PHARMACODYNAMES 0F DEELDRM 1N
DAIRY CATTLE AS AFFECTED BY
PHENOBARBETAL 'FEEDSNG

Thesis for the Degree of M. S.
MECHEGAN STATE UNIVERSITY
KIM ALYN WlLSON ,
1970

IHF 51F

 
 
   

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Michigan State
University

{J LIBRARY

u ‘7'”! ’77."

ABSTRACT

PHARMACODYNAMICS OF DIELDRIN IN DAIRY CATTLE
AS AFFECTED BY PHENOBARBITAL FEEDING

By

Kim Alyn Wilson

This study was undertaken to investigate the meta—
bolic rate of HEOD, the major compound in the pesticide
dieldrin, when administered orally alone and in combina—
tion with phenobarbital to lactating dairy cows.

The experiment included analyzing milk, feces,
blood, urine and body fat for HEOD content. The analyses
were made in order to determine the sites of normal stor-
age and excretion of HEOD. In addition, the investiga-
tion was designed to ascertain the effect of phenobarbital
on the metabolism of HEOD. I

HEUD residues in body fat were lower in animals
receiving phenobarbital during contamination. In addi—
tion, the concentration of HEOD in the body fat of those
animals receiving phenobarbital was lower during decon-
tamination, and declined at a faster rate.

The treatment of dairy cows with HEOD and phenobar-
bital at the levels used did not affect feed intake or

milk production.

Kim Alyn Wilson

Upon cessation of HEOD administration, the level of
the pesticide in the milk fat declined rapidly. During
decontamination, cows receiving the drug produced milk
containing lower concentrations of dieldrin in the milk
fat than animals not treated with phenobarbital. There
was no effect of phenobarbital treatment on the concen-
tration of HEOD in the milk fat during contamination.

The major portion of HEOD was excreted via the
feces. The maximum excretion of the pesticide occurred
during the first week in all groups, which ranged from

2-51 mg HEOD/day. The pattern of residue decline was
similar for all groups. Five days after cessation of the
HHOD treatment the concentration of pesticide in the
feces became undetectable.

The concentration of HEOD in the blood was low and
no effects of treatment were noted. There was no HEOD
detected in the urine.

From 18.6 to 53.1% of the total HEOD given was ac-
counted for in feces. Body fat contained from 7.3 to
11.9% of the HEOD dose, whereas 3.1 to 3.8% of the total
pesticide administered was measured in the milk. Less
than 0.1% of the dose was in the blood and body fluids.
in all tissues analyzed, a range of 29 to 66% of the dose

could be accounted for as HEOD.

PHARMACODYNAMICS OF DIELDRIN IN DAIRY CATTLE

AS AFFECTED BY PHENOBARBITAL FEEDING

By

Kim Alyn Wilson

A THESIS

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

MASTER OF SCIENCE

Department of Dairy

1970

Copyright by
KIM ALYN WILSON

1970

DEDICATED TO

My wife, Dale, and my parents, whose

encouragement has been so meaningful.

ii

ACKNOWLEDGMENTS

The author expresses his gratitude to his major
professor, Dr. Robert M. Cook, for counsel and encourage-
ment during this phase of graduate study. Sincere appre-
ciation is extended to Dr. J. T. Huber and Dr. N. C.
Leeling for their guidance in the preparation of this
thesis.

The contribution of Dr. J. Dalley, College of
Veterinary Surgery and Medicine, for performing surgery
on the experimental animals is appreciated. The author
is grateful for the technical assistance of Mr. Lee Carr,
Miss Diane Robbins and Mr. John Chenault. The milking
and management of the Dairy Barn staff is acknowledged.

Appreciation is extended to Dr. C. A. Lassiter,
Chairman of the Department of Dairy, for financial sup-
port through a Graduate Research Assistantship.

Thanks are extended to Shell Chemical Company for
a grant-in-aid to support this research.

Lastly, but certainly not least, deepest apprecia-
tion goes to my wife Dale for her moral support and sacri-

fice during this study.

iii

TABLE OF CONTENTS

Page

DEDICATION . . . . . . . . . . . . . ii

ACKNOWLEDGMENTS. . . . . . . . . . . . iii

LIST OF TABLES . . . . . . . . . . . . vi

LIST OF FIGURES. . . . . . . . . . . . viii

Chapter

I. INTRODUCTION . . . . . . . . . . I

[1. LITERATURE REVIEW. . . . . . . . . U
A. Pharmacodynamics of Chlorinated

Hydrocarbon Insecticides . . . A

1. Chemical properties of HEOD. A

2. Sources of contamination. 5

3. Absorption . . . . . . 6

A. Transport. . . . . . . 8

5. Storage . . . . . . . 9

6. Biotransformation . . . . l2

7. Excretion. . . . . . . l7

8. Toxicity . . . . . . . 25

9. Decontamination. . . . . 27

III. EXPERIMENTAL PROCEDURES. . . . . . . 30

A. Experimental Design. . . . . . 30

B. Dietary Treatments . . . . . . 31

C. Sample Collection . . . . . . 3A

1. Feed . . _. . . . . . 3A

2. Milk . . . . . . . . 3H

3. Blood . . . . . . . . 35

A. Body fat . . . . . . . 35

5. Feces . . . . . . . . 36

6. Urine . . . . . . . . 36

7. Liver . . . .. . . . . 37

iv

(”under Page

D. Sample Extraction . . . . . . 37
1. Feed . . . . . . . . 3
2. Milk . . . . . . . . 38
3. Blood . . . . . . . . 39
A. Body fat . . . . . . . A0
5. Feces . . . . . . . . A0
6. Urine . . . . . . . . A3
7. Liver . . . . . . . . A3
R. Electron—capture Gas-liquid
' Chromatography . . . . . . AA
1V . RESUL'I‘S . . . . . . . . . . . . A6
.3 Feed intake . . . . 3. . . . A6
R. Body Weight Changes. . . . . . 50
C. Dosage a 51
' Milk Production, and Concentration
and Excretion of HEOD in Milk . 53
E. Conce_ntration of HEOD in Milk Fat
and Milk Fat Production . . 59
F. Concentration of HEOD in the Blood. 61
0.. Concentration of HEOD in Body Fat . 63
H. Concentration of HEOD in Urine . . 6A
1. Feces . . . . . . . . . . 65
”1. Dry matter excretion . L . 65
2.1'Percent dry matter. . . . 66
J. Fecal Concentration and Excretion
of HEOD. . . . , 67
_K. Body Burden and Distribution of
’ HEOD. . . 73

L. Induction of Hepatic Microsomal
Enzyme by Phenobarbital and

Metabolism of HEOD . . . . . 8A
V. DISCUSSION . . . . . . . . . . . 87
VI. SUMMARY AND CONCLUSIONS. .3 . . . . . 101
LITERATURE CITED. . . . . . . . . . . 105
APPENDIX . . . . . . . . . . . . . 121

IO.
11.

12.

13.
1A.
15.
l6.
I7.
18.

19.

LIST OF TABLES

Average age and day of lactation on first
day of experiment . . . . . . . .

Dry matter consumption . . . . . . .

Feed DM intake as a percentage of total
DM intake. . . . . . . .

Dry matter intake as a per cent of body
weight. . . . . . . . .

Per cent dry matter of feedstuffs consumed.
Body weights of experimental animals. . .
HEOD dosage . . . . . . . . . . .
HEOD intake . . . . . . . . . .

