =F =
.72X +N =F =
.78X +N =F =
• 44X +N =F =
0.58118.1
0.5138320
0.9728354
0.4135379
0.36270724
0.392801011
0.344323098
0.344501926
0.286542009
0.304193326
0.282181542
0.483782
9 =
9 =
9 =
9-
9 =
9 =
9 =
9 =
•*.
9 =
9 =
Coolant
0.
2.
2,
2.
2.
2
2
2
1
1
1
-
15X +N =F =
83X +N =F =
60X +N =F =
18X +N =F =
27X +N =F =
44X +N =F =
.55X +N =
.19X +N =F =
.78A +N =F =
.95X 4N =F =
.93X -tN =F =
0.27460
0.56105369
0.5588338
0,44200523
0.422522770
0.373151330
0.345991501
0.365281380
0.282101071
0.27352
• 1 1 2 2
0.29= 336= 2466
- 26 -
observe a decrease in growth as was reported by Cornish (1962) .
This is not surprising since our dose rate, although chronic,
was only about 1/1000 of the dose rate in the 30 day
experiment in which they noted the effect.
The observed slowing of weight gain in all treatments
at the time of transition from milk to solid food, and the
subsequent return to a faster rate, indicate that exposure to
coolant is not detrimental to young mice.
5.4 CONCLUSIONS
Compared to the controls, no decrease in weight at
birth or weaning was seen in animals exposed to coolant at any
litter size. Exposure to coolant, of the concentration used,
therefore does not cause a decrease in fitness, as measured by
weight gain of young, in either the mothers or the young mice.
6. LITTER SIZE, NUMBER OF LITTERS,WEIGHT OF YOUNG AND SEX RATIO
This part of the study describes effects of chronic
ingestion of coolant on lifetime reproductive potential of
Mus. Since a population in the field maintains itself by
keeping natality at least equal to mortality, the rates
measured here are fundamental to a population's ecological
success. Some of the parameters estimated (number of litters
per female, number of young per litter) indicate quantitative
success, whereas weight of the offspring is more a measure of
quality, and is related to the ability of the young to
survive. The sex ratio of the young reflects the proportion
of females available in the next generation to produce young.
- 27 -
6.1 METHODS
Data were recorded for all litters produced in both
replicates of the C2 generation. All pairs were checked each
weekday for births. Weekend births could normally be attributed
to either Saturday or Sunday on the basis of development of
the young. Young were counted and weighed as a group, to the
nearest 0.1 g, when one to two days old. At 20 to 22 days of
age, the young were again counted, weighed and sexed, and then
weaned. Sex ratio was expressed as percent males, and normalized
by an arcsin transformation (arcsin \ percentage). Data were
accumulated in periods 40 days in length, and analyzed primarily
by analysis of variance.
6.2 RESULTS
Ingestion of coolant caused no decrease in the
number of litters per female (Table 7). Females drinking
coolant produced significantly more litters than did the
controls; females drinking 1/10 coolant also produced more
litters. Tables 8 and 9 show the results for birth weight,
number born, weaning weight, number weaned and arcsin percent
males for each 40 day period in each replicate. Although four
significant differences appear in these tables, they pertain
to four different parameters, and there are no apparent trends
that indicate impairment by coolant.
The grand means and overall tests for the five
parameters for the entire 200 day period are shown in Table 10,
There are no indications of impairment, and both of the
significant differences indicate that weight of the young,
at both birth and weaning, is greater in animals exposed to
coolant.
- 28 -
TABLE 7
Number of litters per female in the experimentalgroups. At least two of the means are significantly
different (F = 226.90; 2, 27df; p < 0.05).
to
(da]
iod
u0)&
<D
•H
Replicate
0 - 4 0
41 - 80
81 - 120
121 - 160
161 - 200
Mean
Water
1
0.90
1.00
1.55
1.29
1.47
1.17 :
0
1
1
1
1
t 0
2
.90
.05
.11
.22
.20
.07
1
1.
1.
1.
1.
1.
1.
1/10
00
05
29
23
41
27 ±
2
1.
1.
1.
1.
1.
0.
00
25
56
38
50
06
1
0.
1.
1.
1.
1.
1.
Coolant
79
11
37
61
22
32 :
2
1.
1.
1.
1.
1.
t 0.
00
44
65
47
53
08
TABLE 8
Number born, number weaned and arcsin percent males weaned for both replicatesfor the first 200 days of the experiment. The sample size is in parenthesesand the mean is shown (± SE). An asterisk by an F value indicates p < 0.05.
Rep
Rep
Rep
Rep
Rep
Rep
NUMBER BORN
I
II
NUMBER
I
II
ARCSIN
I
II
Control1/10CoolantF value
Control1/10CoolantF value
WEANED
Control1/10CoolantF value
Control1/10CoolantF value
MALES
Control1/10CoolantF value
Control1/10CoolantF value
0 - 4 0
8.37 ± 08.00 ± 09.59 ± 0
0.13
7.42 ± 09.85 4 07.47 ± 0
*6.96
7.90 ± 07.29 ± 06.94 ± 0
0.51
6.62 4 08.62 ± 07.10 ± 0
2.94
42.63 ± 344.93 ± 342.81 ± 3
0.13
57.43 ± 443.23 4 352.32 ± 1
4.66
days
.56
.69
.73
.68
.33
.53
.63
.63
.76
.55
.62
.59
.40
.51
.70
.11
.88
.59
(19)(18)(18)
(19)(20)(22)
(18)(17)(16)
(16)(16)(19)
(18)(17)(16)
(16)(16)(19)
41 -
6.60 47.35 ±7.32 ±
0.
8.i3 ±8.20 ±7.48 ±
0.
6.21 46.68 ±7.27 ±
0.
7.31 ±8.00 ±6.76 ±
0.
49.68 ±52.64 ±37.12 ±
*7.
45.83 ±43.87 ±52.32 ±
1.
