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Dissertation for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
SUSUMU IWAMOTO

1973 ’

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ABSTRACT

THE EFFECTS OF POLYCHLORINATED BIPHENYLS
AND COHO SALMON ON MINK

By

Susumu Iwamoto

Studies pertaining to reproductive complications and mortality in
mink fed diets that contained Lake Michigan coho salmon or supplemental
polychlorinated biphenyls (PCB's) were conducted at the Michigan State
University Experimental Fur Farm over a four year period. Nine experi-
ments dealing with various aspects of these two associated conditions
were performed to investigate the nature of these disorders, their
etiology and possible remedies.

In Experiment I, conducted to determine the effects of feeding
high levels of supplemental PCB's or coho salmon on mink reproduction
and viability, none of the females that received coho salmon or sup—
plemental PCB's in the diet whelped. Anorexia, bloody stools, fatty
degeneration of the liver and kidneys and hemorrhagic gastric ulcers
were common clinical signs and lesions observed in the animals.

Experiment II was conducted to ascertain the effects of long-term,
low-level consumption of PCB's on mink reproduction and viability, and
to investigate the possibility of interactions between PCB's and some
chlorinated hydrocarbon pesticide contaminants of Great Lakes fish.
Mink growth data suggested that interactions occurred between PCB's

and chlorinated hydrocarbon pesticides in which the action of PCB's

Susumu Iwamoto
or certain chlorinated hydrocarbon pesticides may be potentiated in the
presence of the other. Long-term consumption of PCB's by mink resulted
in decreased body weight gains and increased heart, liver and kidney
weights.

In Experiment III the acute toxicity values (LD ) of some common

50
PCB's for mink ranged from 500 mg/kg for Aroclor 1221 to 2250 mg/kg for
Aroclor 1254.

Experiment IV was conducted to ascertain the effects of coho
salmon and PCB's, fed alone or in combination with chlorinated hydro-
carbon pesticides, on mink reproduction and viability. The results
indicated that chlorinated hydrocarbon pesticides and PCB's do not
appear to have a synergistic effect on mink reproductive performance,
that the toxic factor present in Great Lakes fish is quite heat stable,
and that PCB contamination of the fish is probably the primary cause
of reproductive complications and mortality in mink fed these fish.

The results of Experiment V showed that Aroclor 1254 was much
more detrimental to mink reproduction than Aroclors 1016, 1221 or 1242.
The experiment also indicated that hematologic parameters were net a
good indicator of the toxic effects of PCB's in mink.

In Experiment VI mink fed Aroclor 1254 readily accumulated the
PCB's in the adipose tissue until a plateau level was reached. A 50
percent reduction in PCB residues in the adipose tissue was reached
about 8 weeks after withdrawal of the PCB's.

Experiment VII was conducted to substantiate the results of previous
experiments in which PCB's were implicated as the primary cause of
reproductive problems associated with the feeding of Great Lakes fish
to mink, and to evaluate acetone-hexane extraction of coho salmon as a

practical method of removing PCB's from these fish to render them safe

Susumu Iwamoto
for mink feed. The results confirmed the results of the previous
experiments and showed that the toxic factor present in coho salmon
is stored primarily in the fat and can be removed from the fish by
acetone-hexane extraction.

The results of liver and kidney function tests performed on the
mink in Experiment VIII indicated that impaired liver function was an
early manifestation of PCB and coho salmon toxicity in mink.

The results of Experiment IX showed that reproductive failure
attributed to the feeding of coho salmon and PCB's to mink was not of

a permanent nature.

THE EFFECTS OF POLYCHLORINATED BIPHENYLS

AND COHO SALMON ON MINK

By

Susumu Iwamoto

A.DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Poultry Science

1973

r ‘1‘: ACKNOWLEDGMENTS

The author wishes to express his grateful thanks to Dr. Howard C.
Zindel, Chairman of the Department of Poultry Science, for his advice
and support during my course of study.

The writer also wishes to express sincere gratitude to Dr. Richard
J. Aulerich, adviser, for his excellent guidance, assistance, and
encouragement during the time this work was conducted, and personal
interest throughout the master's program.

I am also indebted to Dr. Robert K. Ringer, Department of Poultry
Science, for his guidance and encouragement.

Appreciation is also extended to Dr. Robert Pittman for serving on
my guidance committee.

To the Department of Poultry Science faculty and staff, including
those at the Teaching and Research Center, I am thankful for their
assistance and cooperation throughout my course of study.

A special word of thanks to Ms. Vida M. Amog, Ms. Susan Asher,

Mr. Sulo Hulkonen and Dr. Matthew Zabik for their technical assistance.

Above all I am indebted to my family for their love and their

encouragement toward making this program worthwhile and successful.

11

TABLE OF CONTENTS

ACKNOWLEDGMENTS. . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . .
LIST OF FIGURES. . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . .

REVIEW OF LITERATURE . . . . . . . .

O O O O O O O O O
O O O O O O O O O
O O O O O O O O
O O O O O O O O O
O O O O O O O O O

Coho SalmonéMink Feeding Studies. . . . . . . .

Mink Reproduction . . . . . .
Factors that Adversely Affect

Stress . . . . . . . .
Microorganisms .'. . .
Feed handling. . . . .
Contaminants . . . . .

Mink.Reproduction

Polychlorinated biph nyl . . . . . . . .

MHODS O O O O O O O O 0 O O O O O O

EXPERmENTS O O O O O O O O O O O O O

Experimerlt I O O O O O O O O 0
Procedure. . . . . .

Results. . . . . . . .

Discussion . . . . . .

Experiment II . . . . . . . .

Procedure 0 O O O O O 0

Results 0 O O O O O O 0

Discussion . . . . . .

Experiment III. . . . . . . .

Procedure. . . . . . .

Results. . . . . . . .
Discussion . . . . . .

iii

Page

vii

CXDONU'IU'ILB

l4

. 17

23

23

30

3O
31
31

Experiment IV . .

Procedure.
Results. .
Discussion

Experiment V. . .
Procedure.
Results. .
Discussion

Experiment VI . .
Procedure.
Results. .
Discussion

Experiment VII. .
Procedure.
Results. .
Discussion

Experiment VIII .
Procedure.
Results. .
Discussion

Experiment IX . .

Procedure.
Results. .

Discussion .

CONCLUSIONS 0 O O O O O 0

LITERATURE CITED . . . .

iv

Page
32
32
33
33
38
38
41
45
50
50
50
54
54
54
56
56
56
58
58
6O
62
63
63
63
65

67

LIST OF TABLES
Table Page
1 Composition of basal (control) diet . . . . . . . . . . . . 13

2 Average body weight change (gm : S.E.) of female mink
fed a control ration or experimental diets (Experi—
ment I) O O O O O O O O O O O O O O O O O O O O O O O O O 0 l6

3 Reproductive performance and mortality of female mink
fed a control ration of experimental diets (Experi-
ment I ) O O O C O O O O O O O O O O O O O O O I O O O O O O 16

4 Gross and histologic lesions commonly observed in tis-
sues from mink that died while receiving.diets that
contained Lake.Michigan.coho salmon or supplemental
PCB's (Experiment I). . . . . . . . . . . . . . . . . . . . 20

5 Mean organ weights of female mink fed a control ration
or experimental diets (Experiment I). . . . . . . . . . . . 21

6 Average PCB content of tissues (meg: S.Ei) from mink
that died or were sacrificed (Experiment I) . . . . . . . . 21

7 Average.body weight change (gm_i S.E.).of female mink
fed a control ration or experimental diets (Experi-
ment II) C C O O O O O O O O O O O O O O O O O O 0 O O O O O 24

8 Reproductive performance and mortality of female mink
fed a control ration.or experimental diets (Experi-
ment II) C O O O O O O O O O O O O O O O O O O O O O O O O O 26

9 Average.PCB content of tissue (ppmft S.E.) from mink
that died or were sacrificed (Experiment II). . . . . . . . 27

10 Mean organ weights of female mink fed a control ration
or experimental diets (Experiment II) . . . . . . . . . . . 28

11 Acute toxicity (LDSO) of PCB's when administered IP
to mink (Experiment III). . . . . . . . . . . . . . . . . . 31

12 Reproductive performance and mortality of female mink
fed a control ration or experimental diets (Experi—
ment IV) C O O O O O O O O O O O O O O O O O O O O O O O I O 34

13 Average body weight change (gm3:.S.E.) of female mink
fed a control ration or experimental diets (Experi—
ment IV) 0 o o o o o o o o o o o o o o o o o o o o o o o o o 35

V

Table

14

15

l6

l7

l8

19

20

21

22

23

24

Page

Mean organiweight and mean PCB content (ppm.i;S,E.) of
tissue from mink that died.while.receiying diets that
contained 15 ppm supplemental PCB (Experiment IV) . . . . . 36

Average body weight change.(gmgtS.Er) of female mink
fed a control ration or experimental diets-(Experi-
ment V) O O O O O O O O O C O O O I O C C O O C 0 O O O O O 42

Hematocrit values for mink fed a.control diet and
rations containing supplemental PCB's (Experiment V). . . . 43

Average hemoglobin (Hb) content and mean corpuscular
hemoglobin concentration (MCHC) for mink fed a control

diet and rations containing supplemental PCB's (Experi-

ment V) . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Reproductive performance and mortality of female mink
fed a control ration or experimental diets (Experi-
ment V) O O O O O O O O O C O O C O O O C O O C O O I O C O 48

Mean organ weight of mink:fed a control diet or experi-
mental rations (Experiment V) . . . . . . . . . . . . . . . 49

Average PCB residues in adipose tissue of mink fed a
control diet or a ration that contained 5 ppm supple-
mental Aroclor 1254 (Experiment VI) . . . . . . . . . . . . 51

Reproductive.performance and mortality of female mink
fed a control ration or experimental diets (Experi-
mwt VII) 0 O O O O O O O O O C O O O O O O O O O O O O O O 57

Urine analysis values at lO—day intervals for mink fed

a diet that contained 30% coho salmon or 5 ppm Aroclor
1254 (Experiment VIII). . . . . . . . . . . . . . . . . . . 59

Average serum alkaline phosphatase (SAP), serum glutamic-
pyruvic transaminase (SGPT) and blood urea nitrogen

(BUN) levels at lO—day intervals for mink fed a diet

containing 30% coho salmon or 5 ppm Aroclor 1254

(Experiment VIII) . . . . . . . . . . . . . . . . . . . . . 61

Reproductive performance.of female mink fed a control

diet following failure to whelp the previous year while
receiving a ration that contained either coho salmon

or supplemental PCB (Experiment IX) . . . . . . . . . . . . 64

vi

Figure

LIST OF FIGURES

Page

Visceral surface of mink stomach showing a pyloric
ulcer frequently observed in mink that died while
receiving diets supplemented with PCB's (Experiment I). . . 19

Relationship between consumption of Aroclor 1254 and

mortality in mink. Calculation based on mortality data

from Experimental groups II — 2, II - 3, IV - 4,

IV - 6 and V - 5 and an assumed feed consumption of

150 g/day . . . . . . . . . . . . . . . . . . . . . . . . . 40

Accumulation and depletion of PCB in adipose tissue
of mink fed 5 ppm Aroclor 1254 (Experiment VI). . . . . . . 53

vii

INTRODUCTION

Mink GMusteZa vison) were first raised in captivity for the produc-
tion of fur in the United States in 1866 (Kellogg at al., 1948), and
toward the end of the nineteen-twenties were exported from North America
to Europe (Aitken, 1963) where they are raised extensively today. In
1972 the estimated world production of mink exceeded 22 million pelts
(Anon., 1972a).

In the United States, the majority of mink ranches are concentrated
in the upper Midwest where there is a suitable climate for raising mink,
as well as abundant supplies of fish from the Great Lakes and meat by-
products from packing plants to feed the animals (Travis and Schaible,
1960). In the United States 10 to 40 percent of the total mink diet
is usually composed of fish (Travis and Schaible, 1960). For many years
ranchers in the Midwest fed fish from the Great Lakes as a major pro-
tein source. The utilization of Great Lakes fish by the mink ranching
industry, however, has constantly diminished since the mid 1960's due
to reproductive complications when mink were fed these fiSh (Hartsough,
1965).

An acute problem was evident in 1968 when coho salmon (Oncorhynchus
kisutch), taken from tributaries of Lake Michigan during the spawning
run of 1967 and fed to mink before and during the breeding and whelping
season, caused an unusually high incidence of kit mortality on several
commercial mink farms. Kit mortality as high as 80 percent was reported
(Aulerich at al., 1971). The cause of the problem was demonstrated to

1

2
be due to the use of coho salmon in the diet. Microbiological toxins,
thiaminase activity in the fish, rancidity, as well as mercury and
chlorinated hydrocarbon pesticide contamination were all suspected as
being responsible for the problem. None of these factors, however,
were substantiated as being the cause of the disorder (Aulerich at aZ.,
1971).