Cumulative HEOD dose administered.- . .

Average daily milk production . . . . .
Percentage decline in milk production . .

Ratio-milk produced per day vs dry matter
consumed . . . . . . . . .

Concentration of HEOD in whole milk . . .
Daily excretion of HEOD via milk . .

HEOD excreted in milk. . . . .

HEOD excreted in milk.

Average percentage milk fat. . .

Milk fat production . . . . . .

HEOD concentration in milk fat. . . . .

vi

Page

A7
A7

A8

149
50.
51‘
52
52
53
514
55

56
57
58
58
59
60
61
62

l'l

able

29.
5”-
31.

32.

A.5,

Concentration of HEOD in blood. . . . .
Mean HBOD concentration in shoulder fat.
Daily urine production . .

Excretion of dry matter in the feces.
Extraction of HEOD from wet and dried feces
Concentration of HEOD in fecal dry matter
HHOD excretion via feces. . .

Percentage of weekly HEOD dose excreted in
feces that week. . . . . . . . .

Cumulative excretion of HEOD as a percentage
of the total amount administered

Total HEOD excretion in feces . . . . .
Cumulative excretion of HEOD in feces

Dry matter digestibility.

Distribution of the total HEOD dose

Nitrogen demethylase activity of liver
microsomes . . . . . . . .

Relative differences in extraction efficiency.

Daily milk production. .

Percentage milk fat. . I. .

Concentration of HEOD in whole milk . . .
Fecal dry matter excretion

Concentration of HEOD in feces. .

vii

Page
62
6A
65
66
67
68
7O

70

71
72
72
73

'3

L-

86
92

123
12A
125

126

Figure

LIST OF FIGURES

Experimental design

Concentration of
taminated for

Concentration of
taminated for

Concentration of
taminated for
phenobarbital

Concentration of
taminated for

HEOD in feces
six weeks. .

HEOD in feces
three weeks .

HEOD in feces
six weeks and
for six weeks

HEOD in feces

of cows con-

of cows con-

of cows con-
treated with

of cows con-

three weeks and treated
with phenobarbital for six weeks. . .

viii

Page
32

7A

76

78

80

CHAPTER I

INTRODUCTION

Pesticide contamination of livestock is a serious
problem in animal agriculture. Concentrations of chlori—
nated hydrocarbon insecticides in meat and milk exceeding
established "safe" levels result in severe economic loss
to the producer, for these compounds persist not only in
the environment, but in livestock for months and even
years.

In the l9A0's, organic chemicals were synthesized
which were not degraded rapidly, enabling lower levels to
be used than was possible with their botanically-derived
predecessors. But, the residual character of the new
compounds, though beneficial against the target organism,
also became their nemesis when non-target organisms be—
came contaminated.

Xenobiotics, compounds foreign to a biological sys-
tem, have become of greater concern recently both because
of their preponderance and also the ability to detect
ever smaller amounts. Millions of tons are added to the
biosphere annually. Chlorinated hydrocarbon insecticides
are easily detected at levels of 0.01 ppb. This concen-

tration is analogous to one second in 32 centuries.

Concentrations this low have been detected in almost
all tissues of animals studied. But, although pesti-
cide residues may be present everywhere, the low levels
observed may not be metabolically significant. For
example, the acceptable level for dieldrin in milk is
0.3 ppm in fat which is 30,000 times higher than de—
tectable limits.

Demand for greater productivity in agriculture has
necessitated the increased use of pesticide and herbi-
cide chemicals. Nutrient levels and their availability
per land unit have been improved by controlling insect
and weed infestations, but continued pesticide use has
caused leaching into ground water and recycling at great
distances from the original application sites. A

It is necessary to study the metabolism of many
representative chemicals in the environment in order
that new chemical classes be understood for their effect
not only on specific targets but on the biosphere as a
whole. Pressure has been applied to reduce the use of
organochlorines and change to "bio—degradable" products.
None of these are completely degradable to carbon dioxé
ide and water-~their degradation products may be as
harmful as organochlorines. Also, metabolic products
of chlorinated hydrocarbons may be beneficial as insect—

icides, perhaps more than current compounds utilized.

The objectives of this work are to account for
the accumulation and excretion of the total dose of
HEOD and to study the effects of phenobarbital on HEOD
clearance and metabolite formation in cows. This is
the first part of a study having the overall objective
of determining the metabolic fate of dieldrin in

ruminants.

CHAPTER II

LITERATURE REVIEW

A. Pharmacodynamics of Chlorinated
Hydrocarbon Insecticides

 

Pharmacodynamics has been defined, ". . . absorp-
tion, accumulation, and elimination and changes in
physiological function consequent to those processes

." (82). In particular, the pharmacodynamics of
dieldrin have been investigated in many biological sys-
tems, as has the interaction of dieldrin with other
factors (53, 82, 85, 1A0). Recent reviews on the status
of pesticide contamination and metabolism have appeared
(20, 29, 56, 83, 8A, 110, 123, 127, 131, 132).

1. Chemicalgproperties
of HEOD

 

Dieldrin is a light brown crystalline material
containing not less than 85% of the active ingredient
(1,2,3,A,10,lO—hexachloro-6,7—epoxy-l,A,Aa,5,6,7,8,8a-
octahydro-l,A—gndg,gx9-5,8-dimethanonaphthalene), a
white crystalline compound commonly abbreviated HEOD.
For the purposes of this thesis, both because the con~
taminant employed was HEOD (99+% pure) and because the

analytical method primarily detects HEOD, the abbreviation

will be used to distinguish from the technical grade
material, dieldrin.

Dieldrin has a melting point near lA5°C. HEOD
melts at 175-17600 (7). The molecular weight of HEOD
is 380.93. The compound is readily soluble in benzene
and acetone and less soluble in hexane and methanol.
HEOD solubility in water is less than 0.185 ppm (126).
It is stable in strong base but labile in strong acid.

Photodecomposition occurs in sunlight to form
dechlorinated products with additional cyclic rearrange-
ments (77, 133, 135). Dieldrin is the epoxide form of
aldrin. This epoxidation of aldrin is catalysed

readily by sunlight, microbes or in mammals (68).

2. Sources of contamination

 

The primary route of livestock contamination by
pesticides is via oral ingestion of feeds containing
pesticide residues (78, 137, 157). Direct contamination
of animals or their environs with pesticides for in-
sect control has been of secondary importance. Aerial
movement (drift) of insecticides applied to areas ad-
jacent to pasture or croplands is a significant source
of feedstuff contamination in many areas (78, 152).

Feeds can become contaminated by the direct appli-
cation to crops of either excessive amounts of pesti-
cide or by the use of incorrect pesticides (10A, 1A8,

162). The insecticides can also be translocated from

soil to the crop which serves to concentrate these ma-
terials prior to consumption (1, 105, 137, 15A, 155).
Accidental contamination of livestock appears to
be more prevalent than previously thought (19, 28, 119,
121, 150) and represents a severe economic loss to the
producer. Often the loss is three—fold: (1) death
losses, (2) levels in meat and/or milk prevent disposi-
tion of the animals and/or products, and (3) the period
of decontamination is long during which no income from

the sale of meat and/or milk is received.