80
000
40
100
17
00077
000
80
343
21
424
46
days
.59
.84
.60
.73
.65
.71
.53
.71
.59
.72
.64
.73
.65
.42
.27
.00
.59
.26
(20)(22)(22)
(21)(20)(23)
(19)(19)(22)
(19)(20)(21)
(19)(22)(22)
(19)(20)(21)
788
987
788
787
464244
524647
81 - 120 days
.35 ± 0
.77 i
.50 i0
.47 i
.91 i
.81 i0.
.42 i
.00 4
.32 ±0.
.36 ±
.88 ±
.37 ±2.
.37 ±
.93 ±
.10 ±0.
.46 ±41 404 41.
: 0: 058
200
59
00047
000
41
2.2.2.
41
3.3.2.
16
.58
.60
.74
.09
.52
.61
645781
694755
802090
640928
(26)(22)(22)
(19)(24)(27)
(26)(21)(22)
(19)(25)<27)
(26)(20)(22)
(19)(23)(27)
889
888
789
798
464549.
43.46.42.
121
.14
.27
.22C
.05
.18
.190
.75
.00
.391
.63
.0583
1
900366
0
048726
0
- 160
± 0± 0± 0.68
± 04 0± 0.01
± 04 0± 0.61
± 0± 04 0.24
4 3.4 3.± 2..61
4 3.4 2.4 3..65
.79
.68
.73
.78
.66
.80
.62
.6877
546771
603030
663506
days
(21)(22)(27)
(21)(22)(21)
(20)(22)(23)
(19)(21)(23)
(20)(22)(23)
(19)(20)(23)
678
887
678
887
47.47.49.
45.44.46.
161
.76
.63
.19
- 200
± 0± 04 0
1.05
.64
.88
.95C
.60
.45
.001
.233150
0
504520
0
396761
0
± 0± 0± 01.59
± 0± 0± 0.13
± 0± 04 0.52
± 3.± 3.4 4..07
t 3.4 1.4 3..12
.58
.74
.80
.65
.54
.68
.55
.71
.76
585272
043229
199938
days
(25)(24)(21)
(22)(26)(22)
(25)(22)(21)
(22)(26)(20)
(25)(22)(21)
(21)(26)(20)
TABLE 9
Weight of young born and weaned for both replicates for the first 200 daysof the experiment. The sample size is shown in parentheses and the mean
is shown (± S E ) . An asterisk by an F value indicates p < 0.05.
Rep
Rep
Rep
Rep
BIRTH
I
II
WEIGHT
Control1/10CoolantF value
Control1/10CoolantF value
WEANING WEIGHT
I
II
Control1/10CoolantF value
Control1/10CoolantF value
131413
131815
686659
627166
0 •
.13
.50
.470
.83
.58
.992
.58
.37
.600
.03
.86
.730
- 40
± 1.± 1.± 1..37
± 1.± 1.± 1..54
± 5.± 4 .± 5..75
± 4.± 5.± 5..84
days
222407
161520
609048
135853
(19)(18)(18)
(19)(20)(21)
(18)(17)(16)
(16)(16)(19)
41 -
12.9714.7016.17
1
14.8017.2817.28
0
60.9059.7472.49
2
72.0079.2473.42
0
- 80
± 1.± 1.+ 1..40
± 1.± 1.± 1..94
± 3.± 5.+ 4..52
± 5.± 5.± 7..38
days
226026
511962
905706
791331
(20)(20)(22)
(21)(20)(23)
(19)(19)(22)
(19){20)(21)
161818
171818
698277
769779
81 -
.34
.30
.990
.10
.90
.600
.57
.34
.361
.85
.15
.78*3
• 120 days
+ 1± 1± 1.01
± 1± 1± 1.44
± 4± 4± 4.80
.00
.16
.29
.52
.11
.50
.79
.73
.85
± 5.78+ 4± 5.78
.93
.74
(26)(22)(22)
(19)(24)(27)
(26)(21)(22)
(19)(23)(27)
121
17.6318.8420.45
18.4520.4?20.70
80.8887.4594.05
81.2995.4999.60
- 160 days
± 1.90± 1.41± 1.62
0.75
± 1.84± 1.61+ 1.60
0.53
+ 5.91+ 6.14± 6.550.70
±5.59± 5.13± 6.64
2.54
(21)(22)(27)
(21)(20)(23)
(20)(22)(23)
(19)(20)(23)
161 -
15.6217.1818.05
0
18.8419.3818.41
*7
72.3478.5378.08
0
86.1675.6571.86
1
200 days
± 1.57± 1.66± 2.00.51
± 1.92± 1.30± 2.18.47
+ 5.90±•6.12± 6.32.19
± 6.84± 4.84± 6.88.40
(25)(24)(21)
(22)(26)(22)
(25)(22)(21)
(22)(26)(20)
I
o
- 31 -
TABLE 10
Grand means for both replicates for the first 200 daysof the experiment. Sample size is in parentheses,
and the F resulting from analysis of varianceis shown (Vindicates p < 0.05).
Birth weight
Number born
Weaning weight
Number weaned
Arcsin percentmales
(
15
7
73
7
47
Control
.94
.93
.34
.41
.53
(213)
(214)
(203)
(199)
(202)
17
8
80
8
46
1/10
.91
.37
.01
.06
.31
(216)
(219)
(205)
(209)
(205)
(
17
8
78
7
46
-oolant
.98
.02
.25
.69
.32
(226)
(225)
(214)
(218)
(214)
8
0
3
2
0
F
.55*
.88
.06*
.45
.76
- 32 -
6.3 DISCUSSION
The observations on this group of animals will be
continued for the entire life of the females; therefore these
results are tentative. There is, however, no indication of
any detrimental effect of coolant on any of the parameters
estimated. On the contrary, animals exposed to coolant appear
to not only produce more litters in a given time, but also
produce heavier offspring both at birth and weaning. That
this is a real effect of ingestion of coolant cannot be
accepted as demonstrated until the experiment has been completed.