Because of the tremendous human health implications, as well as
the economic importance of Great Lake fishery to the mink ranching and
pet food industries, this study was initiated to determine the factor
or factors responsible for the reproductive failure and mortality

associated with the feeding of Great Lakes fish to mink.

REVIEW OF LITERATURE

Coho SalmonrMink Feeding Studies

 

A series of mink feeding experiments were conducted by Aulerich at
al. (1970a, 1971, 1972 and 1973) in an effort to investigate the repro-
ductive problems attributed to the feeding of Great Lakes fish to mink.
In these trials, whole, raw, sexually mature coho salmon taken from
tributaries of Lake Michigan were fed to mink at 30% of the diet from
two months before breeding through gestation. Several other species
of marine and Great Lakes fish were fed at the same level for comparison.
In one study coho salmon canning by-products were used instead of whole,
raw fish.

All of the 15 mink that received the Lake Michigan coho salmon
canning by-products in their diet died between the beginning of breeding
season and the end of the whelping period (Aulerich at al., 1971). In
another feeding trial four out of 10 females fed the diet that contained
the ground, whole, Lake Michigan coho salmon also died; the rest failed
to whelp (Aulerich at aZ., 1971). Anorexia, bloody stools, fatty
livers, and hemorrhagic gastric ulcers were common clinical signs and
lesions observed in these mink. Antibiotic treatment and injections of
iron, vitamins A, D and E, as well as the B-complex vitamins, had no
beneficial effect in arresting the mortality. Reduced reproductive
performance and/or excessive kit mortality were also observed in mink
fed diets that contained Lake Michigan bloater chub, Lake Michigan
yellow perch, and mature, whole, raw coho salmon from Lake Erie. Mink

3

4
rations that contained West Coast coho salmon and Lake Erie yellow
perch did not impair reproduction nor result in excessive kit mortality
GAulerich at aZ., 1971).

No correlation was found between the degree of oxidative rancidity
or mercury contamination of the fish and the reproductive performance
of the mink QAulerich at al., 1971). The level of pesticide residues
(DDT and dieldrin) in the fish and the degree of reproductive decline
and/or kit mortality was, however, directly related, but based on the
results of previous studies by Aulerich and Ringer (1970b), chlorinated
hydrocarbon pesticide residues were discounted as the cause of the
problem.

These experiments demonstrated that coho salmon per as does not
cause the reproduction and mortality problems, but that the disorder
is associated with other species of Great Lakes fish and appears to be
dependent upon the species of fish and its environment (Aulerich at aZ.,

1971).

Mink Reproduction

 

Mink have one breeding season per year, which extends from late
February through March. Both spermatogenesis and estrus are controlled
by environmental light conditions (Bowness, 1957; Travis and Schaible,
1960; Holcomb at al., 1962; Aulerich at aZ., 1963; and Bostrom et al.,
1968). Due to delayed implantation in this species there is a wide
variation in the gestation period. Gestation averages about 50 days
but varies between 40 and 70 days (Hansson, 1947).

Female mink may produce offspring by different males (superfecunda—
tion) and/or from two different ovulations more than a week apart (super-

fetation) (Travis and Schaible, 1960). Litter size may vary from 1 to

5
8, or more, but generally averages about 4 kits at birth. The average
litter size at four weeks postpartum of Standard Dark and Pastel female
mink at the Michigan State University Experimental Fur Farm in 1970

was 3.6 kits per female (Aulerich, 1970b).

Factors that Adversely_Affect Mink Reproduction

Stress:

Animals are frequently under stress of one kind or another, caused
by chilling, overheating, poor ventilation, disease, parasites, vaccina-
tion, medication, dehydration from water supply failure, improper
handling, inadequate nutrition and unusual or excessive noises. All
of these stresses may cause reproductive complications with mink

(Schaible, 1969, and Travis, 1968).

Microorganisms:

Spoiled feed or toxic microorganism contamination, which may be
the result of improper handling of feed, are frequent sources causing
reproductive complications in mink. Salmonella and botulism organisms
have been reported to cause infectious abortion in mink (Travis and

Schaible, 1960).

Feed handling:

Improper handling and freezing of mink.feed ingredients may result
in loss in flavor, produce dangerous peroxides, and destroy essential
vitamins (Schaible, 1969). A problem of this nature which caused
heavy kit losses shortly after weaning was first described by McDermid
and Ott (1947). The disease was termed "yellow fat" by McDermid and
0tt (1947), but later referred to as fatty degeneration of the liver

by Chaddock (1948), and steatitis by Hartsough and Gorham (1949).

6

This condition in mink is associated with the feeding of diets high in
unsaturated fatty acids and low in vitamin E. The source of the highly
unsaturated fat has been traced to rancidity in imprOperly stored fish
and/or horsemeat. The use of vitamin E or antioxidants in the diet
protects mink from the disease (Lalor et al., 1951, and Mason et al.,
1951).

Sodium nitrite, frequently used in the preservation of fish, has
been shown by Stout at al. (1968) to react with trimethylamine forming
the toxic compound, dimethylnitrosamine, which causes mortality when

fish treated with this compound are fed to mink.

Contaminants:

The infiltration of drugs, chemicals and hormones into mink feed
ingredients can also cause severe reproductive problems. In recent
years many new substances have been introduced in the agricultural
area. These substances occasionally get into cereal or slaughter house
by-products used in mink diets (Schaible, 1969). Diethylstilbestrol
(DES), an estrogen-like compound previously used in poultry and livestock
production to stimulate growth and enhance carcass quality, has been a
common source of reproductive failure in mink. As little as 10 pg of
DES/mink/day completely inhibits mink reproduction (Travis at al., 1956).

Chlorinated hydrocarbon pesticides such as DDT and dieldrin are
frequent contaminants of mink feed since they are common pollutants of
many fresh water fish used for mink feeding (Hartsough, 1965).

Since 1965, the U.S. Bureau of Commercial Fisheries has been
monitoring insecticide residues in Great Lakes fish. 0n the basis of
the DDT and dieldrin levels in these fish, the rank of the Great Lakes,

in the order of highest to lowest concentration of insecticides was:

7
Michigan, Ontario, Huron, Erie and Superior (Reinert, 1970). Among 12
Lake Michigan species of fish examined, bloater chubs had the highest
whole fish averages for DDT (8.61 ppm) and dieldrin (0.23 ppm) (Reinert,
1970). Reinert (1970) also demonstrated that both the DDT and dieldrin
concentrations in Great Lakes lake trout increased in direct proportion
with total length of the fish; a 30-inch lake trout from Lake Michigan
may be expected to contain between 20 and 30 ppm DDT. Analyses by the
National Marine Fisheries Service, as reported by Aulerich at al. (1971)
showed that Lake Michigan coho salmon contained 18.23 ppm total DDT and
0.12 ppm dieldrin residues, but Lake Erie coho salmon contained only
2.76 ppm total DDT and 0.03 ppm dieldrin residues.

The physiological effect of chlorinated hydrocarbon pesticides,
such as DDT and dieldrin on reproduction in many birds (Rubin at al.,
1947; Dewitt, 1955; Genelly and Rudd, 1956; Albert, 1962; Azevedo et al.,
1965; Locke et al., 1966; and Atkins and Linder, 1967), and mammals
(Kitselman, 1953; Ball at al., 1953; Bernard and Gaertner, 1964;
Mestizova, 1966; and Ware and Good, 1967) has been well documented.

In studies with mink, Gilbert (1969) reported that as little as 0.58
ppm DDE contamination of mink diets caused early kit mortality. Mink
rations supplemented with 100 ppm DDT (Duby, 1970) or 100 ppm DDT plus
50 ppm DDD (Aulerich and Ringer, 1970b), however, had no adverse effect
on mink reproduction or kits mortality.

The mercury content of the various fish fed to the mink in an
experiment associated with this problem (Aulerich at al., 1971) ranged
from 0.08 ppm in ocean whiting to 0.36 ppm in the Lake Erie cOho salmon,
but no association was evident between the mercury content of the fish

and the reproductive performance or mortality observed in the mink.

8
Since the Great Lakes fish-mink feed problem did not appear to be
due to any of the above mentioned causes of reproductive complications,
yet based on the clinical signs and lesions reported by Aulerich
(1971) seemed to be due to some toxic substance in the fish, emphasis
in the present study was placed on the influence of other contaminants

of Great Lakes fish on mink.

Polychlorinated biphenyls: .

Polychlorinated biphenyls (PCB's) are chlorinated hydrocarbon
compounds that have been employed in industry as dielectric fluids in
transformers and capacitors, as well as for a multiplicity of less
essential uses over the past two decades (Anon., 1972b). These com-
pounds were not thought of as contaminants until environmental problems
were attributed to them in Sweden (Jensen, 1966). Today, they are
considered to be widespread environmental pollutants (Holden and
Marsden, 1967; Holmes at al., 1967; and Risebrough at al., 1968).

PCB's are fat soluble and persistent compounds that tend to accumu-
late in oils and fats, and have been detected in milk, eggs, meat,
fats. oil and cereal products (Anon., 1972b). They have directly or
indirectly found their way into animal feed and animal products through
water, paint, heat transfer fluids and plastic and cardboard food
packing materials (Anon., 1972b).

Nisbet and Sarofim (1972) reported PCB levels in fish as high as
213 ppm from industrialized rivers in the U.S. They further reported
PCB levels of 19, 20, 0.01 and 0.03 ppm in fish from Lake Ontario, Lake
Michigan, and Atlantic Ocean and the Pacific Ocean, respectively.
Analysis of Lake Michigan coho salmon used in the mink feeding trials

concerned with this investigation showed that these fish contained 10

9
to 15 ppm PCB's (Aulerich at al., 1971). Bachezet al. (1972) found as
high as 30.4 ppm PCB in trout from Cayuga Lake, New York.
The basic structure of PCB's is shown below. Any of the atoms
appearing at the positions marked with an "X" can be substituted for by

a chlorine atom (Jensen, 1970). The PCB's produced commercially contain

X X X X
KO Ox
X X X X

approximately 50 of the 210 different compounds which are theoretically

possible when biphenyls are chlorinated. The most common mixtures contain

between 40 and 60 percent chlorine.(Anon., 1972b). In the United States

these compounds are identified by the percent of chlorine by weight in the

total product and sold under the trade name AroclorR* (Anon., 1972b). The

last two digits of the Aroclor number indicate the percent chlorine.
Despite the recent interest in these compounds the LDSO of PCB's

is rather poorly defined in comparison with the chlorinated hydrocarbon

pesticides. Acute oral or dermal toxicity of chlorinated biphenyls on

mice, rats, rabbits and guinea pigs have been studied by Miller (1944)

and Tucker and Crabtree (1970). Sublethal effects of PCB's on mammals

and birds were summarized by Peakall and Lincer (1970). The most striking

pathological changes in the organs in mammals are alterations in the

liver (Bennett at al., 1938; Miller, 1944; Nishizumi, 1970; Koller and

Zinkl, 1973; Grant at al., 1971a; and Cecil at al., 1973).

 

*
Trade name for PCB's manufactured by Monsanto Company, St. Louis,
Missouri.

10

Like other organochlorine compounds, such as DDT, PCB's have been
shown to have an adverse effect upon reproduction in birds, mice, rats
and dogs (Deichmann and MacDonald, 1971). Pheasants given a capsule
containing 50 mg of Aroclor 1256 weekly for 17 weeks produced fewer
eggs than controls, and although a high percentage of the chicks piped
the shell they did not hatch (Dahlgren and Linder, 1971).

In a study by the FDA (Anon., 1972c), weanling male and female rats
were fed diets that contained Aroclor 1254 at levels of 0, 100 or 500
parts per million for 67 days and then pair-mated. Reproduction in the
rats fed 100 ppm PCB was comparable to the controls in the numbers of
litters produced and the number of pups per litter. However, 76.8 per—
cent of the pups in the group fed 100 ppm PCB survived to weanling
compared with 95.5 percent for the controls. The mean body weight of
the rats fed 100 ppm PCB at weanling was 31.4 gm and that of the control
group was 39.2 gm. Only two of the ten females fed 500 ppm Aroclor 1254
had litters and these pups died within 3 days after birth.

Platonow and Karstad (1972) reported on the effects of PCB's on
mink. Feeding female mink rations that contained 3.57 ppm or 0.64 ppm
of total PCB's resulted in either reproductive failure or total kit
mortality within 24 hours postpartum. Adult female mink fed the ration
that contained 3.57 ppm PCB all died by the 105th day, although clinical
signs in animals were either absent or non-specific. The gross lesions
observed at necropsy were emaciation, gastrointestinal and intra-
abdominal hemorrhages, and a yellowish discoloration of the liver.

Based on these results, Platonow and Karstad (1972) suggested that mink

were among the more sensitive species to PCB poisoning.