3. Absorption

 

Absorption of chlorinated hydrocarbon insecti-
cides occurs primarily in three ways: (1) dermal or
percutaneous, (2) inhalation (respiratory), and (3)
oral ingestion (alimentary). Dermal application causes
a rapid rise in blood levels (72, 73, 125, 1A0). The
rate of absorption is rapid and depends on the solvent
used. The rate is greatest in acetone, less in benzene,
slower in corn oil. The structure of the compound
also affects absorptive rate, the rate being highest
for dieldrin followed by malathion, carbaryl and DDT
(77). Inhalation of DDT by dairy cows produces lower
levels of DDT and DDE in milk than does alimentary con-
tamination (162).

Oral ingestion of chlorinated pesticides is the

most common environmentally encountered absorptive route

and results in the rapid elevation of blood levels.
Rapid absorption of a single dose of DDT from the gut
produced high blood levels initially which tended to
reach equilibrium within 1-3 days (A2, 90). In one
trial the half-life of a single dose of DDT intrave—
nously in cows initially was 60-80 minutes. The half-
1ife of DDT increased until equilibrium was reestab-
lished in 2A hours (161). Retention of dieldrin in the
gut or the regulation of absorption appears to be af—
fected by gut contents (7A).

Keane et_al. (93) demonstrated that the degree
of obesity is directly related to the time required to
produce poisoning in animals receiving chronic doses
of HEOD. They suggested that this relationship should
be valid regardless of whether absorbed orally, by inhala-
tion or through the skin. If the "adipose sink" works
in this manner, then the work by Morgan and Roan (116)
would suggest that the toxicity of DDT should be af-
fected in a similar manner, and to an even greater
degree, by obesity. Chronic doses or low level con-
tamination results in initially high blood levels which
continue to rise with contamination, dependent upon

dosage level (10A).

A. Transport

 

Pesticides are transported bound to blood lipo-
protein (69, 116), are solubilized by blood components,
e.g., fatty acids, and have been shown to be, in the
case of dieldrin, A,000 times more soluble in serum
than water (68, 118). Dieldrin in blood is located
primarily in plasma and erythrocytes, at a ratio of 2:1,
respectively. A rapid initial rate of dieldrin removal
from blood has been shown to be followed by a logarith—
mic rate of removal (118). Absorbed pesticides are
transported to the liver via the portal vein and their
toxicities are controlled by their low solubilities in
water and high solubilities in lipoidal material. Slow
infusions are necessary in order to permit dieldrin to
be absorbed by adipose tissue, because rapid infusion
overloads the central nervous system. Shortly after
absorption, considerable dieldrin appears not in the fat,
but in brain and other organs, and the faster the rate
of absorption, the greater the proportion found in the
brain (7A). In addition, dieldrin is more extensively
distributed to non-lipid cellular and blood components
than are DDT and DDE (116). This relation conflicts
with the relative LD levels of dieldrin and DDT. The

50

lower LD50 of dieldrin implies a rapid association with

the neural system, especially the lipoidal fraction.

9

A recent report on the effects of dietary methio-
nine on DDT metabolism in rats clearly demonstrated that
accumulation of DDT and its metabolites in liver tissues
was dependent upon dietary methionine level (1A9).

With the knowledge that limiting the availability of
methionine reduces the formation of chylomicra thus im—
pairing lipid transport (86), the authors (1A9) suggest
that the opposite may also be true, that stimulation of
lipid transport may in fact be responsible for increased

tissue storage of DDT.

5. Storage

 

Studies have attempted to correlate body fat levels
of pesticides with blood levels and storage as a measure
of environmental exposure (AA, 80, 87, 91, 92, 120, 130,
13A). Robinson §£_§ln have measured tissue concentra-
tion of HEOD in rats at 12 weeks and found that exact
levels depend on the type of tissue studied, with adi-
pose > liver > brain > blood (13A). The half—life of
HEOD was 10.3 days in adipose tissue of rats. Corre—
lations of 0.81-0.96 were noted between levels of HEOD
in blood and its concentration in the heart, liver,
kidney, lung and fat (130). A high correlation (r=>0.8)
was found for dieldrin concentration in different tis-
sues from the same person of workers occupationally ex—
posed to the pesticide (71). Concentrations of diel-

drin in body fat and liver are correlated with levels

IO

in the blood (50). Keane and Zavon suggested that
"under conditions of equal and constant oral intake,
the concentration of a chlorinated insecticide per gram
of fat is inversely related to the total body burden

of the insecticide and also inversely related to the
degree of obesity of the individual sample" (92).

Their data also supported the contention of Hunter (82)
that the blood concentration of a chlorinated insecti-
cide can be used as an index of the concentration of
that insecticide in the fat. Long—term surveys of human
residue levels suggest that dieldrin is more extensively
distributed to non-lipid and blood components than are
DDT and DDE (116).

When aldrin is fed to livestock it is rapidly con—
verted to dieldrin which is stored in adipose tissue up
to four times the level ingested (0.25 to 10.0 ppm in
diet) (87). In an extensive review (78), Henderson con—
cluded that the concentration of pesticides in the body
fat increases rapidly upon ingestion of a pesticide,
then levels off at a plateau characteristic for the
amount of the insecticide in the feed. The lower the
concentration in the feed, the sooner the plateau is
reached. The dietary level is concentrated 10-20 fold
in milk fat and 5-10 fold in body fat when heptachlor
epoxide is fed for 12 weeks, whereas dieldrin is only

concentrated 2-fold in each tissue at 12 weeks (27).

11

Dietary levels of Telodrin of between 0.02-0.15
ppm caused a A-fold concentration in tail head fat at
88 days in dairy cattle. After A0 days on treatment,
milk levels of Telodrin were 30% of those in the ra-
tion (8). A series of studies by Gannon gt_al. (58,
59, 60) showed that dieldrin fed from 0.1—2.25 ppm in
the diet was concentrated 2-3 fold in the various body
fats of dairy cows after 12 weeks. In the same period,
milk levels rose to l/10-1/5 that in the diet. Accu-
mulated intakes for the 0.1 and 2.25 ppm level were
0.293 and 6.556 mg/kg body weight. The same levels of
pesticide were given to steers, hogs, lambs and poultry.
Dieldrin concentration in tissues of these species was
highest in hens and steers and lower for hogs and lambs.
Although hens had the greatest level in body fat, their
eggs contained very little dieldrin. After 12 weeks
hens concentrated the dietary level lO-fold, steers
2-fold, hogs and cows < l—fold and lambs to about 1/3
the level in the ration. At A2 days of feeding 10 and
50 ppm dieldrin to dairy cows, the ratio of dietary
level to concentration in the whole milk was 6:1 and
appeared to be dose dependent. The ratio reached a
maximum of 5:1 at 16 weeks. At 50 ppm in the diet the
body fat level was 11 times higher than the milk level
at 16 weeks (58, 59, 60). Dieldrin at l and 10 ppm
concentrates in adipose tissue of rats to 15 and 67.5

ppm, respectively, in 10 weeks (1A2, 1A5).

l2

DcMott gt_al.(5l) noted a 0.97 correlation between
the amount of heptachlor and heptachlor epoxide consumed
and the concentration of heptachlor epoxide in milk fat.
The ratio between milk fat and body fat levels decreased
as time before parturition increased in dairy cattle
(15). Contamination of pregnant dairy heifers early
rather than late in gestation produced significantly

higher body fat levels (15).