7. SURVIVAL FOLLOWING X-IRRADIATION
It is well known that many factors, including general
3tate of health, diet and endocrine status influence the
radiosensitivity of mammals. It may be that any factor, with
the exception of hypoxia, hypothermia and the radioprotective
drugs, tending to disturb the homeostasis of an animal would
increase its radiosensitivity. Fundamentally, this implies
that resources utilized to maintain homeostasis against one
stress cannot be deployed against another stressor. In the
present experiment, exposure to organic coolant is the first
stress, and to X-irradiation the second. If exposure to
organic coolant at the levels used in this study is a stress
its severity can be measured by the decrease in the amount
of radiation required to kill 50 percent of the test animals,
over a 30 day period.
- 33 -
7.1 METHODS
Male C_ mice housed individually were irradiated at
approximately 50 days of age. On the day of irradiation, they
were removed from their cages, placed in individual 1/2 pint
cylindrical cardboard containers, moved to the X-ray machine,
irradiated, and then returned to their home cages. All
irradiations were conducted between 0900 and 1100 hrs, and
the animals were held for one to two hours in the containers.
During irradiation, mice from each of the 3 treat-
ments were put into individual cartons and the 12 cartons
placed on a turntable which revolved at 1.1 rpm. The rotation
of the table compensated for any inhomogeneity in the radiation
field, and the dose rate averaged 50 rads/minute at the centers
of the cartons. The radiation dose was given by a Westinghouse
X-ray therapy unit operated at 250 kV and 15 mA with 1 mmCu
and 1 mmAl filtration, and irradiation was to the dorsal surface
of the mice. Dose delivered to the mice was measured by a
Victoreen integrating meter with a remote ion chamber placed
in a constant position relative to the cartons.
Irradiated animals were checked daily for 30 days,
and deaths were recorded. Animals dying overnight were scored
as dead on the day of discovery.
7.2 RESULTS
Ages and weights of the animals at the time of
irradiation showed no systematic differences between treat-
ments or doses (Table 11). The animals exposed to coolant
had a higher percentage surviving than those exposed to
water at all radiation doses except 400 rads. Other than in
the 400 and 500 rad groups, time to death was relatively
constant in the three treatments.
TABLE 11
Sample size, mean (± SE) age and weight, percent survival andtime to death for animals irradiated at various dosages.
DOSE(rads)
400 500 600 700 750 800 900
Numberof
Animals
Age(Days)
at irradiation
Weight(g)
at irradiation
PercentSurvival(30 days)
Time toDeath(Days)
W1/10C
W
1/10
C
W
1/10
C
w1/10c
w1/10
c
14
13
12
47.6 ± 0.6
48.6 ± 0.4
49.0 ± 0.5
24.4 ± 0.8
24.1 ± 0.9
24.0 ± 0.7
100
100
91.7
15
14
13
49.6 + 0.7
49.6 + 0.7
49.6 ± 0.6
24.1 + 0.8
24.5 + 0.8
23.5 + 1.0
93.3
100
100
13
14
15
15
48.6 ± 0.6
49.0 ± 0.6
49.2 ± 0.4
22.8 + 0.8
24.3 ± 0.8
22.3 ± 0.6
78.6
86.7
93.3
12.3 + 0.7
19
13
27
28
29
50.9 ± 0.4
50.8 ± 0.4
50.0 ± 0.4
23.2 + 0.5
23.6 + 0.3
23.9 + 0.5
63.0
78.6
93.1
13.9 ± 0.6
12.2 ± 0.4
8.0 ± 0.2
13
14
14
51.1 + 0.4
49.8 ± 0.5
50.4 + 0.4
22.5 + 1.4
24.1 ± 0.6
24.0 ± 0.7
30.8
42.9
35.7
13.8 + 1.0
11.9 + 1.1
15.9 ± 1.9
25
28
26
49.8 + 0.4
50.4 + 0.3
49.8 ± 0.4
22.5 ± 0.5
23.7 ± 0.4
23.6 ± 0.7
24.0
39.3
50.0
12.8 ± 0.4
12.3 ± 0.8
12.8 * 0.5
15
14
16
50.3 ± 0.6
50.4 + 0.7
49.5 ± 0.5
23.0 ± 0.7
24.8 ± 1.0
23.3 ± 0.9
13.3
14.3
18.8
13.5 ± 0.6
13.8 + 1.2
12.5 * 0.6
- 35 -
Probit analysis showed coolant animals had the
highest LD 5 0 / 3 Q value and water the lowest, but the 95 percent
confidence limits for all treatments overlapped (Table 12 and
Figure 7).
7.3 DISCUSSION
The LD5Q ,3Q estimates for both 1/10 and coolant
treatments were higher than for water, but the 95 percent
confidence intervals overlapped. The groups exposed to
coolant had a higher percentage surviving at all doses except
400 rads. There is thus no indication of a deleterious effect
of coolant on survival after X-irradiation. Chronic ingestion
of coolant did not, therefore, stress the animals, as expressed
by their ability to survive X-irradiation.
7.4 CONCLUSIONS
No deleterious effect of coolant on post-irradiation
survival was observed. On the contrary, mice exposed to
coolant had somewhat higher LD5- ,__s than those exposed to
water.
TABLE 12
Slope, intercept, and L^g/on valuesfrom probit analysis.
Slope(b)
Intercept
LD50/30 ± 95%confidencelimits (rads)
Water
0.00723
-0.25173
726 ± 50
1/10
0.
-1.
764
Coolant
00836
38945
± 36
Coolant
0
-0
00664
30284
799 ± 56
- a e -
60
5 soo
&
3 400 1
CO
o
2 30o<E111
a.
20
10
WATER
1/10 COOLANT
COOLANT
_L300 400 800 900 1000500 600 700
DOSE (rods)
Figure 7.