METHODS

The animals used in these experiments were from the Michigan State
University experimental mink ranch. The mink were confined in individual
pens of uniform size and each pen was equipped with a water cup and
nest box. Except where stated otherwise in the text, the following
procedures pertain to all the experiments:

Standard ranch procedures were followed in feeding, breeding, and
caring for the animals. The mink were vaccinated for canine distemper,
botulism and virus enteritis each year in July.

In alloting the mink into various groups for experimentation,
littermates were divided between groups in an effort to balance genetic
differences in growth, reproduction and response to treatments.

The mink were weighed individually at the start of the experiments
and at specified intervals thereafter, except during the gestation
period (March-May). All matings were verified by taking a vaginal
smear after copulation and observing it microscopically for the presence
of normal appearing motile sperm. The females were checked daily for
the birth of young during the whelping period. Kits were counted and
weighed the day of birth and at 4 weeks of age.

Since the results of previous experiments in which PCB's and coho
salmon.were fed to mink showed no adverse effects on spermatogenesis
(Aulerich, 1970), only reproductive data pertaining to female mink are

reported in this study.

11

12

Mink kits found dead the day of birth or that died during the
eXperiment, as well as tissue from the adult animals that died or that
were sacrificed while receiving the various dietary treatments, were
submitted to the M.S.U. Pesticide Research Center for PCB analysis.

The tissue samples were stored in glass vials in a freezer (0°C) until
processed for analysis.

The animals that died during the experiments were necropsied, organ
weights recorded, and selected tissues collected for histopathologic
examination. Routine histologic procedures were employed in preparing
the tissue for histopathologic examination. The tissues were stained
with hematoxylin and eosin.

‘In the preparation of the eXperimental diets employed in this study,
the PCB's or pesticides were dissolved in acetone and incorporated into
the experimental diet by blending the solution with a small quantity of
ground commercial mink cereal. The acetone was evaporated off and the
premix containing the PCB's or pesticide was mixed with the other ingredi—
ents of the diet to yield a ration that contained the desired amount of
the various chlorinated compounds.

The composition and proximate analysis of the basal (control) diet
is shown in Table 1.

All experimental data were subjected to analysis of variance. The
differences among the dietary group means were tested using the Duncan's

Multiple Range Test (Duncan, 1955).

13

Table 1. Composition of basal (control) diet

 

 

Ingredients Percent
Supplemented cereall 15.0
Tripe 20.0
Liver (beef) 5.0
Horsemeat 15.0
Chicken 15.0
Ocean fish2 30.0

 

lKel-Centrate #1002, W. K. Kellogg Co., Battle Creek, Michigan.

2Mixture of cod, haddock and flounder.

 

Proximate analysis of basal diet ("as-fed" basis)

 

Crude Ether N-free Crude
protein extract extract Ash fiber H20
Percent 11.16 7.14 7.71 2.41 0.89 70.69

1973.

EXPERIMENTS

The research reported in this thesis was conducted between 1971 and

It consisted of the following experiments:

Experiment

Experiment

Experiment

Experiment

Experiment

Experiment

Experiment

Experiment

I.

II.

III.

VI.

VII.

VIII.

Effects of PCB's and Lake Michigan coho

salmon on mink reproduction and viability

A. Effects of long-term, low-level consumption
of PCB on mink

B. Investigation of interactions between
PCB's and chlorinated hydrocarbon
pesticides

LDSO of PCB's for mink

Effect of various levels of PCB's and PCB's

plus chlorinated hydrocarbon pesticides on

mink reproduction and viability

Comparison of various PCB's on mink growth,

reproduction, hematologic parameters and viability

Rate of accumulation and depletion of PCB's

in adipose tissue of mink

Evaluation of acetone-hexane extraction as a

practical method of removing toxic factors

from Great Lakes fish

Investigation of the mode of action of PCB's

in mink

l4

15
Experiment IX. Impermanence of reproductive failure in mink

fed PCB's or Lake Michigan coho salmon

Experiment I
This experiment was conducted to determine the effects of feeding

high levels of supplemental PCB's or coho salmon on mink reproduction

and viability.

Procedure
The experiment was conducted from January 1, 1971, to June 30,
1971. Thirty-six female (proven breeder) mink were allocated in three
groups consisting of twelve females per group, and were fed a basal
diet (Table l) supplemented or modified as follows:
Group I - 1 Unsupplemented, unmodified basal (control) diet
Group I - 2 Basal diet except for the substitution of 30 per-
cent ground, whole, raw, Lake Michigan coho salmon
for 30 percent ocean fish
Group I - 3 Basal diet supplemented with 30 ppm PCB's (10 ppm

each of Aroclors 1242, 1248 and 1254)

Results

No significant differences were noted between the mean body
weights of the females on the three dietary treatments (Table 2) during
the first three months of the feeding trial.

The reproductive performance of the mink is shown in Table 3. None
of the females that received Lake Michigan coho salmon (Group I - 2) or
supplemental PCB's (Group I - 3) in the diet whelped.

All the mink in Group I — 3 (30 ppm PCB) died between February 28

and May 2, 1971. Six of the 15 mink in Group I - 2 died between March

16

Table 2. Average body weight change (gm : S.E.) of female mink fed a
control ration or experimental diets (Experiment I)

 

Weight change

 

 

Dietary Initial wt. 1 mo. 2 mo.

treatment (Dec. 27) (Jan. 27) (Feb. 25)

I - 1 (control) 1067 i 42.3 - 9 i 17.6 —145 i 24.0
(n=12)

I - 2 (30% coho salmon) 1015 i 41.9 -15 i 21.8 -120.i.22-5
(n=12)

I - 3 (30 ppm PCBl) 976 i 55.1 -32 i 13.0 -155 i 16.4
(n=12)

 

1The PCB supplementation consisted of 10 ppm each of Aroclors
1242, 1248 and 1254.

Table 3. Reproductive performance and mortality of female mink fed a
control ration of experimental diets (Experiment I)

 

 

 

 

 

Adult females , Kits

Dietary Z No. No. No. born Whelped/¥ INo. alive
treatment mortality mated whelped Alive Dead mated at 4 wks.
I - 1 (control) 0 12 11 35 19 4.5 29
I - 2 (30% coho 25 12 0 - - - -

salmon)
I - 3 (30 ppm 100 112 0 — - - -

PCBl)

l

The PCB supplementation consisted of 10 ppm each of Aroclors 1242,
1248 and 1254.

2One female died prior to the mating season.

17
10, 1971, and June 1, 1971, when the feeding trial was terminated.
Anorexia, bloody stools, fatty degeneration of the liver and kidneys and
hemorrhagic gastric ulcers (Figure 1) were common clinical signs and
lesions observed in the animals that died in Groups I - 2 and I — 3.
The clinical signs and lesions observed in these two groups of mink
were strikingly similar. The gross and histopathologic lesions observed
in the tissues from these mink are summarized in Table 4. Three of the
six females in Group I - 3 that died during the latter stages of gesta-
tion had fetuses in their uteri. No fetuses were observed in the one
female from Group I - 2 that died during the whelping period.

At necropsy, heart, liver, kidney, spleen and brain weights were
recorded for the mink that died during the feeding trial. The mean organ
weights (expressed as a percent of brain weight) for the female mink in
Groups I - 2 and I - 3 are shown in Table 5, along with comparable data
for seven control animals. Analysis of variance of the data presented
in Table 5 showed no significant differences between the organ weights
of the mink from the various dietary groups.

PCB tissue residue analyses from control mink and from the animals
that died while receiving diets that contained coho salmon or supplemental
PCB's are shown in Table 6. Similar PCB residue levels were noted in

the tissues from mink fed the two experimental diets.

Discussion

 

The results of this experiment confirmed the findings of a study by
Aulerich et a1. (1971), who reported that reproductive failure occurred
from feeding Lake Michigan coho salmon to mink.

Although no significant differences were observed in the weight

gains of the mink on the various dietary treatments, these results may

18

Figure l. Visceral surface of mink stomach showing a pyloric
ulcer frequently observed in mink that died while receiving diets
supplemented with PCB's (Experiment I).

 

19

 

Figure I

Table 4 .

20

Gross and histologic lesions commonly observed in tissues from

mink that died while receiving diets that contained Lake

Michigan coho salmon

or supplemental PCB's (Experiment I)

 

 

Dietary
treatment Tissue Pathologic observations
I - 2 (30% coho Liver Gross: pale, fatty
salmon) Histologic: cloudy swelling and
fatty degeneration
Stomach Gross: gastric ulcers (pyloric)
Cervical Gross: enlarged and hard
lymph nodes
Kidneys Histologic: cloudy swelling and
fatty degeneration
Lungs Histologic: diffuse hyperemia
Adrenals Histologic: cortical hyperplasia and
hypertrophy, cortical adenomas,
medullary atrophy
I - 3 (30 ppm Liver Gross: pale, fatty, focal hemorrhages
PCB'sl) Histologic: cloudy swelling, fatty
degeneration, diffuse hemorrhages
Stomach Gross: gastric ulcers (pyloric)
Kidneys Gross: pale
Histologic: cloudy swelling, hydropic
degeneration, diffuse hemorrhages
Lungs Gross: pale
Histologic: diffuse hyperemia
Adrenals Histologic: cortical hyperplasia,
cortical adenomas
Brain Histologic: focal cortical chromo-
philic areas
Spleen Histologic: amyloidosis

 

lThe PCB supplementation consisted of 10 ppm each of Aroclors 1242,

1248 and 1254.

21

Table 5. Mean organ weights1 of female mink fed a control ration or
experimental diets (Experiment I)

 

Dietary ' Organ
treatment Heart Liver Kidneys Spleen

 

I - 1 (control) 75.8 1 4.75 399.5 1 48.68 65.31 3.09 35.5 1 5.92
(n=7)

I - 2 (30% coho 76.3 1 18.51 383.8 1 19.04 67.6_1 5.76 32.3 1 11.78
salmon) (n=3)

I - 3 (30 ppm 72.9 1; 4.23 397.2 1_24.1 70.2 1_3.94 28.3 + 6.45
P082) (n-11)

 

1Expressed as percent of brain weight :_S.E.

2The PCB supplementation consisted of 10 ppm each of Aroclors

1242, 1248 and 1254.

Table 6. Average PCB content of tissues (PPm‘:_S.E.) from mink that died
or were sacrificed1 (Experiment I)

 

 

 

Dietary Tissue

treatment Brain Liver Kidney Spleen Lung Muscle Heart
I - 1 (con— <0.01 <0.01 <0.01 <0.01 <0.01 N.D.3 N.D.

trol) (n=4)2

I - 2 (30% 8.91 11.43 6.37 6.19 6.84 6.24 3.13

coho zalmon) (12.19) (16.22) (10.19) (10.07) (11.70) (11.98) (10.70)
(n=4)

I — 3 (30 11.00 4.95 4.47 4.79 4.48 4.88 3.26
ppm PCES) (11.43) (10.57) (10.41) (10.39) (10.57) (10.54) (10.54)
(n=12)

 

IAnalyses by M.S.U. Pesticide Research Center
2Sacrificed

3None detected

4Died

5The PCB supplementation consisted of 10 ppm each of Aroclors
1242, 1248 and 1254.

22
be misleading, since the animals were fed to condition them for optimum
reproduction during this period, i.e., lean animals were "full—fed"
while obese mink received restricted amounts of feed prior to and
during the mating season.

If it is assumed that an adult female mink consumes 150 g of feed
per day (Schaible, 1969), the average total intake of PCB's by the mink
in Group I — 3 that died while receiving the diet that contained 30 ppm
PCB's (for an average of 106 days) would have been about 477 mg. Since
the Lake Michigan coho salmon used in this experiment contained PCB
residues ranging from 10 to 15 ppm (Aulerich at al., 1971), the diet
fed to the mink in Group I - 2 (30% coho salmon) contained approxi-
mately 3 to 5 ppm of PCB's. The females in Group I - 2 that died and
whose tissues were analyzed for PCB content survived an average of 68
days on the experiment and, thus, would have consumed approximately 51
mg of PCB's. The PCB residue levels shown in Table 6 do not reflect the
variation in PCB consumption of the mink in Groups I - 2 and I - 3.
This, however, may be due to differences in the toxicity and metabolism
of the PCB's fed to the mink in Groups I - 2 and I - 3.

Studies by Bennett et a1. (1938), Miller (1944), Grant at al.
(1971a) and Cecil et a1. (1973) have demonstrated degenerative liver
changes, as well as an increase in liver size and lipid content in rats
exposed to PCB's. Vos and Notenboom-Ram (1972) observed a similar
increase in the weight of liver of rabbits exposed to Aroclor 1260.
Although no significant increase in liver weights was noted in the
mink that died while receiving diets that contained PCB's in this
experiment, fat accumulation and other degenerative changes (as shown

in Table 4) were evident.

23
The results of this eXperiment support the supposition that PCB's
may be, at least in part, responsible for the reproductive failure and

mortality observed when Lake Michigan coho salmon are fed to mink.