6. Biotransformation

 

The behavior of the parent compound, HEOD, has
been reviewed in the preceding sections. HEOD is
rapidly absorbed, transported and assimilated by
lipoidal material. Almost immediately upon absorp-
tion of a toxin the body begins a detoxification process.
Detoxification can include any or all of these alter-
natives: direct elimination of the toxin in the urine
as a conjugate; enzymatic alteration and conjugation;
absorption into the adipose tissue either as the parent
compound or as a metabolite; or recycling the toxin to
the gut from the blood via the saliva, bile, gastric
or pancreatic juices.

The logical action by the liver is a structural
alteration of the compound in order to reduce its
toxicity to the host. Often the liver is stimulated by
the drug (or other chemical) to increase its capacity

for detoxification. However, it is obvious that if

13

liver action results in a metabolite more biologically
active than the parent compound, the net effect has

been an enhancement rather than a reduction of toxicity
(35). Examples of this toxification have been demon—
strated in mammals (21, 22, 23, 26, A7, A8, 160). The
classic toxification reactions are the conversion of
aldrin and heptachlor to their respective epoxides,
dieldrin and heptachlor epoxide. Bann gt_al. (6)

found that aldrin is converted to dieldrin in pigs,
poultry, sheep, rats, beef and dairy cattle regardless
of the route of entry. The change occurred whether ad-
ministered orally or subcutaneously. Christensen (32)
showed a 30% change of aldrin to dieldrin in rats and
dogs by 2A hours. In addition, a photoconversion prod—
uct of HEOD has been shown to exhibit increased toxi-
city to rats, mice, guinea pigs and pigeons, but is less
toxic than HEOD to domestic fowl or a species of fish, and
no discernible difference in toxicity was noted for
beagle hounds (26). Likewise, another photo-product

of HEOD is more toxic to houseflies and mice (76).

Other photo-products have been reported (A0, 133, 135).
These are readily formed by the action of sunlight on
HEOD applied to plants, soil and glass, and by artifi-
cial light (mercury lamp) on glass and in solution. The
metabolism of one photo-product has been studied (5).
Microorganisms in soil degrade dieldrin rapidly (111,

112).

1A

That dieldrin may be degraded to other forms was
first suspected when rats fed dieldrin excreted a com-
pound similar to HEOD in the urine (102). From 1962 to
1965 reports appeared on the presence of degradation
products which were apparently derived from HEOD but
these were not identified (32, A3, A7, 7A, 108, 109,
117).

British researchers found that when chlorine—
labelled HEOD was fed to rats, very little of the label
was excreted as HEOD. The amount of the dose which
appeared in the feces probably was excreted as a
glucuronide in bile and then reconstituted in the gastro—
intestinal tract to be incorporated into the feces (7A).
HEOD is considered to be stable in KOH, yet in alkaline
conditions metabolites are readily changed (A7). Pre-
liminary trials suggested that less metabolites were
excreted in urine from an intravenous dose than from an
oral dose (A7). The trans—diol was found to be unstable
at greater than 200°C (108). Almost all of a dose of
aldrin was accounted for in the tissues of rats and
dogs at 2A hours. However, only A0% of a dieldrin dose
could be accounted for primarily in the fatty tissues.
None was found in urine or feces. Since much of the
dieldrin was not recovered, it was assumed that it was
partially converted and rapidly excreted. It was also

suggested that the metabolites of dieldrin are polar in

nature and, as such, are not likely to be located in
fatty tissues (32).

The first identification of an HEOD metabolite
occurred in 1965 (101). These workers isolated a di-
hydroxylated form of HEOD (108), which differed by the
addition of one oxygen and two hydrogen atoms across
the epoxy group. Rabbits receiving dieldrin—luc per
os for 21 weeks excreted about 1/3 of the dose in the
urine. 0f the total radioactivity excreted in the
urine, 86% occurred as one of six metabolites, which
was shown to be the compound resulting from the hydro-
lytic cleavage of HEOD. The toxicity of this compound
is lower than dieldren for mice. When administered
intravenously to rats, it was excreted unchanged in the
feces. As was the case in subsequent work, the ini-
tial steps of isolation proved as difficult as those
of identification. It appeared that the metabolite
had been present in earlier experiments, but methods
for isolation of the compound had not been established.
Further studies in the pharmacodynamics of dieldrin
have centered around the development of techniques de-
signed to isolate metabolic degradation products of
organochlorines in tissues and excreta of animals.

Since the identification of the dihydroxy meta-
bolic conversion product of dieldrin, several other
metabolites have been isolated and the structures

elucidated (5, 22, 2A, 100, 111, 112, 113, 11A, 128,

16

129, 13A). Richardson and coworkers identified a rat
urinary metabolite as a ketone which retains the epoxide
ring but is monodechlorinated and the carbon skeleton
rearranged (128, 129). The fecal metabolite of HEOD

from the rat appears to be altered only by hydroxyla-
tion at the Aa or 5 carbon. Recent work has confirmed

a fecal metabolite hydroxylated at carbon Aa, and the
urinary product as a ketone at carbon 3 and an additional
carbon—carbon bond between positions 2 and 9 (100, 113).
Proposed reaction mechanisms for the formation of each
metabolite of dieldrin have been reported by Robinson
et_al. (13A). Damico et_al. (A6) confirmed the identity
of the ketone metabolite from rat urine. They have also
established the structure of a ketone metabolite of
aldrin in rat urine which differs from dieldrin only

by the endo- or exo-orientation of the epoxide ring.

The most recent metabolite of dieldrin to be
identified is 9—hydroxy dieldrin (5A). Extensive studies
on the extraction and isolation of this compound (75)
suggested that the Aa or 5-hydroxy dieldrin may have
appeared during feeding trials with sheep. But subse—
quent analysis demonstrated that the 9-carbon of the
5,8—methylene bridge is hydroxylated. This compound,
as well as trans-dihydroxy-dihydro-aldrin, was extract-
able from sheep urine with hexane. Eight metabolites

were isolated from sheep urine, four of which were

17

hexane-extractable. The remaining four were ether and
ethyl acetate-soluble, but their structures were not
established. Isolation was complicated by the narrow
pH range which permitted extraction. A change from
pH 3 to pH 5 caused a decline in yield from 100% to

15%.

7. Excretion

 

a. Normal patterns.—-The excretion of unchanged

 

pesticide occurs after residues circulating in the blood,
and those mobilized from adipose tissue, are taken up
by the liver, kidney and mammary gland to be excreted
in the bile, urine and milk. When biotransformation
occurs, the compound is usually made more polar, thus
it is more readily conjugated and excreted in the bile
and urine (7A, 75, 11A, 13A). The primary excretory
routes are in feces and urine and in milk during lacta-
tion. The extent to which each route contributes to
total excretion depends on the obesity, sex, stage of
lactation or gestation, nutritional status, species,
age, type of contaminants, and duration and intensity
of exposure to the contaminant.

Many studies have attempted to provide information
on the normal excretion pattern of herbicides and in-
secticides in livestock (9, 15, A2, 51, 52, 58, 60, 65,
66, 75, 81, 96, 120). The excretion equilibrium among

feces, urine and milk is highly variable. The chlorinated

18

hydrocarbon insecticides have been shown to be excreted
primarily in the milk and feces (as the parent compound)
and in the urine as metabolites. Any physiological change
which affects lipoidal material, such as adipose tissue,
should also affect the pesticides incorporated there.
This is not always the case. For example, a substantial
decrease in milk fat production of dairy cattle produced
a corresponding increase in concentration of dieldrin in
the milk fat, such that the total excretion of the pesti—
cide was nearly the same (15). A representative sampling
of experiments which examined excretion patterns show
that thyroprotein feeding caused no effect on the rate of
decline of DDT or dieldrin from lactating cows treated
for 30 days and 56 days, respectively (15, 115).
Heptachlor fed at 0.28 — 0.95 ppm on hay for 30 days
caused a rapid rise in milk levels of heptachlor epoxide,
reaching a plateau at 10 days, which lasted 20 days.