Relationship between probability of death and X-radiation dose formice exposed to the three treatments.
- 37 -
8. KIDNEY FUNCTION
Since components of irradiated HB-40 have been
reported to cause renal pathology at high dose rates, this
aspect of the study was designed to investigate effects of
chronic low doses of coolant on renal water retention. The
simple method of giving the experimental animals progressively
more concentrated solutions of sodium chloride in water was
chosen. This has the same effect on the animal as the
procedurally more difficult method of restricting water intake,
since as the salt concentration increases, higher percentages
of the water ingested must be used to form urine containing
the salt. Eventually, a point is reached at which the
solution contains so much salt, that water intake is
insufficient to permit excretion of the salt load as well as
normal metabolic products. At this point, the animal will
either continue drinking the solution and make up the water
deficit needed to produce urine from its body water, thereby
losing weight, or it will cease drinking and use body water
to excrete other materials, and also lose weight. In some
cases, animals will also cease eating, and maintain themselves
on stored fat, but this fast also results in weight loss. If
maintained for a sufficiently lengthy period, any of these
responses result in death.
The rationale of the experiment was, therefore, that
animals with coolant-impaired kidneys would be able to excrete
less salt into each gram of water lost as urine. They would
therefore lose weight, and die at an earlier date than the
controls.
- 38 -
8.1 METHODS
Male C^ mice housed individually were weighed at
1530 hr on the Thursday nearest their 45th day of age and,
regardless of experimental group, given a bottle containing
a weighed amount of water. On the following Monday at 0900 hr,
the animals and water bottles were reweighed. The animals
were then given weighed amounts of 7.94 g/l NaCl solutions
(0.25 osm) to drink. The following Thursday at 1530 hr the
same procedure was followed, except that the animals were
given a drinking solution of 15.88 g/£ (0.50 osm). This
procedure was repeated (Table 13) until the animals had been
exposed to a NaCl solution of 48.44 g/£ (1.50 osm), at which
time the experiment was terminated. The death dates of
animals that died during the experiment were recorded. Only
the data pertaining to animals that survived the entire period
were used to calculate weight loss, and fluid consumed.
3.2 RESULTS
The mean ages of the animals in the three treat-
ments did not vary significantly (water: X = 43.67 ± 0.35
days; 1/10: 44.26 ± 0.29 days; coolant: 43.53 ± 0.28 days;
F = 1.59; 115, 2df; p > .05). Approximately 20 percent of
the animals in each experimental group died (Table 14), and
it is apparent that there was no increase in death rate due
to coolant ingestion.
The peak weight of most of the animals was reached
when they were drinking 0.75 osm solutions (Table 15). This
indicates that salt solutions up to, and including this
concentration, contain enough water for growth and body
maintenance. The animals in the three treatments did not
reach peak body weights at different salt concentrations
(X between water and 1/10 = 2.42, 3 df, p > 0.05), indicating
no treatment effect.
- 39 -
TABLE 13
The concentrations of salt in the drinking solutionsand the amount of time the animals were
exposed to each solution.
Drinking
g NaCl/Jl H2
0
7.94
15.88
23.82
32.29
40.36
48.44
Solution
0Osmolarity
osm
0
0.25
0.50
0.75
1.00
1.25
1.50
Time started
Thurs.
Mon.
Thurs.
Mon.
Thurs.
Mon.
Thurs.
1530
0900
1530
0900
1530
0900
1530
Time
Time
Mon.
Thurs
Mon.
Thurs
Mon.
Thurs
Mon.
ended
0900
. 1530
0900
. 1530
0900
. 1530
0900
Hoursexposed
89:30
78:30
89:30
78:30
89:30
78:30
89:30
TABLE U
Number and percentage of animals from the different treatmentsthat survived »arious salt concentrations
in their drinking water.
Drinking Solution
Osmolarityosm
0
0.25
0.50
0.75
1.00
1.25
1.50
Number of Animalsat start
Water
No.
49
49
49
49
47
44
38
49
%
100
100
100
100
96
90
78
1/10
No.
48
47
47
47
45
43
37
48
%
100
98
98
98
94
90
77
Coolant
No.
53
53
53
53
53
46
43
53
%
100
100
100
100
100
87
81
- 40 -
TABLE 15
NaC"! concentrations at which animals from thedifferent treatments reached their peak weight.
Drinking Solution
Osmolarityosm
0
0.25
0.50
0.75
1.00
1.25
1.50
Number of Animals
Water
No.
0
5
4
39
1
0
0
49
%
0
10
8
80
2
0
0
1/10
No.
0
5
9
33
1
0
0
48
%
0
10
19
69
2
0
0
Coolant
No.
0
6
5
40
2
0
0
53
%
0
11
9
75
4
0
0
Considering the amount of each salt solution drunk
by each experimental group (Table 16), it was found that
consumption increased through the three solutions of lower
osmolarity (0 - 0.50 osm), and decreased thereafter. The
1/10 mice drank significantly more of the low salt concentra-
tions, and significantly less of the high salt concentrations,
than did the other two groups (Table 16).
As expected, changes in body weight (Table 17)
paralleled the results for the amount of salt solutions
consumed (Table 16). Both parameters peaked at 0.50 osm, and
decreased at higher salt concentrations. There was, however,
no significant difference in body weight among the three
groups.
TABLE 16
Grams of salt solution (X ± SE) consumed by mice from the three treatmentsSample sizes are in parentheses. The means were compared with analysis
of variance, and * indicates an F with p < 0.05 and ** indicates p < 0.01.
Drinking Solution
Osmolarityosm
0
0.25
0.50
0.75
1.00
1.25
1.50
Water
30.30 ± 0
(37)
35.48 ± 1
(37)
54.90 ± 1
(35)
39.83 ± 1
(34)
32.64 ± 1
(36)
17.59 ± 1
(36)
11.26 ± 1
(36)
.82
.61
.83
.68
.74
.14
.23
34
36
54
37
25
11
7
1/10
.52 ± 1.