Experiment II
This experiment was started on August 24, 1971, to ascertain the
effects of long-term, low-level consumption of PCB's on mink repro-
duction and viability and to investigate the possibility of interactions
between PCB's and some chlorinated hydrocarbon pesticide contaminants

of Great Lakes fishes.

Procedure
In this experiment, 30 natural dark female mink kits approximately
three and one—half months of age were allocated into five groups and

placed on the following dietary treatments.

Group II - l Unsupplemented, unmodified basal (control) diet

Group II - 2 Basal diet supplemented with 5 ppm.Aroclor 1254

Group II - 3 Basal diet supplemented with 10 ppm Aroclor 1254

Group II - 4 Basal diet supplemented with 10 ppm Aroclor 1254
plus 10 ppm DDT

Group II - 5 Basal diet supplemented with 10 ppm Aroclor 1254

plus 0.5 ppm dieldrin
Individual mink body weights were recorded at the beginning of the
feeding trial and at monthly intervals throughout the experiment (except

during the gestation period).

Results
The average initial body weights and average weight gains of the
mink on the various dietary treatments during the growth period are

shown in Table 7.

24

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muofio anuaoafiummxo Ho gwumu Hobaoo m mam Mada Imfimaow .05 Aemem H 85 omqmfio H.303 boon owmuo>< .m manna.

25

Five ppm supplemental PCB's (Group II - 2) in the ration did not
significantly affect the weight gains of the mink. Ten ppm PCB's alone
(Group II — 3) or in combination with 0.5 ppm dieldrin (Group II - 5)
resulted in significant differences (P<0.05) in body weight gains after
four months feeding. The mink fed the ration that contained 10 ppm
PCB's plus 10 ppm DDT (Group II - 4), however, showed a significant
reduction in weight gains after only two months feeding and weighed
less than their initial body weights after 4 months on the diet.

No clinical signs of PCB or chlorinated hydrocarbon poisoning
(other than the reduced weight gains) were observed in the treated
animals during the growth period (Aug. 25 to Dec. 22). Considerable
mortality, however, occurred on some diets during the maintenance and
reproductive periods (after the animals had consumed the experimental
rations for at least 160 days). None of the animals fed the ration
supplemented with 10 ppm PCB plus 0.5 ppm dieldrin survived to the
termination of the experiment (June 1, 1972), and only 1 of 6 mink
survived on the diet that contained 10 ppm PCB. Although DDT in combi-
nation with PCB appeared to reduce mortality (Table 8), none of the
females that survived to term on the rations that contained supplemental
PCB whelped.

The PCB treated mink that survived to June 1, 1972, were sacri-
ficed, their organs weighed, and selected tissues from those in Groups
II - 2 and II - 3 were analyzed for PCB content. The PCB tissue
residues and mean organ weights from these animals are shown in Tables
9 and 10. The mean weights of the kidneys, liver and heart of the PCB

treated mink were significantly (P<0.05) greater than the controls.

26

Table 8. Reproductive performance and mortality of female mink fed a
control ration or experimental diets (Experiment II)

 

 

 

 

 

Adult females Kits A
Dietary % No. No. No. born Whelped/¥ No. alive
treatment mortality mated whelped Alive Dead mated at 4 wks.
II — l 17 5 3 l7 8 5.0 8
(control)
II - 2 (5 ppm 33 4 - - — - -
PCB)1
11 - 3 (10 ppm 83 1 - - — - -
PCB)
II - 4 (10 ppm 33 4 - - - - —
PCB + 10 ppm DDT)
II - 5 (10 ppm 100 l - - - — —
PCB + 0.5 ppm
dieldrin)

 

1Aroclor 1254

27

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28

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29
Discussion

Although no significant differences in body weight gains were
observed in the short-term feeding trials (Experiments I and IV) involv-
ing PCB's, long-term administration of PCB's, as noted in this experi-
ment, resulted in significant body weight changes among the various
dietary treatments. The lack of significant differences in weight
gains during the fifth and sixth months of the experiment may be due
to the feeding regime employed to condition the animals for breeding.
Based on these results it would appear that body weight changes are
not a sensitive measurement of PCB exposure in mink.

As shown by the data presented in Table 7, there appeared to be
an interaction between PCB's and the chlorinated hydrocarbon pesticides
on the growth of the mink. In a study by Lichtenstein at al. (1969)
it was found that adding PCB and DDT together produced 100% mortality
in flies at concentrations that produced only slight mortality when
the compounds were tested alone. In this experiment and Experiment IV
(Table 12) adult mink mortality was not increased by the addition of
DDT to the PCB's but in both experiments the addition of dieldrin to
the PCB's resulted in increased mortality. Dahlgren er al. (1972)
suggested that effects of PCB's and dieldrin together are additive, not
synergistic. Whether or not reproduction would be influenced by the
interaction or potentiation posted in the growth of the mink was not
ascertained, as none of the PCB-treated females whelped.

The differences in the PCB tissue residues of the mink in Experi-
ments I (Table 6) and II (Table 9) are marked and may be attributed to
the different levels of PCB's fed, duration of feeding, as well as the
fact that the PCB tissue residues in Experiment II were from sacrificed

animals whereas those in Experiment I were from mink that died while

30
receiving diets that contained PCB's. Dahlgren at al. (1972) stated
that brain levels of PCB were generally higher in birds that died than
in birds that were sacrificed, as was also observed in mink in Experi-
ment II (Table 9) in this study.

PCB's are fat soluble and persistent compounds that tend to accumu—
late in oils and fats (Anon., 1972b) and residues are usually found to
be most abundant in the tissues with the greatest concentration of
adipose tissue (Grant at aZ., 1971a, Curley at al., 1971, and Platonow
at al., 1972).

The significant differences in the organ (heart, kidneys, and
liver) weights of the PCB-treated and control mink observed in Experi-
ment II (Table 10) and the relative similarity in organ weight of the
mink on the various treatments in Experiment I (Table 5) might be due
to the variation in the length of the two feeding trials, the longer
exposure to PCB in Experiment II being manifested by an increase in the
organ weights as was reported in rats by Bennett at al. (1938), Grant
et al. (1971a), Miller (1944), Cecil at al. (1973) and in rabbits by

Vos and Notenboom—Ram (1972).

Experiment III
This experiment was conducted during December, 1971, in an attempt
to determine the LD50 (lethal dose for 50% of the animals) of some
common PCB's (Aroclors 1221, 1242 and 1254) for mink.

Procedure
Fifty adult mink not previously fed Great Lakes fish nor exposed
to any supplemental PCB's or chlorinated hydrocarbon pesticides were

allocated to the experiment.

31
The PCB's were administered intraperitoneally (IP) since oral
administration of the compounds resulted in vomiting shortly after they

were administered.

Results

The acute toxicity values (lethal dose for 50 percent of the
animals within 96 hours after administration) of Aroclors 1221, 1242
and 1254 for mink are given in Table 11. The toxicity of the PCB's
varied inversely with the chlorine content (last two digits of the
Aroclor number) of the compounds. The values ranged from 500 mg/kg

for Aroclor 1221 to as high as 2250 mg/kg for Aroclor 1254.

Discussion

 

Lewin and McBlain (1972) reported an acute IP LD50 (5 days)
toxicity of PCB's in mice of 880 to 2840 mg/kg for Aroclor 1254°

Although some investigators (Risebrough and Brodin, 1970, and
Prestt at al., 1970) have reported a positive relationship between the
percentage of chlorine in PCB's and their relative toxicity, the toxicity
of the Aroclors tested with the mink in this study varied inversely with
the percentage of chlorine in the compounds.

Table 11. Acute toxicity (LD50)l of PCB's when adminisrered IP to
mink (Experiment III)

 

PCB LD

 

50
Aroclor 1221 > 500 < 750 mg/kg
Aroclor 1242 1000 mg/kg
Aroclor 1254 > 1250 < 2250 mg/kg

 

1Lethal dose for 50% of the anImals within 96 hours.

32

Experiment IV

This experiment consisted of a series of dietary treatments involv-

ing PCB's, chlorinated hydrocarbon pesticides, and coho salmon fed to

mink in an attempt to ascertain the effects of these compounds on mink

reproduction and viability.

Procedure

The experiment was conducted from January 1, 1972, to May 10, 1972.

Eight groups consisting of twelve adult female (proven breeder) mink per

group were placed
Group IV - 1
Group IV - 2
Group IV - 3
Group IV - 4
Group IV — 5
Group IV - 6
Group IV - 7
Group IV - 8
The fish fed

and 15 p.s.i. for

was formulated in

content in the 30

in Group IV - 2.

on the following dietary treatments:

Unsupplemented, unmodified basal (control) diet

Substitution of 30 percent 1971 Lake Michigan

coho salmon for 30 percent ocean fish in the

basal diet

Same as Group IV - 2 except that the coho salmon

was cooked
Basal diet
Basal diet
Basal diet
Basal diet
plus 6 ppm
Basal diet
plus 6 ppm

to the mink

prior to substitution

supplemented with 1 ppm Aroclor 1254

supplemented with 5 ppm Aroclor 1254

supplemented with 15 ppm Aroclor 1254
supplemented with 5 ppm Aroclor 1254

DDT

supplemented with 5 ppm Aroclor 1254

DDT plus 0.2 ppm dieldrin

in Group IV - 3 were autoclaved at 120°C

30 min. The diet fed to the mink in Group IV - 8

an attempt to duplicate the PCB, DDT, and dieldrin

percent Lake Michigan coho salmon fed to the animals

33
Results

The reproductive performance and mortality of the mink on the
various dietary treatments are summarized in Table 12. Supplementation
of the control diet with 1 ppm PCB (Aroclor 1254) caused a slight reduc-
tion in reproduction; 5 ppm PCB resulted in a marked reduction in repro-
duction; and 15 ppm supplemental PCB totally inhibited reproduction and
resulted in 31 percent adult mortality when fed for four months. Repro-
ductive failure also occurred on the diets that contained 30 percent
autoclaved coho salmon (Group IV - 3).

The average body weight change of mink fed the various dietary
treatment in Experiment IV is shown in Table 13. There were no sig-
nificant differences in mink weight gains among the dietary treatments.

Table 14 shows the mean organ weights (expressed as a percent of
brain weight) of the mink in Group IV - 6 that died during the experi-

ment, as well as the PCB content of their tissues.

Discussion

 

The reproductive performance of the females fed coho salmon in
this experiment was similar to those fed coho salmon in Experiment
I - 2, as none of the animals whelped. The reproductive failure of the
females fed the autoclaved coho salmon (IV - 3) demonstrated that the
toxic factor present in the fish is quite heat stable. Supplementing
the basal diet with equivalent amounts of PCB, DDT and dieldrin, as
contained in the diet fed to the mink in Group IV — 2, did not impair
reproduction to the extent noted in the animals in Group IV - 2. The
reproductive performance and mortality data of mink fed the diet that
contained 5 ppm PCB (IV - 5) and 5 ppm PCB plus DDT (IV - 7) or DDT and

dieldrin (IV - 8) indicated that PCB's are the major source of

34

Table 12. Reproductive performance and mortality of female mink fed a

control ration or experimental diets (EXperiment IV)

 

 

 

 

Adult females Kits
Dietary % No. No. No. born Whelped/¥ No. alive
treatment mortality mated whelped Alive Dead mated at 4 wks.
IV - l 8.3 11 ll 56 10 6.0 46
(control)
IV - 2 (30% raw 8.3 11 0 0 0 0 0
coho salmon)
IV - 3 (30% 0 12 0 0 0 0 0
cooked coho
salmon)1
IV - 4 (1 ppm 8.3 12 8 35 8 3.6 18
PCB)2
IV - 5 (5 ppm 0 12 3 3 6 0.8 2
PCB)
IV - 6 (15 ppm 33.0 11 0 0 0 0 0
PCB)
IV - 7 (5 ppm 0 ll 4 5 6 1.0 0
PCB + 6 ppm
DDT)
IV - 8 (5 ppm 8.3 11 5 15 7 2.0 0

PCB + 6 ppm DDT
+ 0.2 ppm
dieldrin)

 

lAutoclaved at

2Aroclor 1254

120°C and 15 p.s.i. for 30 min.

35

 

 

 

Table 13. Average body weight change (gm :_S.E.) of female mink fed a
control ration or experimental diets (Experiment IV)
Weight change

Dietary Initial weight 1 mo. 2 mo.

treatment (Jan. 1) (Feb. 3) (Mar. 1)

IV - 1 (control) 1034 1 78.6 -38 1 33. -200 1 47.
(n=12)

IV - 2 (30% coho salmon) 996 i 45.4 -58 :_10. -l30 : l9.
(nelZ)

IV - 3 (30% autoclaved 1028 i 27.7 -60 :_l3. -l42 : 28.
coho salmon) (n=12)

IV - 4 (1 ppm PCB)1 1070 1 49,5 -72 1 51. -181 1 30.
(n=12)

IV - 5 (5 ppm PCB) 979_: 31.9 -33 :_l9. -l79 : l9.
(n=12)

IV - 6 (15 ppm PCB) 1058 :_33.9 -91 i 26. -219 j 30.
(n=12)

IV - 7 (5 ppm PCB + 6 ppm 961 i 60.5 -66 i 24. -l68 : 32.
DDT) (n=12)

IV - 8 (5 ppm PCB + 6 ppm 1031 i 45.3 -68 i 15. -189 i 29.