Upon withdrawal of the contaminated feed there was a
rapid decline in heptachlor epoxide levels. Twenty to
twenty—nine percent of the total intake was excreted in
the milk (81). The carbamate TemikCDwas concentrated

in milk to only 1/100 that in the feed, with an excre-
tion distribution of l, 92, and 3% in milk, urine and
feces respectively (52). The herbicide BromacilCDis
excreted only in the milk, with highly variable daily
concentrations, but 10 times more is excreted in the

evening milking than the morning (66). The excretion of

19

HEOD is reported to occur primarily in the milk (15).
Dairy cows fed 10 ppm HEOD for 112 days contained 1.78
ppm HEOD in the whole milk. At one day past withdrawal
the level had declined to 1.26 ppm followed by 0.A7 ppm
at 21 days (60). A single dose of DDT caused a rapid
initial rise to 3A0 ppm in milk fat, then declined to a
6.0 ppm plateau to reach 2.5 ppm in 15 weeks (A2).
Some of the "metabolically unreactive" compounds such as
DDT, DDE and dieldrin have been shown to be excreted in
milk at about 1% per day (103). These data suggest that
the excretion of foreign compounds is a complex phenome—
non. Furthermore, the body is able to adapt to an in-
finite variety of conditions such that detoxification
and eventual elimination of those compounds not essen-
tial to basic metabolic process is accomplished.
Braund (15) found that contrary to previous reports when
animals are contaminated with dieldrin prior to lacta-
tion they excrete little of the dose. At parturition
the levels in milk are extremely high, reflecting the
storage of the high levels which are mobilized upon
calving (15). Thus it was reported that milk is the pri—
mary excretory route of stored dieldrin in the lactating
dairy cow.

There are examples of other pesticide excretion
experiments which are of a more basic nature. The amount
of dieldrin appearing in milk depends upon the daily in-

take and the existing concentration in the fat of the

20

animal (58 . The ratio of HEOD concentration in body fat
to that in milk is 12 - 18:1 during feeding, but upon
withdrawal, milk depends on stored HEOD in body fat for
the HEOD and the ratio increases to 55:1. Body fat con—
centration of HEOD declined 30%, HEOD in milk by 80% in
six weeks of decontamination. Correlations have been
established, respectively, (51, 96) for body fat and milk
fat concentrations (r2 = .96) and pesticide consumed and
milk fat concentration (r2 = .97).

The above experiments did not investigate the mech—
anisms of metabolism. However, there are two good ex-
amples of a systematic approach to the mechanism of me-
tabolism of xenobiotics. First, Casida (30) presented a
comprehensive account of the use of radioisotopes to
study the metabolism, degradation and mode of action of
radiotracer chemicals. The use of labelled chemicals is
essential to the examination of the complete metabolic
fate of a xenobiotic. Second, Robinson et_al. proposed
a two-compartment pharmacokinetic model to simulate the
elimination of HEOD from the rat (13A). Partitioning of
HEOD between blood and tissues is dependent upon the
lipophilicity of HEOD. The model proposes the circu-
lating blood and liver constitutes one compartment. The
:Pate of transfer from the compartment is dependent upon
the concentration of HEOD in the compartment. The peri-
pheral compartment is conceived to be passive storage

areas, contrasted with the central compartment where

21

biotransformation occurs. The authors conclude ".

the rate-controlling factor for the elimination of HEOD
from the whole body, after the initial period of rapid
change in the central compartment, is the transfer of

HEOD from the peripheral compartment (of which the adipose
tissue is part) to the central compartment." This simpli-
fied model of a highly complex series of events may be
confounded by the inductive effect that the xenobiotic

may have on the liver, thereby altering storage and ex-
cretion rate constants.

Also, Hayes (70) and Heath and Vandekar (7A) have
suggested that most of the excretion of dieldrin, dieldrin-
derived material, and most residual chlorinated hydro-
carbon insecticides is via the feces. In addition, re-
gardless of route of administration, DDT excretion in
feces exceeds that in the urine (70). All metabolites
of HEOD in rats (113) and sheep (75) were present in
several-fold greater amounts in feces than were the same
forms in the urine. Biliary excretion of the compounds
most likely accounts for the substantial amount appearing
in feces. Thirteen percent of an intravenous HEOD dose
appeared in the small intestine in the form of hydro-
philic compounds (117). Enterohepatic circulation of
dieldrin was demonstrated by total cannulation of the
bile duct, which increased the amount of HEOD excreted
from 3 up to 10% of the total HEOD-derived material

excreted (7“)-

22

More recent work has shown that dieldrin metabolites
are conjugated in the liver and the conjugates are ex—
creted in the bile (7A, 75, 113, 11A). Trans-dihydroxy—
aldrin was conjugated by liver microsomes (AG—50% of the
total label). The metabolites formed in vitro were shown
to be identical to those produced in vivo (113). Others
have shown that over 90% of labelled dieldrin injected
i.v. appeared in the feces of rats and in the bile of
bile—fistulated animals, suggesting that most of the
intravenously administered dieldrin (0.25 mg/kg body
weight) in the feces was hepatically derived (33). In
intact animals, 50% of the dose was excreted within
three days (33). In support of the above, another re-
cent study has indicated that HEOD degradation does occur
resulting in up to 18% of the dose being excreted per day
in feces compared to 8% in the urine. In all animals
studied, fecal excretion of the dose was greater than
urinary (75).

b. Induced biotransformation and excretion.——Many

 

chemicals are known which induce hepatic microsomal drug
metabolizing enzyme systems and also cause a prolifera-
tion of smooth endoplasmic reticulum in the cell (16, 31,
3A, 35, 36, 39, 62, 63, 12A, 1A2, 1A5, 163). Of primary
concern to this author is the effect of drugs and/or
pesticides on the induction of hepatic mixed—function
oxidases that metabolize xenobiotics. An excellent review

of the mechanism and consequences of enzyme induction has

23

been presented by Conney (35). Some of the drugs which
have caused induction are the barbiturates, diphenyl-
hydantoin, tolbutamide, phenylbutazone, aminopyrine,
methylcholanthrene and benzpyrene. This group includes
hypnotics, analgesics, tranquilizers, anticonvulsants,
alkaloids and antihistiminics. In addition, some steroid
hormones induce liver enzymes.

in addition to the above compounds which affect
liver enzymes, the chlorinated hydrocarbon insecticides
have also been shown to induce microsomal mixed-function
oxidases (36, 55, 61, 62, 63, 6A, 67, 9A, 1A3, 1A5). For
example, DDT is known to induce hepatic drug metabolizing
enzyme systems which will degrade DDT, the inducer. The
similar effect by drugs is borne out by the decreased
hexobarbital sleeping times noted in animals chronically
administered a drug (16, 3A, 35, 57, 1A6). This effect
on the duration of drug action was used in early studies
as the measure of microsomal enzyme induction. The "no
effect" level of DDT induction of microsomal epoxidation
enzymes has been established at 2.0 ppm in the diet of
6—week old male rats fed 1A days (61). Street eEJiL.
established that oxidase and O—demethylase levels were
induced by between 1 and 5 ppm DDT or dieldrin in the
diet (1A6). This study was conducted to better under-
stand the potential hormonal imbalance which may be

affected by low levels of chlorinated pesticides in the

1”!