(32)
.79 ± 1.
(32)
.36 ± 2.
(33)
.92 + 2.
(35)
.91 ± 1.
(36)
.91 ± 0.
(37)
.95 ± 0.
(35)
26
43
03
03
72
97
91
30
33
53
37
28
16
11
Coolant
.28 ± 0.
(43)
.62 ± 1.
(42)
.35 ± 1.
(40)
.45 ± 1.
(41)
.97 ± 1.
(41)
.32 ± 1.
(42)
.36 ± 1.
(41)
98
04
86
72
29
18
46
5
1
0
0
4
6
2
F
.35*
.39
.18
.47
.37*
.97**
32
I
TABLF 17
Mean w e i g h t (g ± S E ) , of animals from the three t r e a t m e n t s after drinking saltw a t e r . The sample sizes are in p a r e n t h e s e s . The means were compared by
analysis of v a r i a n c e . None of the dif f e r e n c e s were s i g n i f i c a n t .
Drinking Solution
Osmolarityosm
starting weight
0
0.25
0.50
0.75
1.00
1.25
1.50
25
27
27
28
27
25
23
20
Water
.93 ± 0
(37)
.73 ± 0
(36)
.59 ± 0
(37)
.45 ± 0
(37)
.16 ± 0
(37)
.69 ± 0
(37)
.97 ± 0
(37)
.80 + 0
(37)
.47
.48
.44
.47
.40
.44
.46
.50
26.
27.
28.
29.
27.
25.
23.
20.
1/10
63 ± 0
(37)
73 ± 0
(37)
1 9 + 0
(37)
14 ± 0
(37)
68 ± 0
(37)
86 ± 0
(37)
93 ± 0
(37)
03 ± 0
(37)
.63
.82
.71
.65
.66
.50
.51
.51
Coolant
25.97 ± 0.46
(44)
27.54 ± 0.42
(43)
27.72 ± 0.43
(43)
28.83 ± 0.43
(43)
27.43 ± 0.43
(43)
25.99 ± 0.42
(43)
23.93 ± 0.41
(43)
20.49 ± 0.46
(43)
F
0.55
0.04
0.33
0.42
0.25
0.11
0.00
0.63
to
I
- 43 -
8.3 DISCUSSION
Approximately 20 percent of the experimental animals
died, indicating that the stress applied (hypertonic drinking
solutions) was severe enough to cause any differences in
ability to concentrate urine to be observed. The observed
weight gain while drinking solutions as concentrated as 0.50
osm, and only slight weight loss at 0.75 osm, indicates that
the stress was applied at a slow enough rate to allow the
animals to respond physiologically. The failure to demonstrate
a difference in death rate, peak body weight, or body weight,
indicates that at the concentrations used, coolant has no
major effect on the ability of the kidney to concentrate urine.
The results for the grams of solution drunk by the
three treatments are more difficult to interpret. There are
no consistent differences between mice raised on water
and those exposed to coolant, thus showing no indications of
coolant effect. The mice exposed to 1/10 coolant, however,
drank more of the salt solution at low osmolarities, and less
at high osmolarities. This is the pattern of water consumption
that would be expected if urine concentrating ability was
impaired. It would be expected, however, that if this were the
case, these animals would have lost more weight than ones with
unimpaired kidney function. An alternate possibility is that
the 1/10 animals were exhibiting a superior ability to
concentrate urine at the high osmolarities as demonstrated by
their ability to maintain the same weight as the other groups,
with a lower water intake. This hypothesis implies that the
larger fluid intake of the 1/10 group at low osmolarities was
voluntary intake to minimize the amount of metabolic work
spent in concentrating urine. Since this intake would be
voluntary (not a physiological necessity), the fact that the
1/10 mice chose this strategy and the other treatments did
not, indicates some stimulus or cue was present. The only
- 44 -
known variable that was present between the animals was their
previous exposure to organic coolant. We have no data that
would allow testing of either of these possibilities. If the
effect on the 1/10 mice is real, it would be unlikely to
jeopardize their survival in a natural situation, since
mortality rate and weight loss were not affected.
8.4 CONCLUSIONS
Previous chronic exposure to organic coolant in
drinking water does not affect the ability of mice to survive
or maintain their weight when restricted to drinking hypertonic
NaCl solutions, and thus has not apparently resulted in kidney
malfunction.
9. BEHAVIOURAL EFFECTS
The overt behaviour of an organism can be regarded
as the final product of a complex chain of musculo-neuro-
physiological events. As such, it can be used as an indicator
of the comparative effects resulting from the exposure of
organisms to different experimental conditions. The three
parameters chosen for testing in this study are important to
an individual mouse relevant to its role in a population. By
measuring an animal's activity, we presume to have an
indication of its mobility, of importance in obtaining food
and moisture, in escaping from predators, and in communicating
with other members of the population. An open-field test,
involving confinement in a novel location illuminated by
bright lights, measures the individual's responses to a stress,
- 45 -
Although the results do not apply directly to the field
situation, they probably give an indication of an animal's
ability to meet the manifold stresses present in a natural
situation. Inter-male aggression has recently been implicated
in the dynamics of population regulation of several small
rodent species (Sadleir, 1965; Healey, 1967) , and the ability
of one mouse to be dominant over another has ecological
ramifications. To examine mice exposed to chronic ingestion
of organic coolant suspensions, we have therefore chosen
three tests which estimate the above ecologically important
behavioural parameters.
9.1 METHODS
Two groups of male C~ mice were tested, with c. span
of 15 weeks between the completion of tests on the first group
and the start of second group testing. The mice were 37-44 days
of age at the start of testing, and were divided into weekly
groups of same-age mice. Overnight activity of all mice was
recorded, with weekly groups starting on Monday, and
continuing at a maximum of 10 mice per night until the weekly
group was finished. On the following Monday, open-field tests
were performed on the same individuals, followed by aggression
tests the next day, on Grov^ 1 mice only.