DDT + 0.2 ppm dieldrin)
(n=12)

 

1Aroclor 1254

36

umuaoo cosmomom MUHUHummm .D.m.z mfiu hp mothmam mom

.m.m unustms aHmun mo ucmoumd mm vommouaxm

emNH poHoon<m

N

 

 

 

 

 

H
Amo.eaflv Hoo.mwv Aom.mmu Amm.ouu Amo.muv Amo.mmo AoN.HHo Amuov
mm.mm~ HN.oH cH.mH sa.m Ho.mH Hm.om mo.oH sm.m Amen non mHV o I >H
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uszos nmwmo swoz
osmmHu omome< oHomsz mesa onum puma: uo>HA hocwHM cooHam uooaummuu
onmmHH annuoHn

 

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A>H uaoaHHoaxmv mmum kuaoamHaqam Bed mH puaHmucoo umsu muon
umau Jame Eoum momma» mo Aem.m.HfBenv Nuaouaoo mom name new HuanoB.qmwuo omoz .qH oHan

37
reproductive complications in mink and that the pesticides (DDT and
dieldrin) did not appear to have a synergistic effect on mink repro-
ductive performance, as was suggested by mink growth in Experiment II.

Many pesticides are known to stimulate the synthesis of the
hydroxylating liver enzymes which aid in the digestion of foreign com-
pounds (Morello, 1965). These enzymes, however, also catabolize
endogenous substances, such as the steroid hormones, which may influence
sexual function by disturbing the metabolism of the sex hormones
(Peakall, 1967). Like the organochlorine insecticides, PCB's have
also been shown to induce hepatic hydroxylating enzymes (Risebrough
et al., 1968; Lincer and Peakall, 1970; and Villeneuve at al., 1971a).

Following the administration of high dosages of PCB's to boars,
Platonow et al. (1972) noted lowered levels of dehydroepiandrosterone
and estrogen in the excreted urine and also suggested that PCB compounds
have a deleterious effect upon reproductive activity. Studying the
effects of PCB's and DDT on the estrus cycle of mice, Orbert et a1.
(1972) reported single injections of DDT or PCB's increased the length
of the cycle, resulting in less frequent periods of sexual receptivity
in the female, thereby causing a decline in the reproductive capacity
of the animal.

In a study by Villeneuve et al. (1971b) it was demonstrated that
Aroclors 1221 and 1254 administered orally to female rabbits during
gestation crossed the placental barrier. Grant et al. (1971b) and
Villeneuve at al. (1971b) reported Aroclor 1254 induced abortion and
was fetotoxic to rabbits.

Undoubtedly the adverse physiological effects on reproduction
induced by PCB's, DDT and dieldrin could also account for the repro-

ductive impairment observed in mink fed these compounds.

38
Based on the results of the experiments conducted in this study,
adult mink mortality on diets that contained PCB's was in general
directly proportional to the total intake of these compounds. Assuming
the mink that died in Experiments II, IV and V had consumed 150 g of
feed per day (Schaible, 1969) and weighed 700 g, the oral lethal dose

(LD ) of Aroclor 1254 was calculated to be about 470 mg/kg, and the

100
oral LD was approximately 350 mg/kg (Figure 2). An oral LD of

50 50
Aroclor 1254 for rats was reported by Tucker and Crabtree (1970) to
be 500 to 1000 mg/kg. According to Miller (1944), two oral doses of
69 mg of 42 percent chlorinated biphenyl a week apart were fatal to
guinea pigs.
Although it may be inexpedient to compare the toxicity of compounds

under different experimental conditions, it would appear from these

results that mink are quite sensitive to PCB's.

Experiment V
This long-term feeding trial was conducted to investigate and
compare the effects of four different PCB's fedanza low level on growth,

reproduction, hematologic parameters, and viability of mink.

Procedure

This experiment was initiated on August 8, 1972. Fifty pastel
mink kits approximately three months of age were allocated into five
groups (consisting of 10 females per group) and were fed a basal diet

supplemented or modified as follows:

Group V l Unsupplemented, unmodified basal (control) diet
Group V - 2 Basal diet supplemented with 2 ppm Aroclor 1016

Group V — 3 Basal diet supplemented with 2 ppm Aroclor 1221

Group V 4 Basal diet supplemented with 2 ppm Aroclor 1242

 

‘1'” »

39

Figure 2. Relationship between consumption of Aroclor 1254
and mortality in mink. Calculation based on mortality data from
Experimental groups II - 2, II - 3, IV - 4, IV - 6 and V - 5 and
an assumed feed consumption of 150 g/day.

 

i Mortality

40

100 .
9o "

70 o’
60

so
4o

30 '9'“

20 e’

 

10 I.

 

J 4’ f *
100 200 300 400

Aroclor l254 consumption (mg. I
Figure 2

500

41

Group V - 5 Basal diet supplemented with 2 ppm Aroclor 1254

Females that did not whelp by May 30, 1973, were removed from the
experimental diets; those that whelped were continued on the experimental
rations until their kits were four weeks old.

Samples of blood were collected from the mink (by clipping a toe-
nail) at the beginning of the trial and at monthly intervals thereafter
(except during April). These samples were analyzed for hemoglobin
content (acid hematin method, Dennington and Lucas, 1955) and hemato-
crit values.

On June 1, 1973, three female mink fed each of the diets in Experi-
ment V were sacrificed, the organs weighed, and selected tissue collected

for PCB residue analyses.

Results

The average initial body weights and average weight gains (through
Feb. 19, 1973) of the female mink are shown in Table 15. Although the
weight gains of the mink fluctuated considerably during the course of
the experiment, no significant differences were noted except during the
first month after the start of the feeding trial. After November, 1972,
a trend toward reduced weight gains was observed in the groups fed
supplemental PCB's while the control animals maintained a fairly constant
body weight, except just prior to the breeding season (March) when all
animals were fed to "condition" them for optimum reproduction (thin
mink continued to be fed ad Zibitum but obese females received restricted
amounts of feed).

The average hematocrit values for the mink are shown in Table 16°
The hematocrit values for all the groups dropped considerably after the

whelping period (April 24-May 15). The average hemoglobin content and

42

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mEmm ma UMSOHHOM mosam>a

 

 

 

mwamno unwfi m3

I I I I I I I Amuse

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m sea m oNH .m mma m maa m sea a ONH m ens see we m I >

I I I I I I I ans

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m as m Ha a sea m mmm a eaH am mm m mmn see me e I >

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a wee m ama m mafi m wwN a mem a emu m own age No N I >

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AmH .nmev “NN .qmev Ana .omav Aha .>ozv AH .uoov AH .ammv s .m=< unmaummuu
.08 o .08 m .08 q .08 m .08 N .08 H uswfim3 HmfiuHaH humuown

 

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woman HmucoEHumaxm Ho aowumu HOHucoo m mom ona mamemm mo A.m.m H Emv mwcmao unwfioB hvon ammuo>< .mH manms

43

Table 16. Hematocrit values for mink fed a control diet and rations

 

 

 

Average
Dietary Initial 1 mo. 2 mo. 3 mo.
treatment (Aug. 7) (Sept. 5) (Oct. 2) (Oct. 30)
V - 1 control 51.8 56.0 56.3 57.5
(basal diet) (10.82) (:0.22) (:0.48) (10.58)
V - 2 2 ppm 51.1 54.8 54.6 56.2
Aroclor 1016 (10.81) (i0.88) (10.91) (:0.87)
V - 3 2 ppm 51.7 55.5 55.0 56.8
Aroclor 1221 (:0. 76) (_+_O.81) (:0.67) (:0. 59)
V - 4 2 ppm 51.5 53.8 54.1 54.2
Aroclor 1242 Qtl.06) (:0.76) (:0.74) (:2.43)
V - 5 2 ppm 51.0 55.0 54.7 56.5
Aroclor 1254 (£0.65) (£1.06) (£0.65) 0:0.66)

 

44

containing supplemental PCB's (Experiment V)

 

hematocrit values (Z_i S.E.)

4 mo. 5 mo. 6 mo. 7 mo. 8 mo. 10 mo.
(Nov. 27) (Dec. 27) (Jan. 22) (Feb. 19) (Mar. 19) (May 15)

 

59.7 55.4 57.1 57.9 57.3 52.4
(10.41) (10.80) (10.89) (10.88) (10.74) (12.06)
60.4 58.1 58.1 56.5 58.9 54.2
(10.80) (10.54) (11.18) (10.78) (10.76) (11.21)
59.8 56.4 58.9 54.8 58.1 51.3
(11.01) (10.70) (10.72) (12.83) (11.06) (12.46)
56.4 53.7 51.9 56.4 57.8 52.6
(11.19) (12.92) (13.33) (11.33) (11.26) (11.47)
56.5 54.8 57.8 57.1 56.1 52.5

(10.56) (11.10) (11. 36) (11.02) (10.71) (11.06)

 

45
mean corpuscular hemoglobin concentration for the animals at monthly
intervals are shown in Table 17.

The reproductive performance, adult mortality and average kit
weights at birth are shown in Table 18. The one live kit whelped by
the females fed Aroclor 1254 weighed much less than the average weight
of the kits whelped by females on the other rations (Table 18). Three
adult female mink on EXperiment V died during the study, one each from
Groups V - 3, V - 4 and V - 5. There were no statistical significant

differences among the diet or organ weight (Table 19).

Discussion

 

Based on the previous mentioned assumption concerning feed consump—
tion, the female mink in Group IV - 4 (1 ppm PCB; EXperiment IV) con-
sumed about 18 mg of Aroclor 1254 during the four months they were on
experiments prior to whelping, which did not significantly impair
reproduction. The females in Groups IV - 5 (5 ppm PCB; Experiment IV)
and V - 5 (5 ppm PCB), however, consumed approximately 90 and 81 mg
of Aroclor 1254, respectively, during the same period which severely
impaired reproduction. As noted in the results of these experiments
(Table 18), Aroclor 1016, 1221 and 1242 did not adversely affect repro-
duction, which is in agreement with the results of studies by Villeneuve
at al. (1971a and 1971b). Koller and Zinkl (1973) also reported
Aroclor 1221 was less toxic than Aroclor 1254 and did not induce
enzyme activity in fetuses or placentas of rats and rabbits.

The reduced weight of the kits whelped by the only female that
reproduced in Group V - 5 is in agreement with the work of Villeneuve
(1971a), who reported a significant reduction in litter weights of

rabbits fed Aroclor 1254 at 100 mg/kg/day.

46

Table 17. Average hemoglobin (Hb) content and mean corpuscular hemoglobin
supplemental PCB's (Experiment V)

 

Average hemo-

 

 

Dietary Initial 1 mo. 2 mo. 3 mo.
treatment QAug. 7) (Sept. 5) (Oct. 2) (Oct. 30)
V - 1 Hb 18.610.47 10.0:0.22 20.110.19 20.410.33
control
(basal MCHC 35.910.70 35.610.33 35.610.19 35.4:0.36
diet)
V — 2 2 ppm Hb 18.610.49 19.5:0.20 19.410.24 20.610.34
Aroclor
1016 MCHC 36.310.53 35.8f0.35 35.6:0.32 36.710.25
V - 3 2 ppm Hb 17.8:0.40 19.910.33 19.410.24 21.0:0.13
Aroclor
1221 MCHC 34.410.59 35.810.89 35.310.73 37.010.40
V - 4 2 ppm Hb 18.610.24 19.310.25 19.619.36 20.410.93
Aroclor
1248 MCHC 36.110.41 35.9:0.20 36.219.33 37.310.39
V - 5 2 ppm Hb 18.919.41 20.1:0.29 20.110.18 20.410.20
Aroclor
1254 MCHC 37.110.73 36.610.43 36.810.28 36.010.28

 

47

concentration (MCHC) for mink fed a control diet and rations containing

 

globin (g z i S.E.) and MCHC (% 1 S.E.)