environment. Street has also established that the inter-
action of one pesticide with one or more others can
greatly alter normal metabolism (1A2, 1A5). The inter-
action of DDT with dieldrin to minimize storage and/or
accelerate metabolism of HEOD (31, 1A2, 1A5) demonstrates
a toxification effect by DDT which results in a subse—
quent detoxification of HEOD. Enhanced levels of mixed-
function oxidases appear to be responsible for the hy-
droxylation of several compounds and their subsequent
increase in polarity, which enables conjugation and ex-
cretion to occur. The effect that each pesticide has on
the stimulation of its own degradation is difficult to
measure, but Richardson e£_al. (110) suggested that
100 ppm HEOD in the diet used in their experiments may
have caused proliferation of smooth endOplasmic reticulum,
hence oxidizing activity. It can be seen from previous
work (1A6) that 1—5 ppm dieldrin induces several enzyme
systems, hence their hypothesis would appear to be correct.
Phenobarbital administered at the level of 75 mg/kg
body weight i.p. has been shown to be the most effective
stimulus for increasing bile flow. The drug caused a
50% increase in bile flow in rats which was higher than
all drugs tested (97). The acceleration of biliary flow
causes a concomitant increase in biliary excretion and
plasma disappearance of several classes of drugs (97,

98, 99). This effect occurs after one day of treatment,

25

is maintained for at least three weeks, and remains
elevated for five days after withdrawal of phenobarbital.
A dose as low as 30 mg/kg body weight daily in rats pro—
duced in two days a biliary flow which was significantly
greater than controls. In addition to the increase in
bile flow, there was an increase in the wet weight of the
liver (97). Klaassen and Plaa (99) suggest, that in the
case of sulfobromophthalein (SBP) the effect of pheno-
barbital on SHP plasma disappearance is not mediated
through its biotransformation by the hepatic microsomal
enzyme system. Instead, the effect was accomplished by
the combination of the acceleration of biliary flow and
the stimulation of glucuronide conjugation. Also, en—
hanced maximum rates of transport of iodocyanin green
and bilirubin via increased conjugation have been demon-
strated.

Thus, it is evident that drugs and pesticides play
important roles in affecting homeostasis of a variety of
OPganisms. In particular, the enhancement of normal bio-
transformation by stimulating degradative enzymes in
several tissues, especially in the liver, offers a prom-
ising means for effectively dealing with pesticide con-

tamination.

§;__Toxicity

 

Acute toxicity of various chlorinated hydrocarbon

pesticides is presented as the minimum dose required to

26

cause death in 50% of the test population, usually mice

or rats (LDr0)° Excellent summaries have been reported
J
by O'Brien (61) and Soto and Deichmann (1A1) for LDEO
J
values of cyclodiene insecticides. The LD for HEOD

50
given orally to rats ranges from 38-87 mg/kg body weight,

and 7A—l20 mg/kg for the mouse, the lower values apply—
ing to females, the higher levels to males. The same
relationship was observed by Heath and Vandekar (7A)

but with levels of 50.3 mg/kg for females and 6A.5 mg/kg
for males. A comprehensive study of the toxicity not
only of HEOD, but of a photoisomer of HEOD in several
species revealed that although the photo—isomer was more
acutely toxic than dieldrin, the extent of its presence
in the environment "does not represent an overall in-
crease in the toxicological significance of residues from
the use of dieldrin (26)."

Threshold levels of pesticides in blood for toxi-
city have been difficult to establish, primarily because
of the paucity of agreement of the measure to be used to
define toxicity (91). Levels of HEOD in blood which
produce a convulsion in man and animals were in the range
of 0.15 — 0.20 ppm in the blood (25). Blood concentra-
tion of HEOD which caused a A0% reduction in food consump-
tion by dogs ranged from 0.37 - 0.39 ppm; a 10% decrease
in body weight, 0.38 — 0.50 ppm; and, at the time of
first observed convulsion, 0.7A — 0.8A ppm (91). Dif—

ferences between the levels in these studies (25, 91) are

1’7

attributed in part to differences in time intervals be-
tween dosing, intoxication and blood sampling. It was
concluded that "a threshold level of dieldrin in the
blood which, when exceeded, results in an intoxication,
appears to be reasonable . . . regardless of the type or
duration of exposure to dieldrin" (91).

The interaction of nutritional status and suscepti-
bility to insecticide poisoning has been investigated
extensively (10, ll, 12, 13, 1A, 1A9). In essence, the
less satisfactory the nutritional status, e.g., protein-
deficiency, the more susceptible the subject is to toxic
effects of insecticide chemicals. In addition, increased
levels of pesticides mobilized from fat during reduced
food intake can cause toxicity, but rarely do permanent

lesions result (7A).

9. Decontamination

 

A review of normal rates of decontamination or
depletion of body stores in several species has been pre—
sented above. Several attempts have been made to accel-
erate excretion and clearance of stored and circulating
pesticides from mammals (9, 15, l6, l7, l8, 19, 31, A9,
57, 79, 103, 1A7, 1A9, 158, 159). The reader is referred
to three excellent reviews on the subject (106, 1AA, 151).

Two general approaches have been taken. The first
was through the action of drugs on the liver which in-

duces detoxification enzyme systems which should enhance

28

excretion. Studies have been conducted using drugs such
as heptabarbital (16, 17, 31), hexobarbital (57), pheno-
barbital (19, 159), aminopyrine (31), dephenylhydantoin
(A9), methionine (1A9), vitamin A (79), thyroprotein

(15, 18, 103, 1A7), and inositol (150). The second ap—
proach to decontamination was the use of an absorbent

such as activated carbon to trap pesticides in the gastro-
intestinal tract, prevent reabsorpticn, and thereby in~
crease fecal excretion.

The discovery in this laboratory that dieldrin,
subsequent to intravenous administration, was recycled
from the blood to the gastro—intestinal tract via saliva,
bile and pancreatic juice in several species, illustrated
that the clearance of a pesticide from the bory would not
entirely follow normal routes of excretion (37, 38).
Heabsorption of pesticides from the gut may then prolong
their half-life in the body, but there are also advan—
tages to recycling. First, recycling means that portion
is not being stored, but rather, is mobilized from tissue
storage. The compound should then be able to be adsorbed
within the gut for excretion in the feces. This would
shift the equilibrium even further away from storage and
thereby accelerate the decline in total body burden.

Activated carbon has been used successfully to ad-

sorb recycled and exogenous dieldrin in the gut of the

bovine, sheep and goat (158, 159). The combination of

29

phenobarbital and activated carbon has been shown to
accelerate the decline of dieldrin in milk from cows
accidentally contaminated (19).

The purpose of this study was to obtain information
on the effect of phenobarbital on the elimination of HEOD
from the lactating dairy cow and to account for the total
dose of the contaminant. Braund (15) could account for
50% of a chronic dose of dieldrin as HEOD, and speculated
that the majority of the remainder was converted to
metabolites. The second part of the study to be re-
ported later will examine the extent to which HEOD is

metabolized and identify the major forms produced.