Overnight activity of each individual was recorded
with Ultra-Sonic Motion Detectors (Model 6, Alton Electronics,
Archer, Fla.) connected to an Esterline Angus event recorder.
The mouse was contained by.a 28 x 17 x 17 cm wire cage (6 mm
spacing between wires) inverted into a 31 x 21 x 2.5 cm sheet
metal pan. Each of these containers was housed in a 60 x 45 x
45 cm plywood cell with a transparent lucite door. All cells
were lined with 2.5 cm of fiberglass insulation to decrease
sound interference between cells. The metal pans were bedded
- 46 -
with shavings; whole grain oats were provided, with a slice of
carrot for moisture. The mice were placed in the cells at
approximately 1600 hr, and removed at 0900 hr the following
morning. Cells were used randomly with respect to treatment
group. Illumination was through windows at one end of the
room, providing a natural photoperiod. Activity recorded
between 1800 hr and 0800 hr the following morning was analyzed.
All bars of activity longer than 2 mins were scaled, and the
number of such periods, their total duration, and their mean
duration and standard deviation were computed. Also counted
was the total number of 10 min periods in which there was less
than 2 mins activity.
The open-field was a 60 cm diameter circle painted
on the underside of a plate glass table top. A 25 cm high
sheet metal hoop, painted flat black, was placed on this
circle. Within the hoop, 2 equidistant concentric circles
and 3 diameters (except through the inner circle) partitioned
the area into 1 inner, 6 middle and 12 outer segments, for
a total of 19. A piece of white cloth sheeting was stretched
across the top of the hoop, to diffuse illumination provided
by three 200 W white bulbs suspended 37 cm above the open-field
floor. Behaviour of the mouse was observed from in front of
the open-field table, via a mirror set at an angle below the
open-field floor, to avoid the variable stimulus of movements
by the experimenter which occur when the subject is viewed
from above. Each mouse was placed in the middle circle, and
its path for the following 2 mins was drawn on a map of the
open-field. Locations and frequencies of freezing, urinating
and defecating were noted. Also analyzed were: the number
of different segments entered (maximum 19), separate totals
for the number of times the inner segment, middle segments,
and outer segments were entered, and the grand total of all
segments entered. Following each trial, the hoop was removed
and the surtace of the glass washed with a detergent solution,
- 47 -
to reduce the bias of olfactory cues (Whittier and McReynolds,
1965) . All observations were made by the same experimenter
between 0900 hr and 1600 hr. The order in which individuals
were observed was random with respect to treatment group.
Aggression of pairs of mice was observed in a
55 x 41 x 25 cm glass-walled arena, illuminated by a 40 H red
bulb suspended 64 cm above a silica sand substrate. Each
mouse was tested only once. Coolant mice were always paired
against controls; 1/10 coolant mice were paired only with
others from the same treatment, so that the three treatments
would have a similar history for future experiments.
Observations of each pair continued until the observer was
convinced which mouse was the winner, or to a maximum of
5 mins, at which time the mouse which had exhibited greater
relative amounts of activity and approaching was selected as
the winner. Behaviour considered in choosing the winner
included attacking, tail lashing, vocalizing, following, and
grooming-other. Winners were decided using criteria similar
to those of Levine, Diakow and Barsel (1965) , and Clark and
Schein (1966). Identities of the mice were unknown to the
observer. All observations were made between 0900 and 1200
hr. For each encounter, the winner was recorded, together
with a brief description of the'criteria used to make the
choice in that particular encounter, and the elapsed time
from introduction of the mice until the choice was made.
9.2 RESULTS
9.2.1 ACTIVITY
The total activity (Table 18) of both 1/10 and
coolant treatments of Group 1 mice was somewhat less than
that of the controls, and paralleling this was the finding
TABLE 18
Activity parameters (X + SE) of all m i c e , showing calculated F values resulting fromanalysis of variance. There were no significant differences among treatments.
Parameter
Total minutes active
Number of periods active
Mean duration (minutes) ofperiods active
Mean standard deviation ofperiods active
Number of 10 minute periodswith < 2 minutes activity
Group
GroupGroup
GroupGroup
GroupGroup
GroupGroup
GroupGroup
12
12
12
12
12
WaterGroup 1: N = 87Group 2: N = 99
483.8 ±512.0 ±
13.2 ±12.3 ±
44.4 ±57.0 ±
47.8 +59.9 ±
30.7 ±27.9 ±
11.712.5
0.50.6
2.63.5
3.64.2
1.11.0
1/10GroupGroup
453.9499.9
13.212.1
44.156.9
41.655.4
33.429.2
Coolant1: N = 842: N = 96
± 14.8± 11.5
± 0.6± 0.6
± 3.4+ 4.1
± 3.5± 4.1
± 1.4± 0.9
CoolantGroup 1: N = 86Group 2: N = 102
437.0506.1
14.711.8
38.458.7
40.556.5
34.728.9
± 14.7± 12.4
+ 0.6± 0.6
± 2.9± 3.6
± 3.7± 3.9
± 1.3± 1.0
P
2.990.24
2.230.19
1.280.08
1.200.34
2.690.48
00
I
- 49 -
that the mice of both treatments were inactive for more 10 min.
periods. However, analysis of variance indicated that there
were no significant differences in any of the 5 parameters
measured.
In Group 2, treatment means were very similar, as
shown by the low F values, although total activity was
slightly reduced in both 1/10 and coolant treatments. All
Group 2 mice showed increased activity, compared to
Group 1.
9.2.2 OPEN-FIELD
Analysis of variance of Group 1 results (Table 19)
revealed significant differences among treatments in all
parameters except the number of inner segments entered.
However, the only consistent trend was the reduced number of
segments entered by the 1/10 coolant treatment, in all five
parameters. Conversely, in three of the five parameters, the
coolant treatment mice showed more activity than the controls,
indicating there was no consistent trend in coolant effects
when the two experimental groups were considered together.