 

 

4 mo. 5 mo. . 6 mo. 7 mo. 8 mo. 10 mo.
(Nov. 27) (Dec. 27) (Jan. 22) (Feb. 19) (Mar. 19) (May 15)
20.910.20 20.410.32 20.210.32 20.310.35 20.5:0.51 l8.2:0.96
34.910.34 36.810.34 35.4:0.20 35.110.18 35.810.15 34.810.46
20.810.27 20.9:0.34 20.410.43 20.110.32 20.610.29 19.610.36
34.4fi0.31 36.01flal7 35.010.18 35.510.32 35.010.27 36.310.43
21.4:0.53 20.5:0.28 20.5:0.29 l9.2:1.ll 20.210.36 18.310.34
35.810.66 36.110.66 34.210.34 35.010.46 35.710.21 36.2:1.73
20.110.48 20.0:1.02 18.2:1.21 l9.8:0.55 20.410.53 18.210.64
35.610.39 37.510.36 35.0:0.34 35.110.36 35.3:0.l3 34.910.25
20.5:0.34 20.310.33 20.410.41 20.110.43 19.9:0.30 l7.Z:0.52
36.110.44 37.110.27 35.410.28 35.210.65 35.510.24 33.710.48

 

48

 

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I AHNNH SHOE...
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I GHOH 6892
2.0 + Na mm m... mm m w o mum ES 8 N I >
NCO H m5 2 m... a m w 0 28588 H I >
songs um A.m.m u” .mxB q um wouma 0>HH< voaaona vmuma hufiamuuoa uaoaumouu
mv .uB owwum>< m>HHm w\vomam:3 anon .02 .oz .02 N humumwm

 

moamfiom uH3v<

 

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muofiv Hmucmaaummxm no Goaumn Houudoo m vow Mafia mamamm mo hufiamuuoa was moamauomumn 0>Huoavouamm .mH canny

Table 19. Mean organ weightl
mental rations (Experiment V)

of mink fed a control diet or experi-

 

 

 

Dietary Organ

treatment Spleen Kidney Liver Heart

V - 1 (control) 35.7 58.6 260.4 68.8
(n85) (13. 38) (12 .84) (118.11) (11.26)

V - 2 (2 ppm PCB 30.1 58.9 280.7 61.8
Aroclor 1016) (13.15) (14.21) (122.54) (14.74)
(n=3)2

V - 3 (2 ppm PCB 27.5 58.5 331.6 65.2
Aroclor 1221) (11.28) (12.37) (120.45) (12.36)
(n=3)3

V - 4 (2 ppm PCB 29.6 57.2 276.6 70.9
Aroclor 1242) (11.26) (12.81) (122.07) (15.71)
(n=3)2

V - 5 (2 ppm PCB 31.9 52.8 313.9 65.3
Aroclor 1254) (13.32) (12.47) (114.64) (11.99)

(n=4)3

 

1Expressed as percent of brain weight 1 S.E.

2Sacrificed

3One mink died, the rest were sacrificed

50
Koller and Zinkl (1973) reported that hematological values in rats
and rabbits were not altered by exposure to PCB's, which is consistent

with the results of this experiment.

Experiment VI
This experiment was conducted to determine the rate of PCB accumu-

lation and depletion in adipose tissue of mink.

Procedure

The experiment was conducted from August 8, 1972, through December
26, 1972. Nine natural dark male mink kits were fed a basal ration
supplemented with 5 ppm Aroclor 1254 from August 8, 1972, through
October 17, 1972. From October 18, 1972, through December 26, 1972,
the mink were fed a basal (control) diet that contained no supplemental
PCB's. Three males were fed the basal (unsupplemented) diet from
October 3, 1972, through December 26, 1972, and served as controls.

At biweekly intervals the mink were anesthetized (CI-744)* and
an incision made in the inguinal area. A sample (1 g) of adipose
tissue was removed and the incision stitched closed. The biopsies
were taken alternately from each side of the inguinal area.

The fat samples were stored frozen (0°C) in glass vials prior to
analysis for PCB residues by the Michigan State University Pesticide

Research Center.

Results
The average PCB residues recovered from the adipose tissue samples

are shown in Table 20 and Figure 3. Mink fed 5 ppm Aroclor 1254 in

 

*
Experimental anesthetic, Parke, Davis & Company, Detroit, MI.

 

51

mumu UQUQU OfiOZ

 

 

 

N

«mud uoaoou¢a
no.8“ mH.Qfl HN.QH umHe
.o.z .a.z .o.z H.o N.o «.0 ~.o.z m aHouucouV Hem...MH
mmmH .ou .omo
nwsou£u uoHv
Aaouuaoov Hmmmn
sum was .msmH
I. I. I. I. I. .NH .uoo umfiv
on.m+ mo.m+ co.m+ mH.m+ mo.n+ Housmaoammsm
H.Ho m.mo 0.05 0.8m «.mHH a mom Scum wm>osmm
cmma
uoaoou< Baa m
~w.~+ mm.o+ so.mfl NN.6H Ha.nu. NN.QH euHs emuamamHa
m.mNH w.mNH H.mm «.mn w.oe s.H a Imam uoHe Hammm
on HA mm «H Hm 5H m mH m Hm w xafia uaoaummuu
.009 .002 .>oz .>oz .uoo .uoo .uoo .uaom .unmw .m=< .w=< .oz humuowa

msmmwu mmomwpm a“ A.m.m H Eamv vacancy Hmom rw><

 

AH> unmeauoaxmv enma HoHoou< Amocoaoaamsm
Ema n woafimuaoo umnu coaumu m no omen Hahuaoo m paw Mafia mo mammflu omoafifim GA mwavfimou mom ommum>< .om mHnMH

52

Figure 3. Accumulation and depletion of PCB in adipose
tissue of mink fed 5 ppm Aroclor 1254 (Experiment VI).

PCB's residue (ppm)

150

125

100

53

 

 

 

 

a."

I r \\
/ ‘Q
/ \
/ \
’ ‘ 5 Aroclor I254
. ’l \ ppm
\
\

r: \\

i .\

s ‘4,

E “-

a:

8

Control
L W
Aug. Aug. Sept. Sept. Oct. Oct. Oct. Nov. Iov. Doc. Doc.
8 21 s 19 3 0:7! 31 14 28 11 26
c

Figure 3

54
the diet rapidly accumulated PCB residues in the fat during the.first 8
weeks. At about the eighty week a plateau of approximately 125 ppm in
PCB residue was reached.
Upon withdrawal of the PCB's from the diet (Oct. 18, 1972) the resi—
dues in the adipose tissue decreased. A 50% reduction in PCB tissue

residues was reached about the eighth week after withdrawal.

Discussion

Curley et al. (1971) suspected that the PCB levels in adipose tissue
of rats fed Aroclor 1254 for 8 weeks were not representative of steady
state values because after 240 days on the diet the animals stored sub-
stantially more PCB in their adipose tissue. Although the mink in this
experiment accumulated approximately 125 ppm of PCB in the adipose tissue
when the level plateaued at 8 weeks, the mink in EXperiment IV that
received 15 ppm Aroclor 1254 stored about 250 ppm of PCB in their adipose
tissue.

The PCB tissue residue depletion pattern was similar to that

reported for the chicken by Scott at al. (1971).

Experiment VII
This experiment was conducted to substantiate the results of pre-
vious experiments in which PCB's were implicated as the primary cause
of reproductive problems associated with the feeding of Great Lakes fish
to mink, and evaluate acetone-hexane extraction of coho salmon as a
practical method of removing PCB's from these fish to render them safe

for mink feed.

Procedure
The experiment was initiated January 25, 1973. Thirty female
mink (proven breeders) were randomly divided into three groups consist-

ing of 10 mink per group. They were fed a basal mink ration (Table 1)

55
supplemented or modified as follows:

Group VII - l Basal diet consisting of 30% ocean fish extracted
with acetone andhexane and the supernatant and
residue then added back to the diet (control)

Group VII - 2 Substitution of the supernatant from the equiva-
lent of 30%.acetone—hexane extracted Lake Michigan
coho salmon for the supernatant from 30% acetone-
hexane extracted ocean fish in the basal diet

Group VII - 3 Substitution of the residue from the equivalent
of 30% acetone—hexane extracted Lake Michigan
coho salmon for the residue from 30% acetone-
hexane extracted ocean fish in the basal diet

The fish used in these diets were extracted in 15 kg quantities by

mixing in a 1:1 ratio (by volume) with acetone for 24 hrs. The acetone
fish mixture was then put through a press to remove the supernatant from
the residue. The supernatant (extract) was mixed with 5 kg of commercial
mink cereal and the.acetone evaporated off under a hood and the cereal-
extract mixture was saved. The residue from the acetone extraction was
then mixed for 24 hours with an equal volume of hexane and the mixture
was pressed to separate the supernatant from the residue. The residue
was air-dried and the supernatant was combined with 5 kg of cereal and
the hexane evaporated from the mixture under a hood. The acetone and
hexane extracted portions of the fish.(combined with the cereal) and

the residue from the extractions were then incorporated with the other

dietary ingredients (Table 1) to provide the diets outlined above.

 

56

Result

The reproductive performance of the female mink is shown in
Table 21. The extraction process with acetone and hexane did not
adversely affect reproduction as evidenced by an average of 4.1 kits
whelped per female mated when ocean fish was extracted and used in the
diet. The mink fed the diet that contained the coho salmon acetone
and hexane extract (Group VII - 2) showed impaired reproduction, as
only five kits were whelped. These kits were dead at birth or died
within 24 hours postpartum. The diet that contained the residue from
the acetone-hexane of the coho salmon (Group VII - 3) supported satis-

factory reproduction.

Discussion

 

The results of this experiment indicate that the toxic factor(s)
present in coho salmon is stored primarily in.the fat.and can be
removed from the fish by acetone-hexane extraction.

As previously discussed (Experiment II) PCB's are fat soluble
compounds which are stored predominantly in adipose tissue. In addi-
tion to removing PCB's from the fish, the acetone-hexane extraction
procedure should have also removed other potentially harmful chlorinated
hydrocarbon contaminants, such as DDT and dieldrin, since the acetone-
hexane extraction employed in this experiment is similar to the procedure
used by the M.S.U. Pesticide Research Center to remove PCB from tissues

for residue analysis (Zabik, 1972).

Experiment VIII
This experiment was conducted to ascertain the mode of action of
PCB's and the factor(s) present in Lake Michigan coho salmon responsible

for mortality and reproductive complications in mink.

57

Table 21. Reproductive performance and mortality of female mink fed a
control ration or experimental diets (Experiment VII)

 

 

 

 

Adult females Kits
Dietary % No. No. No. born Whelpedl¥ No. alive
treatment mortality mated whelped Alive Dead mated at 4 wks.
VII - 1
Ocean fish
ext. + ocean O 10 8 37 4 4 1 37
fish res.
(control)
VII - 2
Coho salmon 20 9 2 O 5 0.6 0
ext. + ocean
fish res.
VII - 3
Ocean fish 0 10 9 33 14 4.7 33

ext. + coho
salmon res.

 

58

Procedure

The experiment was conducted from January 19, 1973, to May 31,
1973. Thirty-six female.mink were allocated into two groups, and were
fed a basal diet (Table 1) supplemented or modified as follows:

Group VIII — 1 Substitution of 30% Lake Michigan coho salmon

for 30% ocean fish in the basal diet

Group VIII - 2 Basal diet supplemented with 5 ppm Aroclor 1254

Three of the 18 females in each group were placed on each of the
dietary treatments at the beginning of the‘experiment. At 10 day inter—
vals, three additional females from each group received the experimental
diets. The mink were.weighed and blood and urine samples were collected
from the animals at 10 day intervals- Beginning March 1 the females
were mated with males.fed the basal (control) diet. The urine samples

*
were analyzed for pH, protein, glucose and ketones using LabstixR

**
reagent strips. Specific gravity was determined with a refractometer.
The blood samples were used to monitor liver and kidney function and

***
were analyzed for serum alkaline phosphatase (SAP), serum glutamic—

pyruvic transaminase (SGPT)+ and blood urea nitrogen (BUN).++

Results
The urine analyses (Table 22) showed no significant changes in the

parameters measured in the mink on either dietary treatment during the

 

*
Ames Company, Division Miles Lab., Inc., Elkhart, IN.
**
Sargent-Welch Scientific Co., Detroit, MI.
*** R
Sigma 104 , Sigma Chemical Co., St. Louis, MO.
+Sigma 505R, Sigma Chemical Co., St. Louis, MO.

H.Urograth, Warner Chilcott Lab., Morris Plains, NJ.

59

Table 22. Urine analysis values at lO-day intervals for mink fed a
diet that contained 30% coho salmon or 5 ppm Aroclor 1254
(Experiment VIII)

 

 

 

Dietary . .Days on dietary treatment
treatment Parameters Initial 10 days 20 days 30 days 40 days
VIII - 1 (30% le 5.0 5.0 5.0 6.0 6.0
Lake Mich. coho
salmon) Specific -- 1.109 1.063 1.083 1.088

gravity

Protein1 30.0 30.0 38.3 39.2 58.0

(mg/100 ml)

Glucose1 trace amounts

Ketonesl trace amounts

Blood1 values varied from trace to moderate amts.
VIII — 2 (5 ppm le 5.0 6.0 5.7 6.0 6.0
Aroclor 1254)

Specific 1.037 1.066 1.059 -- --

gravity2

Proteinl 50.0 30.0 22.5 31.0 --

(mg/100 ml)

Glucose1 trace amounts

Ketonesl trace amounts

Bloodl values varied from trace to moderate amts.