CHAPTER III

EXPERIMENTAL PROCEDURES

A. Experimental Design

 

Eight lactating cows were selected from the Michigan
State University dairy research herd. Averaging 256 days
of lactation and A7 months of age, the cows were assigned to
treatment groups according to average daily milk yield.
Uniformity of physiological state was thereby achieved,
which would minimize bias of total pesticide excretion
data.

The duration of the experiment was six weeks. This
was divided into two three-week periods (see Figure 1).

Animals were assigned to the following groups:

 

 

Period I - 3 weeks Designation in text
a. Control — dieldrin only - Control

A cows
b. Treatment — dieldrin + pheno- PB

barbital — A cows
Period II — 3 weeks
a. Control - dieldrin only - Control — C

2 cows

Control — no dieldrin — 2 cows Control — D
b. Treatment — dieldrin + pheno- PB - C

barbital - 2 cows

Treatment — no dieldrin + pheno— PB — D
barbital — 2 cows

30

31

During contamination, each animal received the prin—
ciple active ingredient in dieldrin, 1,2,3,A,10,10-
hexachloro—6,7—epoxy—1,A,Aa,5,6,7,8,8a-octahydro—l,
A—gngo-ggg,5,8-dimethanonaphthalene, abbreviated HEOD.
The HEOD was greater than 99% pure and was a gift of
Shell Chemical Co.1

Each day animals were given a gelatin capsule
orally with a balling gun which contained 15.0 gm
chromic oxide. In addition, the following was admini-
stered daily by capsule to the designated cows:

HEOD: 0.10 mg/kg body weight

Phenobarbital: 10.0 mg/kg body weight

Intake of HEOD and phenobarbital was based on body
weights of cows taken for two consecutive days at weekly
intervals. The weights were averaged and then used to
determine the dosages for the following seven days. The

gelatin capsules for each animal were made up for the next

week containing the appropriate doses.

B. Dietary Treatments

 

All cows received a ration of A.5A kg alfalfa hay,
9.08 kg urea~treated corn silage and sufficient concen—
trate to meet requirements for maintenance and lactation.
Concentrate levels were adjusted weekly based on the pre-
vious week's daily milk yield. Daily weights of each feed

offered and unconsumed were recorded for each cow.

 

lShell Chemical Co., New York.

cmfimoo H.ucmswsedxmnu.

4.
r

epzmfim

33

 

new: 2. ~ - .. a

 

 

3.3.0.3:23 + 00m... U

 

 

3.30

 

 

 

 

 

3.3%

w 1i
3.3m o
an enema
- com: o: _ .. a
. now: u
“43m o
u macaw

203mm. ._<._.Zm¢<..~_m._Xm

3 A

All animals were housed in the same stanchion barn
throughout the experiment. With the exception of weeks
2 and 6, when total urine production was measured, the

cows were turned out to exercise 1-2 hours each day.

C. Sample Collection

 

1. Feed

Three times per week, 1-2 kg of each feed were ob-
tained at the time of feeding. For hay, small grab sam—
ples were made from several bales at the time of feeding
and were composited weekly. Each feed sample was placed
in a polyethylene bag, sealed, and frozen at -20°C until

analysed for HEOD and dry matter.

2. Milk

In the evening of each day and the morning of the
next, the milk from each cow was weighed, thoroughly
mixed, and an aliquot (1%) removed for a daily composite
for % milk fat and HEOD analyses. In addition, approxi-
mately 2 liters of milk were saved from each cow at each
milking as a reserve for subsequent HEOD metabolite
analyses. The milking machine buckets and claws were
thoroughly rinsed between cows in order to minimize
cross-contamination. Milk fat percentage was determined
by the Babcock method on each daily composite. Remaining
samples were then treated with 3—A drops of formaldehyde

and stored frozen at —20°C until extracted for HEOD.

3. Blood

 

Five samples from each animal were obtained at
approximately weekly intervals except for samples ob-
tained at the time of fat biopsy. Samples were drawn
from the tail or mammary veins into 15 ml vacuum tubes
containing sodium heparin as an anticoagulant. hey
were then frozen at —20°C until extraction for HEOD

analysis.

A. Body fat

 

External body fat samples were obtained at 0, 2,

A and 6 weeks of the experiment. Biopsies performed in
the scapular region, were alternated at each two week
period between left and right sides in order to minimize
trauma and infection. Weights of 10-30 grams were ob—
tained and attempts were made during surgery to avoid
removing fat from a previous biopsy site. The first
biopsy on each side went high on the scapular fat pad;
the second went low. A local nerve block was achieved
using 2% procaine + epinephrine.

Internal body fat samples were also obtained at
the time of the last biopsy. Samples of omental fat and
abdominal fat from cows in each group were surgically
removed during the laparotomy performed to facilitate a
liver biopsy. Samples were stored in small plastic bags

and frozen at -20°C until extraction for HEOD analysis.

36

5. Feces

 

Daily grab samples of feces were taken from each
cow, placed in plastic bags and stored frozen at -20°C
until extraction for HEOD, dry matter and chromic oxide

determinations.

6. Urine

 

During weeks 2 and 6 of the experiment total urine
collections were made for periods of 5 and 7 days respec-
tively. Two-liter samples were saved from each day's
collection from each cow for later compositing. Urine
collecting bags made of polyethylene were surgically at—
tached to the periphery of the vulva and sealed with
branding cement. At the base of each bag a small funnel
was sealed and connected to a rubber hose which drained
into a polyethylene—lined stainless steel container.
Daily urine production was weighed each morning of the
total collection period.

On the days when urine was not totally collected,
attempts were made to take one urine sample from each cow.
Vulvular stimulation usually induced urination. After
total collection periods the vulva was too tender for
urinary stimulation. Over the experimental period,
urine was obtained on the average every other day from

each cow.

37

Each sample was treated with formaldehyde to contain
0.1% in the final sample as a preservative. All samples
were then thoroughly mixed and stored frozen until later

Hfldl analimais.

'70 1.}.iver.

 

Laparotomies were performed at the time of the last
hiOpsy for shoulder fat. This was the method of choice
to facilitate removal of 20 to 30 grams of liver from
each cow with a minimum of trauma. Each sample was im-
mediately homogenized in 1.15% KCl buffer containing
0.02% nicotinamide (U ml buffer per gram wet tissue).
The homogenate was then centrifuged at 15,300x: for 15
minutes. The supernatant was decanted to cellulose ni—
trate centrifuge tubes for centrifugation at 105,000xg
for 1.5 hours. The supernatant from centrifugation was
decanted from the microsomal pellet. The microsomal
pellets were suspended in 0.1 M sodium phosphate buffer

in 50% glycerol, pH 7.5, at the rate of 1 ml buffer per

pension was made using

U)

1 gm tissue homogenized. The su
a teflon—glass tissue grinder, then decanted into a
screw-capped test tube, gassed with pure nitrogen, sealed

and stored frozen at —20°C until analyzed.

D. Sample Extraction

 

.2

All sample extractions were performed in this
laboratory employing methods previously established and

found applicable to the particular type of samples

?8

studied. References to the procedure are duly noted and
the method as well as the modifications employed are
described. All reagents used were reagent grade; all
solvents were reagent grade or NanogradeO2 and were

routinely checked for interference with HEOD analysis.