The three groups were very similar when the relative amounts
of movement in a given segment were expressed as a percentage
of the total movement for that treatment group, the largest
difference between any two treatments being 2.5 percent.
Freezing was significantly more frequent in 1/10 coolant mice
than in controls (Table 20), and both 1/10 and coolant treat-
ment groups urinated more than did the controls (0.10 > p >
0.05). Differences in defecation were not significant.
Analysis of Group 2 results (Table 19) showed that,
although 1/10 coolant results were the lowest for each of
the five parameters, the only significant difference shown by
TABLE 19
Open-field movement (X ± SE) of all mice, showing calculated Fvalues resulting from analysis of variance.
Parameter
Number of different segmentsentered (maximum 19)
Cumulative total segmentsentered
Cumulative outer segmentsentered
Cumulative middlesegments entered
Cumulative inner segmentsentered
Group
GroupGroup
GroupGroup
GroupGroup
GroupGroup
GroupGroup
12
12
12
12
12
WaterGroup 1: N = 8 6Group 2: N = 98
1717
7167
5650
1314
22
.1 ±
.5 ±
.7 ±
.5 ±
.1 ±
.7 ±
.1 ±
.0 ±
.5 ±
.8 ±
0.20.2
3.02.3
2.21.9
1.10.9
0.20.2
1/10GroupGroup
15.816.8
56.364.3
43.749.3
10.312.5
2.32.6
Coolant1: N = 872: N = 95
± 0.4+ 0.4
± 2.9± 2.9
± 2.4+ 2.4
+ 0.9+ 0.8
± 0.2± 0.2
CoolantGroup 1: N = 87Group 2: N = 102
17.4 ±17.4 +
66.9 ±70.3 ±
50.6 +51.3 +
13.6 ±15.9 ±
2.7 ±3.1 ±
0.20.2
2.52.3
1.91.9
1.01.0
0.20.2
F
7.78**2.05
7.31**1.38
8.09**0.25
3.37*3.44*
0.551.43
oI
* p < 0.05, F > 3.04** p < 0.01, F > 4.70
- 51 -
analysis of variance was between the 1/10 and coolant groups.
All of the parameters reached the same or higher levels in
Group 2 results, with the exception of total and outer segments
entered by the water treatment mice. As in Group 1, relative
movement in a given segment was very similar among treatments.
There were significantly more freezes in the 1/10 treatment,
and significantly fewer urinations by the coolant treatment
mice (Table 20). Defecating was infrequent in all three treat-
ment groups.
9.2.3 AGGRESSION
Of the 86 control x coolant aggressive encounters
observed, 43 were won by each treatment. Only two of the
encounters were decided on the basis of relative activity and
approaching at the end of the 5 min test period, the rest
being decided using the more aggressive criteria of Levine et al.
(1965) , and Clark and Schein (1966) . Since there was no
suggestion of differences between treatments, Group 2 mice were
not subjected to the aggression test.
9.3 DISCUSSION
Open-field responses are regarded as reactions to
the stress of a brightly lighted, unfamiliar and confining
area. Under such conditions, emotionally reactive animals are
typified by little activity, and high defecation and urination
rates. Conversely, animals low in "emotionality" are more
active, and defecate and urinate less (Pare, 1964; Whimbey and
Denenberg, 1967; Smith, 1972). The majority of open-field
studies have used laboratory strains of rats as experimental
animals, but a recent study using laboratory mice concluded
TABLE 20
Open-field freezing, urinating and defecating by all mice.Both 1/10 coolant and coolant results were tested againstthose of the water group; x^ values are in parentheses.
Parameter
FREEZING:
Number of individualsfreezing
Total number of freezes
URINATING:
Number of individualsurinating
Total number ofurinations
DEFECATING:
Number of individualsdefecating
Total number of fecal boli
Group
Group 1Group 2
Group 1Group 2
Group 1Group 2
Group 1Group 2
Group 1Group 2
Group 1Group 2
WaterGroup 1: N = 86Group 2: N = 98
48
79
1317
1518
52
82
1/10 CoolantGroup 1: N = 87Group 2: N = 95
16 (7.06)**13(1.34)
22(7.60)**19(3.90)*
24(3.14)12(0.71)
27(3.30)12 (1.02)
9(1.10)0
13(1.13)0
CoolantGroup 1: N = 87Group 2: N = 102
10 (2.50)6(0.37)
12(1.25)9(0.01)
25(3.66)7(4.57)*
28(3.78)7(5.29)*
3 (0.52)1
6(0.31)1
* p < 0.05, x" > 3-84
** p < 0.01, x2 > 6- 6 3
- 53 -
thctt defecation may be a territorial marking response, and
suggested that ambulation scores are more indicative of
"emotionality" in mice (Brain and Nowell, 1969) .
Group 1 results indicated that chronic ingestion of
the 1/10 coolant suspension had resulted in increased
"emotionality" in the group. Freezing, another stress reaction,
also tended to support this conclusion. Coolant treatment mice
exhibited the same trend in freezing and urinating responses as
the 1/10 animals, but their open-field movement scores were, in
the majority of cases, higher than those of the controls.
Results from Group 2 mice, however, do not support the conclusion
of changes in "emotionality" deduced from Group 1 results. Of
the 15 total means calculated for each Group (five in each of
the three treatments), 12 were larger in Group 2 when compared
to Group 1. Both of the means which decreased were in the
water treatment, leading us to suspect that the Group 1 means
for these two parameters may have been over-estimated. In
comparing freezing and urinating results, the increased freezing
in Group 1 mice of the 1/10 treatment was partially supported
by Group 2 results. However, the decreased urinating of coolant
mice in Group 1 was negated by Group 2 results, which found
urinating least frequent in the water treatment. It is concluded
from the two sets of results, therefore, that chronic coolant
ingestion had no significant effect on open-field behaviour.