 

1Determined by LabstixR reagent strips, Ames Co., Elkhart, IN.

2Determined by refractometer - TS meter, Sargent-Welch Scientific
Co., Detroit, MI.

60

course of the experimentt The results of the blood analyses are shown
in Table 23. No changes were noted in the BUN or SAP values for the
animals fed either diet.during the trial. The BUN values noted for the
mink were within.the.lO to 30 mg/100 ml concentrations usually considered
as normal in the dog (Bernstein, 1965). The SGPT values, however,
increased in both the;PCB and coho salmon fed mink in direct proportion
to the length of time the animals were fed the experimental diets.

Although the last three females in each group were placed on the
dietary treatments during.the mating period (March 1-24), only one
female from Group VIII -.l.whelped, producing one kit. This female had
received the experimental.ration containing 30 percent coho salmon for
67 days prior to whelping. Two females from Group VIII - 2 (5 ppm PCB)
whelped seven kits. Five of the kits, however, died prior to weaning.
The females that whelped in Group VIII - 2 had received the PCB supple-

mented ration for an average of 58 days prior to whelping.

Discussion

 

\

As noted in the results, all tests conducted to.monitor kidney
function showed no adverse effects on the kidneys of the mink fed either
coho salmon or PCB. These results support the findings of Koller and
Zinkl (1973) who reported no changes in BUN levels of rabbits treated
orally with 300 mg of Aroclors 1221, 1242 or 1254 weekly for 14 weeks.

The use of SAP values as a diagnostic test of liver function has
been described by Bodansky et al. (1933), Bessey at al. (1946), Bloom
(1957) and Kaneko and Corneluis (1970). Hoe (1961b) reported that
increased SAP levels were associated with hepatic neoplasms, hepatitis
and fatty degeneration of the liver. Although no changes were observed

in the SAP levels in the mink during this experiment, the values were

61

Table 23. Average serum alkaline phosphatase (SAP), serum glutamic-
pyruvic transaminase (SGPT) and blood urea nitrogen (BUN)
levels at lO-day intervals for mink fed a diet containing
30% coho salmon or 5 ppm Aroclor 1254 (Experiment VIII)

 

 

 

Dietary Days.on dietary treatment
treatment Analysis Initial 10 days 20 days 30 days 40 days
VIII - 1 SAP1 (sigma 1.87 1.56 1.89 2.05 --
30% Lake units/ml)
Mich. coho 2 -
salmon SGPT 46.5 57.0 79.0 104.7 114.0
(units/ml)
BUN3 26.7 21.7 15.0 25.0 22.5
(mg/100 ml)
VIII - 2 SAP1 (Sigma 1.86 1.87 1.83 1.49 1.72
5 ppm Aroclor units/ml)
1254 2
SGPT 48.0 49.9 79.4 125.8 33.3
(units/ml)
BUN3 17.5 15.8 22.0 17.0 20.0

(mg/100 ml)

 

1

Sigma 104 procedure, Sigma Chemical Co., St. Louis, MO.

2Sigma-Frankel procedure, Sigma Chemical Co., St. Louis, MO.

3Urograph procedure, Warner—Chilcott Labs., Morris Plains, NJ.

 

62
considerably greater than the.normal levels reported for the dog.(0.05-
0.55 Sigma units/m1) by Van Vleet and Albert (1968), but within the
normal range (0.8-2.3 Sigma units/m1) for man (Anon., 1963).

The use of serum transaminase activity was first employed for the
diagnosis of hepatic necrosis by Wroblewski and La Due (1955). Accord-
ing to Hoe (1961a), transamination activity, especially SGPT, can play
an important part in the diagnosis of active liver cell damage and
necrosis before any abnormalities are shown by other liver function
tests. In this experiment the'SGPT values of the mink in both Groups
VIII - l and VIII — 2 doubled within 30 days after the animals were
placed on the dietary treatments. These elevated SGPT levels (see
Table 23) were in the range (SO-400 units/ml) of values indicative of
moderate liver necrosis in dongs (Kaneko and Cornelius, 1970). Vos
and Notenboom—Ram.(1972) observed microscopic liver lesions and elevated
serum transaminase levels in rabbits treated with Aroclor 1260. Koller
and Zinkl (1973) reported elevated SGPT and serum glutamic-oxalacetic
transaminase (SGOT) in rabbits treated with Aroclors 1242 and 1254 but
not with Aroclor 1221.

The similarity in results obtained with these liver and kidney
function tests in rabbits and mink suggest that the mode of action of
PCB's in these species are similar. The results of this trial also
support the theory that the toxic factor contained in Great Lakes fish
and responsible for the reproductive failure and mortality in mink is

PCB's.

Experiment IX
This experiment was conducted to ascertain whether or not the repro-
ductive failure attributed to the feeding of coho salmon and PCB's to

mink was of a permanent nature.

63

Procedure

Eleven adult females that received a ration that contained 30 per—
cent whole, raw, Lake Michigan coho salmon (Group IV - 2) and three
adult female that.were.fed a diethupplemented with 5 ppm PCB 1254
(Group IV - 5) during 1972 and failed to whelp were placed on a basal
(control) diet (Table l) on June 2, 1972, and retained on this ration
through the gestation period in 1973. These females were mated with

males (fed the basal diet) during March 1973.

Results

The reproductive performance of the females during 1973 is shown
in Table 24. All three of the females previously fed 5 ppm Aroclor
1254 whelped and the average litter size (433) was comparable to that
obtained from control animals in previous experiments. Seven out of
the 11 females previously fed the coho salmon diet whelped. The average
litter size per female mated was 3.5. Excessive early kit mortality was

not noted in either group.

Discussion

Based on the results obtained in Experiment VI which showed a 50
percent reduction of PCB residues from adipose tissue eight weeks after
PCB withdrawal from.the diet, one would expect a one year withdrawal
period, as employed in this experiment, would eliminate almost all PCB
residues from the mink's bodies. The lesions and cellular changes
ascribed to PCB exposure in mink, as discussed in the previous experi-
ments, were also apparently not.irreparable.

These results of this experiment further suggest that PCB contamina-
tion of Great Lakes fish is probably the primary cause of the reproductive

and mortality problems associated with the feeding of these fish to mink

64
and indicated that PCB (Aroclor 1254) exposure does not permanently

impair reproduction in mink.

Table 24. Reproductive performance of female mink fed a control diet
following failure to whelp the previous*year while receiving
a ration that contained either coho salmon or supplemental
PCB (Experiment IX)

 

Dietary Dietary

 

treatment treatment No. 3 No. 2 Avg. li ter
Jan. 1, '72- Jun. 2, '72- . mated whelped size/
Jun. 1, '72 Jun. 1, '73 (March 1973) (May 1973) mated
IX - l Basal ll 7 3.5
30% Lake (control)

Mich. coho diet

salmon

IX - 2 Basal 3 3 4.3

5 ppm (control)

supplemental diet

Aroclor

1254

 

CONCLUSIONS

This study consisted.of a series of experiments conducted to investi-
gate reproductive complications and mortality in mdnk associated with the
use of Great Lakes fish in mink diets and to isolate the factor(s)
present in the fish that.are responsible for this problem. The follow-
ing conclusions are based on the results of these experiments.

1. Feeding Lake Michigan coho salmon at 30% of the diet to mink
resulted in reproductive failure and/or mortality.

2. PCB's are extremely toxic to mink.

3. The clinical signs and lesions (anorexia, bloody stools, fatty
degeneration of the liver and kidneys and hemorrhagic gastric ulcers)
observed in mink fed PCB's and Lake Michigan coho salmon were strikingly
similar.

4. Lethal dose studies (based on IP injections of PCB's) indicated
that the toxicity of PCB's to mink varied inversely with the chlorine
content of the compounds. PCB feeding experiments, however, showed
that Aroclor 1254 was much more detrimental to mink reproduction than
Aroclors 1016, 1221, or.1242.

5. Mink growth data suggested that interactions exist between
PCB's and chlorinated hydrocarbon pesticides in which the action of
PCB's or certain chlorinated hydrocarbon pesticides may be potentiated
in the presence of the other.

6. Long term consumption.of.PCB's by mink resulted in decreased
body weight gains and increased organ (heart, liver and kidney) weights.

65

66

7. Mink readily.accumulated Aroclor 1254 in adipose tissue until
a plateau level was reached. The depletion rate from the adipose tissue
was 50% in 8 weeks.

8. Impaired liver function was an early manifestation of PCB and
coho salmon toxicity in.mink.

9. The toxic factor(s) (PCB's?) present in Lake Michigan coho
salmon are quite heat.stable.

10. Acetone-hexane extraction of Lake Michigan coho salmon removed
the toxic factor(s) (PCB's?) and rendered the fish safe for use as mink
feed.

11. The reproductive failure in female mink produced by the feed-
ing of PCB's or Lake Michigan coho salmon is not'of a permanent nature.

12. The results of the experiments conducted in this study indi-
cated that the toxicity and.impaired.reproduction encountered in feeding
coho salmon and other Great Lakes fish to mink is probably due to PCB

residues in the fish.

LITERATURE CITED

LITERATURE CITED

Aitken, F. C. 1963. Feeding of Fur—Bearing Animals. Tech. Comm. No.
23. Commonwealth Agricultural Bureau. Farnham Royal, Bucks,
England.

Albert. T. F. 1962. The effect of DDT on the sperm production of the
domestic fowl. Auk 79:104-107.

Anon. 1963. The colorimetric determination of phosphatase acid,
alkaline prostatic in serum or other fluids at 400-420 mu.
.Tech. Bul. No. 104. Sigma Chemical Co., St. Louis, MO.

Anon. 1972a. World production of mink pelts in 1971. Vara Paladjur.
43:104.

Anon. 1972b. Agriculture responsibility concerning polychlorinated
biphenyls (PCBs). U.S.D.A. Ad Hoc.Group on PCBs, Office of
Science and Education, U.S.D.A., Washington, D.C.

Anon. 1972c. Polychlorinated biphenyls and the.environment. Inter-
national Task Force on PCBs, Washington, D.C.

Ambrose, A. M., H. E. Christensen, D. J. Robbins, and L. J. Rather.
1953. Toxicological and pharmacological studies on chlordane.
Arch. Ind. Hyg. Occup. Med. 7:197-210.

Atkins, T. D., and R. L. Linder. 1967. Effects of dieldrin a repro-
duction of penned hen pheasants. J. Wildl. Manage. 31(4):746-
753.

Aulerich, R. J. 1970. (personal communication)

Aulerich, R. J., L. C. Holcomb, R. K. Ringer and P. J. Schaible. 1963.
Influence of photoperiod on reproduction in mink. Mich. Agr.
Expt. Sta. Quart. Bul. 46(1):132-138.

Aulerich, R. J., R. K. Ringer, P. J. Schaible and H. Seagran. 1970a.
An evaluation of processed Great Lakes fishery products for
feeding mink. Feedstuffs 42(42):48—49.

Aulerich, R. J., and R. K. Ringer. 1970b. The effects of some chlorin-

ated hydrocarbon pesticides on mink. Progress Report to Mink
Farmers. Research Foundation, Milwaukee, Wisc.

67

68

Aulerich, R. J., R. K. Ringer, H. L. Seagran, and W. G. Youatt. 1971.
Effects of feeding coho salmon and other Great Lakes fish on
mink reproduction. Can. J. Zool. 49:611-616.

Aulerich, R. J., R. K. Ringer and S. Iwamoto. 1972. Reproductive
failure and mortality in mink.fed coho salmon and other Great
Lakes fish. Proc. Int. Symp. Environment and Reproduction in
Mammals and Birds, Edinburgh, Scotland, March 27-30, 1972 (in
press).

Aulerich, R. J., R. K. Ringer, and S. Iwamoto. 1973. Why Great Lakes
fish are unsafe to use as a feed for mink. U.S. Fur Rancher
52(3):12.

Azevedo, J. A., Jr., E. G. Hunt, and L. A. Woods, Jr. 1965. Physio-
logical effects of DDT on pheasants. Calif. Fish, Game 51(4):
276-2930

Bache, C. A., J. W. Serum, W. D. Youngs and D4 J. Lisk. 1972. Poly-
chlorinated biphenyl residues. Accumulation in Cayuga Lake
trout with age. Sci. 177:1191-1192.

Ball, W. L., K. Kay, and J. W. Sinclair. 1953. Observations on toxicity
of aldrin. 1. Growth and estrus in rats. Arch. Ind. Hyg. Occup.
Med. 7:292-300.

Bennett, G. A., C. K. Drinker and M. F. Warren. 1938. Morphological
changes in the livers of rats.resulting from exposure to certain
chlorinated hydrocarbons. J. Ind. Hyg. Toxicol. 20:97-123.

Bernard, R. F., and R. A. Gaertner. 1964. Some effects of DDT on
reproduction in mice. J. Mammal. 45(2):272-276.