Similarly, Group 2 results for activity showed fewer
significant differences than did Group 1. In comparing the two
Groups, the only evident difference is the increased activity
of all three treatments in Group 2. Activity was recorded
using natural photoperiod, and Group 2 mice averaged 12 hrs
55 mins darkness, but Group 1 mice averaged only 9 hrs 36 mins
darkness. Since Mus is a nocturnally active rodent, the greater
total activity found in Group 2 mice is expected. However, the
longer periods of darkness do not explain why nearly all Group 2
- 54 -
open-field results also showed greater activity as compared to
Group 1, since open-field testing of both Groups occurred under
artificial lighting during the light part of the animal room
photoperiod. The animal room in which the mice were held
except during the experiments reported here was maintained at
a 16L.-8D photoperiod (on at 0600, off at 2200). It can be
seen that Group 2 mice, which were tested mainly in the
shorter day-length months of October and November, had
considerably longer dark periods during the 1800-0800 hr
recording period. However, within Group 2 there were no
significant differences, and it is thus concluded that chronic
coolant ingestion apparently had no effect on voluntary
activity of the mice.
Finally, coolant ingestion did not affect the
ability of the mice to express themselves in an aggressive
context. Considered in their entirety, these results indicate
that the organic coolant suspensions used in this study did
not impair the experimental animals' reactions to stress, their
voluntary locomotory activity, or their responses during
intraspecific aggression. Differences between the 2 groups
may have been due to the fact that Group 1 was produced by
females 1-2 months old, whereas Group 2 mice were produced
by the same females when they were 8-9 months old. Thus,
differences in maternal ages may render inter-Group comparisons
invalid. Similarly, if the differences in open-field response
found in Group 1 were real differences, they may not have
existed in Group 2 due to this maternal age-effect. For this
reason, parts of the study will be repeated. But it is
apparent that if any effects are present, they are not major
mes.
- 55 -
10. CONCLUSIONS
It must be emphasized that some parts of this study,
such as the life span and reproductivity, and behaviour, are
not yet complete. Other parts, such as histopathology, have
not begun. The results presented here, however, show clearly
that no gross effects have been produced by chronic ingestion
of 0.25 or 0.025 mg/kg/day irradiated HB-40. Since an organism
is an integrated unit and damage to one part of its system is
likely to affect other parts, these results probably also
indicate that no major effects will be observed during the
remainder of the study. It is therefore probable that
deleterious effects on wild mammals will not prove to be the
factor limiting the amount of HB-40 tolerable in the environ-
ment. It is entirely possible though, that minor effects may
still be observed and that these will be limiting, particularly
if humans are considered as the population at risk.
- 56 -
11. ACKNOWLEDGEMENTS
This study would not have been possible without
assistance from a great number of sources. We would like to
thank O.E. Acres, W.A. Boivin, J.E. Guthrie and A.M. Wiewel
for providing coolant suspensions and analyzing coolent
contents of the suspensions. A. Beck , D. Robertson ,
V. Tunnicliffe, A. Weeks and M. Wikjord under the day-to-day
direction of K. Severson assisted with management of the
animals. S. Chura, J. Mitchell and 0. Schwartz helped with
aspects of data analysis and report preparation.
Employed under an Opportunities For Youth grant obtainedthrough Manitoba Scientists to Combat Pollution.
2Employed under a Local Initiatives Program grant obtainedthrough the Manitoba Environment Research Committee.
- 57 -
12. LITERATURE CITED
Acres, 0. E. and J. E, Guthrie. 1968. Organic reactorcoolants: an apparatus producing dispersions inwater for toxicity testing. Atomic Energy ofCanada Limited Report AECL-3115, 15 pp.
Adamson, I, Y. and J. L. Weeks. (in press) The LD50 andChronic Toxicity of Reactor Terphenyls. Arch.Environ. Health.
Brain, P. F. and N. W. Nowell. 1969. Some behavioral andendocrine relationships in adult male laboratorymice subjected to open field and aggression tests.Physiol. Behav. 4:945-947.
Clark, L. H. and M. W. Schein. 1966. Activities associatedwith conflict behaviour in mice. Anim. Behav.14:44-49.
Cornish, H. H., R. E. Balor and R. C. Ryan. 1962. Toxicityand metabolism of ortho-, meta-, and para-terphenyls.Amer. Ind. Hyg. Assoc. J. 23:372-37 8.
Healey, M. C. 1967. Aggression and self-regulation ofpopulation size in deermice. Ecology 48:377-392.
Levine, L., C. A. Diakow and G. E. Barsel. 1965. Interstrainfighting in male mice. Anim. Behav. 13:52-58.
Ott, H. and D. Pirrwitz. 1970. The acute hepatic toxicityor organic reactor coolants. EUR 4548e. 13 pp.Commission of the European Communities. JointNuclear Research Center, ISPRA Establishment -Italy.
Pare*, W. P. 1964. Relationships of various behaviors in theopen-field test of emotionality. Psychol. Rep.14:19-22.
Sadleir. R. M. F. S. 1965. The relationship betweenagonistic behaviour and population changes in thedeermouse, Peromysous manieulatus (Wagner). J.Anim. Ecol. 34:331-352.
Smith, R. H. 1972. Wildness and domestication in Musmusaulus: A behavioral analysis. J. Comp.Physiol. Psychol. 79:22-29.
- 58 -
Whimbey, A. E. and V. H. Denenberg. 1967. Two independentbehavioral dimensions in open-field performance.J. Comp. Physiol. Psychol. 63:500-504.
Whittier, J. L. and P. McReynolds. 1965. Persisting odoursas a biasing factor in open-field research withmice. Canad. J. Psychol. 19:224-230.
Additional copies of this documentmay be obtained from
Scientific Document Distribution OfficeAtomic Energy of Canada Limited
Chalk River, Ontario, CanadaKOJ 1J0
Price - $1.50 per copy
1112-73