Bernstein, L. M. 1965. Renal Function and Renal Failure. The Williams
and Wilkins Co., Baltimore, Md.

Bessey, O. A., O. H. Lowry and M. J. Brock. 1946. A method for the
rapid determination of alkaline phosphatase with five cubic
millimeters of serum. J. Biol. Chem. 164:321-329.

Bitman, J., H. C. Cecil, S. J. Harris and G. F. Fries. 1968. Estro-
genic activity of 0, P'—DDT in the mammalian uterus and rat
oviduct. Sci. 162:371-372.

Bloom, F. 1957. The diagnosis and treatment of liver diseases of the
dog. N. Am. Vet. 38(1):l7-27.

Bodansky, A., L. F. Hallman and Rt Bonoff. 1933. Phosphatase studies.
II. Determination of serum phosphatase. Factors influencing
the accuracy of the determination. J. Biol. Chem. 101:93-104.

Bostom, R. E., R. J. Aulerich, R. K. Ringer, and P. J. Schaible. 1968.
Seasonal changes in the testes and epididymides of the ranch
mink. Mich. Agr. Expt. Sta. Quart. Bul. 50(4):538—558.

69

Bowness, R. E. 1957.. Influence of light on mink production. Nat. Fur
News 28(11):18.

Cecil, H. C., S. J. Harris, J. Bitman, and G. F. Fries. 1973. Poly-
chlorinated biphenylrinduced decrease in liver vitamin A in
Japanese quail and rats. Bull. Environ. Contam. Toxicol. 9(3):
179-185.

Chaddock, T. T. 1948. Fatty degeneration of liver in mink. Vet. Med.
43:197-199.

Curley, A., V. W. Burse, M. E. Grim, R. W. Jennings and R. E. Linder.
1971. Polychlorinated biphenyls: .distrihntion.and storage in
body fluids and tissue of Sherman rats. Environ. Res. 4:481-495.

Dahlgren, R. B., and R. L. Linder- 1971" Effects of polychlorinated
biphenyls on pheasant reproduction, behavior, and survival. J.
Wildl. Manage. 35(2):315-319.

Dahlgren, R. B., R. L. Linder, and C. W. Carlson. 19.72.. Polychlorinated
biphenyls: their effects on penned pheasants. Environ. Health
Persp. 1:89-101.

Deichmann, W. B., and W. E. MacDonald. 1971- Organochlorine pesticides
and human health. Food Cosmet..Toxicol. 9:91-103.

Denington, E. M., and.AH.M. Lucas. 1955. Blood technics for chickens.
Poultry Sci. 34:360-368.

DeWitt, J. B. 1955. Effects of chlorinated hydrocarbon insecticides
upon quail and pheasants. J. Agric. Food Chem. 3(3):672-676.

Duby, R. T. 1970. Pesticides vs. reproduction still a puzzle. Amer.
Fur Breeder 43(4):15.

Duncan, D. B. 1955. Multiple range and multiple F tests. Biometrics
11:1_420

Genelly, R. E., and-R. L. Rudd. 1956. Effects of DDT, toxaphene, and
dieldrin on pheasant reproduction. Auk 73:529-539.

Gilbert, F. F. 1969. Physiological effects of natural DDT residues and
metabolites on ranch mink. J. Wildl. Manage. 33(4):933—943.

Grant, D. L., W. E. J. Phillips and D. C..Villeneuve. 1971a. Metabolism
of a polychlorinated biphenyl (Aroclor 1254) mixture in the rat.
Bull. Environ. Contam. Toxicol. 6(2):102-112.

Grant, D. L., D. C. Villeneuve, K. A. McCully and W. E. J. Phillips.
1971b. Placental transfer of polychlorinated biphenyls in the
rabbit. Environ. Physiol. 1:61-66.

Hansson, A. 1947. The physiology of reproduction in mink with special
reference to delayed implantation. Acta 2001. 28:1-136.

70

Hartsough, G. R., and J. R. Gorham. 1949. Steatitis (yellow fat) in
mink. Vet. Med. 44:345-346.

Hartsough, G. R. 1965. Great Lakes fish now suspect as mink food.
Amer. Fur Breeder 38(10):25.

Hoe, C. M. and D. G. Harvey. 1961a. An investigation into liver func-
tion tests in dogs. Part 1. Serum transaminases. J. Small
Anim. Pract. 2:22-31.

Hoe, C. M. and D. G. Harvey. ‘l96lb. An investigation into liver func-
tion tests in dogs. Part 2. Tests.other than transaminase
estimation. J. Small Anim. Pract. 2:109-127.

Holcomb, L. C., P. J. Schaible, and R. K. Ringer. 1962. The effects of
varied lighting.regimes on reproduction in mink. Mich. Agr.
Expt. Sta. Quart. Bul. 44(4):666-678.

t

Holden, A. V., and K. Marsden. 1967. Organochlorine pesticides in
seals and porpoises. Nature 216:1274.

Holmes, D. C., J. H. Simmons, and J. O'G. Tatton. 1967. Chlorinated
hydrocarbons in British wildlife. Nature 216:227.

Jensen, S. 1966. Report of a new chemical hazard. New Sci. 32:612.

Jensen, S. 1970. PCB as contaminant.of the.anvironment-history.
National Swedish Environmental.Protection Board PCB Conference,
Wenner—Cren Center, Stockholm, Sweden, Sept. 29, 1970.

Kaneko, J. J., and C. C. Cornelius. 1970. Clinical Biochemistry of
Domestic Animals, 2nd ed. Academic Press, New York.

Kellogg, C. E., C. F. Bassett, and R..K. Enders. 1948. Mink raising.
Circ. No. 801. U.S. Dept. of Agriculture, Washington, D.C.

Kitselman, C. H. 1953. Long term studies on dogs fed aldrin and
dieldrin in sublethal dosages, with reference to the histopatho-
logical finding and reproduction. J. Am. Vet. Med. Assoc. 123
(916):28-30.

Koller, L. D., and J. G. Zinkl. 1973. Pathology of polychlorinated
biphenyls in rabbits. Am. J. Pathol. 70(3):363-373.

Lalor, R. J., W. L. Leoschke and C. A. Elvehjem. 1951. Yellow fat in
the mink. J. Nutr. 45:183-188.

Lewin, V., and W. A. McBlain. 1972. Acute intraperitoneal toxicity of
DDT and PCB's in mice using two solvents. Bull. Environ. Contam.
Toxicol. 8(4):245-250.

Lichtenstein, E. P., K. R. Schulz, T. W. Fuhremann, and T. T. Liang.
1969. J. Econ. Entomol. 62(4):76l—765.

71

Lincer, J. L., and D. B. Peakall. 1970. Metabolic effects of PCB in
the American kestrel. Nature 228:783—784.

Locke, L. N., N. J. Chura, and P. A. Stewart. 1966. Spermarogenesis
in bald eagles experimentally fed a diet containing DDT. Condor
68(5):497—502.

Mason, K. E., and G. R. Hartsough. 1951. "Steatitis" or "yellow fat"
in mink, and its relation to dietary:fats and inadequacy of
vitamin E. J. Am. Vet. Med. Assoc. 119:72.

McDermid, A. M., and G- L. Ott. 1947. "Yellow fat" as observed in
mink. J. Am. Vet. Med. Assoc. 110:34.

Mestizova, M. 1966. On reproduction studies and the occurrence of
cataracts in rats after long-term.feeding of the insecticide
heptachlor. Experientia 23(1):42-43.

Miller, J. W. 1944. Pathologic changes in animals.exposed to a com-
mercial chlorinated diphenyl. Public Health Rep. 59:1085.

Morello, A. 1965. Induction of DDT-metabolizing enzymes in.microsomes
of rat liver after administration of DDT. Can. J. Biochem.
43(3):1289-1293.

Nisbet, I. C. T. and A. F. Sarofim. 1972. Rates and routes of trans-
port of PCBs in the environment. Environ. Health Persp. 1:21-38.

Nishizumi, M. 1970. Light and electron microscope study of chloro-
biphenyl poisoning.in.mouse and monkey liver. Arch. Environ.
Health 21:620-632.

Orberg, J., N. Johansson, J. E. Kihlstrom, and C. Lundberg. 1972.
Administration of DDT and PCB prolong oestrous cycle in mice.
Ambio. l(4):l48—l49.

Peakall, D. B. 1967. Pesticide-induced enzyme breakdown of steroids
in birds. Nature 216(4):505-506.

Peakall, D. B., and J. L. Lincer. 1970. Polychlorinated biphenyls
another long-life widespread chemical in the environment.
Bioscience 20:958-964.

Platonow, N. S., and L. Karstad. 1972. Distribution, metabolism and
some effects of polychlorinated biphenyls (Aroclor 1254) in mink.
Can. Fed. Biol. Soc., 15th Ann. Congr., Quebec.

Platonow, N. S., R. W. Liptrap, and H. D. Geissinger. 1972. The dis-
tribution and excretion of polychlorinated biphenyls (Aroclor
1254) and their effect on urinary gonadal steroid levels in the
boar. Bul. Environ. Contam. Toxicol. 7(6):358—365.

Presst, I., D. J. Jefferies, and N- W. Moore. 1970. Polychlorinated
biphenyls in wild birds in Britain and their avian toxicity.
Environ. Pollut. 1:3-26.

72

Reinert, R. E. 1970. Pesticide concentrations in Great Lakes fish.
Pestic. Monit. J. 3(4):233-240.

Risebrough, R. W., P. Reiche, D. B..Peaka11, S. G, Herman, and'M. N.
Kirven. 196B. Polychlorinated biphenyls in the gldbal ecosystem.
Nature 220:1098.

Risebrough, R. W., and V. Brodine. 1970. More letters in the wind.
EnVironment 12(1):16.

Rubin, M., H. R. Bird, N. Green, and R. H4 Carter. 1947. Toxicity of
DDT to laying hens. Poult. Sci. 26:410-413.

Sdhaible, P. J. 1969. Nutrition and feeding, Sec. III. In the blue
book:of fur farming. Editorial Service Co., Inc., Milwaukee,
Wisc.

Scott, M. L., D. V. Vadera, P. A. Mullenhoff, G. L. Ramsey, and R. W.
Rice. 1971. Results of experiments on the effects of PCB's
on laying hen performance. Proc. 1971 Cornell Nutr. Conf.

Stout, F. M., J. Adair and J. E..01dfie1d. 1968. Hepatotoxicosis in
mink associated with feeding toxic herring meal. Amer. Fur
Breeder 41(6):12-l4.

Travis, H. F., C. F. Bassett, Ri G. Whrner and Ja.Ka Loosli. .1956.
Influence of diethylstilbestrol on growth.and reproduction of
mink (abstr.). Fed. Proc. 15:175.

Travis, H. F., and P. .J. Schaible. 1960..- thdamentals of mink ranch-
ing. Cir. Bul. 229, Michigan State University, East Lansing, Mich.

Travis, H. F., and P. H. Harman. 1968. Sonic booms and farm raised
mink. Nat. Fur News 41(6):18—20.

Tucker, R. K., and D. G. Crabtree. 1970. Handbook of toxicity of
pesticides to wildlife. U.Sq Dept. of the Interior, Bur. of
Sport Fisheries and Wildlife. Resources Publ. No. 84.

VanVleet, J. F., and J. O. Alberts. 1968. Evaluation of liver function
tests and liver biopsy in experimental carbon tetrachloride
intoxication and extrahepatic bile duct obstruction in the dog.
Am. J. Vet. Res. 29(11):2119-2131°

Villeneuve, D. C., D. L. Grant, W. E. J. Phillips, M. L. Clark, and D.
J. Clegg. 1971a4 Effects of PCB administration on microsomal
enzyme activity in pregnant rabbits. Bull. Environ. Contam.
Toxicol. 6:120-128.

Villeneuve, D. C., D. L. Grant, K. Khera, D. J. Clegg, H. Baer, and
W. E. J. Phillips. 1971b. The fetotoxicity of a PCB mixture
(Aroclor 1254) in the rabbit and in the rat. Environ. Physiol.
1:67-71.

73

Vos, J. G., and E..Notenboom—Ram, 1972. Comparative.toxicity study of
2, 4, 5, 2', 4', S-hexachlorobiphynyl and a.polychlorinated
biphenyl mixture in rabbits. Toxicol. Appl. Pharmacol. 23:
563-578.

Ware, G. W., and E. E. Good. 1967. Effects.of insecticides on repro-
duction in the laboratory mouse. Toxicol. Appl. Pharmacol.
lO(1):54-6l.

White, A. G. 1961. Clinical Disturbances of'RenaZ Function. W. B.
Saunders Co., Philadelphia.

Wroblewski, F., and J. S. LaDue. 1955. Serum glutamic oxalacetic
transaminase activity as an index of liver cell injury a pre-

liminary report. Ann. Internal Med. 43:345-360.

Zabik, M. J. 1972. (personal communication)

"Ilillijillllifl'iiills