1. Feed

a. HEOD.—-Ten to twenty grams of each feed was
placed in a 250 ml ground-glass stoppered erlenmeyer
flask to which was added respectively, 10, 2 and 10 ml
of water, hexane and isopropyl alcohol. This mixture was
shaken for 30 minutes on a Burrell Wrist—Action Shaker,

Model CC3

set at 10. The hexane layer was then aspirated
off, dried over anhydrous sodium sulfate and 1-2 micro—
liters of the extract were injected into the chromato-

g:1‘:1;3}i .

b. Percent dry matter.—-Fifty to one hundred gram

 

samples of each feed were placed in a forced air oven set
at 9500 for 2“ hours, then removed and placed in a des—

sicator to cool and then rapidly weighed.

2. Milk

a. HEOD.--Milk samples were warmed to room temper-
ature then thoroughly agitated and l.Oml of each was im—
mediately removed by volumetric pipet and placed in

lhxl25 mm screw-capped tubes to which was added 2.0 m1

2Mallinkrodt Chemicals, St. Louis, Missouri.
3

Burrell Corporation, fittsburg, Pennsylvania.

39

ethanolic potassium hydroxide (EtKOH) prepared by dis—
solving 25 grams of KOH in 15 ml of distilled water and
diluted with 85 ml of absolute ethanol. The tubes were
then sealed with teflon—lined screw caps and placed in

a water bath at 75°C for 15 minutes during which time the
tubes were agitated 2-3 times to insure complete mixing
for hydrolysis. The tubes were removed and cooled to
room temperature. After cooling, 1 or 2 ml of hexane was
added, the tubes shaken vigorously by hand for three
l-minute periods permitting the layers to separate be-
tween shakings. All samples were then routinely centri-
fuged at 1700 xg for 15 minutes to remove any emulsions
that may have formed and to provide complete separation.
One to three microliters of the hexane layer were then
injected into the chromatograph for HEOD analysis. This
procedure is similar to that reported by Crosby and
Archer (41).

b. Percent milk fat.--The standard method of milk

 

fat percentage determination is the Babcock procedure.
This technique was used on all daily samples, and is the

same method used by DHIA.

3. Blood

 

The extraction of blood was essentially the same as
the extraction of milk, except that 1.0 ml blood were
used, and 2 ml distilled water were added after extrac-

tion with hexane. The tubes were shaken again and then

U0

centrifuged at 1700 xg for 15 minutes. One to three
microliters of the hexane layer were injected into the

chromatograph for HEOD analysis.

A. Body fat

 

hody fat samples were warmed to room temperature
and 1—2 grams were weighed and placed in a teflon—glass
tissue grinder. The sample was then ground in hexane
and transferred to a 16x125 mm screw-capped test tube to
a final dilution of 5 ml hexane to 1 gm of fat. Approx—
imately 0.5 gm anhydrous sodium sulfate were added and
the tube shaken vigorously for 30 seconds. The tubes
were then centrifuged at 1700 xg for 15 minutes to clarify
the solution. Appropriate dilutions were made as 1-2
microliters of the hexane layer were injected into the

chromatograph without prior cleanup.

5. Feces

 

a. HEOD.——Upon thawing, each fecal sample was
thoroughly mixed to provide a uniform sample, then 1—5
grams were weighed into a 16x125 mm screw—capped tube.
A 1:1 mixture of isopropyl alcohol:distilled water was
pipetted into the tube at the rate 2 ml per gram of wet
sample. The tube was capped and shaken vigorously for
3—5 minutes. One to two milliliters of hexane were
pipetted into the tube which was then capped and shaken

vigcrously for 3—5 minutes with 2-3 interruptions to

U1

permit layers to separate. A11 tubes were then centri-
fuged for 20 minutes at 1700 xg. One to three micro—
liters of the hexane layer were injected into the gas
chromatograph after appropriate dilutions, in order for
the values to fall within the linear range of the
instrument.

h. Percent dry matter.—-A 20—10 gram aliquot of

v

 

each daily sample of wet feces was plaCed in an aluminum
drying dish, weighed and dried in a forced air oven at
100°C for U8 hours. After drying the sample was removed
and placed in a dessicator to cool, then rapidly re-
weighed for dry weight. Samples were stored at 60°C
until subsequent chromic oxide analysis.

c. Chromic oxide determination.«-One to two grams.

 

of each sample used for percent dry matter determination
were placed in a 100 m1_volumetric flask to which was
added 5.0 mg sodium molybdate (0.1 m1 of 50 mg/sodium
molybdate/ml double—distilled water) and 10.0 ml concen4
trated nitric acid. 'The flask was heated slowly on a.
hot plate until the initial foaming stopped and vigorous
boiling occurred. Boiling wfis then continued for 10
minutes, the flask was cooled and 5 ml 70% perchloric
acid added. This second digest was continued until 1-2
minutes past the point when the predominantly green color
changed to yellow, orange or red (the color at this point
is concentration dependent). During each digestion, the

flasks were frequently swirled. Total time for the second

A2

digest was usually around 20 minutes. The flask was then
cooled and made up to volume with water and mixed well.
The precipitate formed was permitted to settle, then each
sample was pipetted into spectrophotometer tubes and read
at AAO mu wave length on a Coleman Jr. 11 spectrophoto—
meter.

Standards were prepared by digesting 0, 10, A0,
70, and 100 milligrams of Cr203 in nitric and perchloric
acids, and making final concentrations of O, 10, A0,
70, and 100 ug/ml. These levels were checked against the
same levels of potassium dichromate in double-distilled
water. The zero level was used as the reagent blank; all
samples were read against this solution. Recovery data

indicated that standards gave satisfactory results.

Cr2O3
Standards prepared this way provided constant readings

for as long as A months, and were linear with each analy—
sis. The concentration of each sample was divided by its
dry weight, the result of which was divided into the 15
grams CrQO9 given each cow to obtain the dry matter excreted

J

per day. Hence, concentration of Cr2O3 was inversely
proportional to the dry matter excretion. The method em-
ployed was that reported by Kimura and Miller (95). A
mcre recent investigation of the absorption maxima for
the color reaction reported that a wave length of 350 mu

was more sensitive than AAO mu (A5). This difference was

also noted in the present study. However, AAO mu gave

113

more repeatable results. The sensitivity of the method
using 350 mu was not needed in this experiment due to the

high concentration of chromic oxide used.

6. Urine

 

One-hundred milliliter aliquots of urine were
tracted three times with 50 ml portions of hexane.
The hexane extracts were concentrated on a rotary evapo—
rator under vacuum at 50°C and brought up in l—lO ml of
hexane. The alkaline hydrolysis procedure for milk and

blood was also used.

7. Liver

 

a. HEOD.—-Liver tissue was analyzed for HEOD ac-
cording to the alkaline hydrolysis procedure used with
milk and blood. One to two ml of Uzl homogenate were
added to B—U ml ethanolic KOH and the mixture was heated
for 15 minutes at 75°C and cooled and extracted with 1—2
ml pentane. The pentane extract was injected into the
,as chromatograph with no prior cleanup.

b. Hepatic drug metabolizing enzyme activity.—-The

 

enzyme activity was determined by measuring formation of
formaldehyde from aminopyrine by nitrogen-demethylase
(122). The enzyme incubation mixture contained 0.1M phos-

phate buffer pH 7.“, aminopyrine (10 mM), MgCl (6 mM),

2
isocitric acid (10 mM), NADP (0.5 mM), isocitric acid
dehydrogenase (0.05 units per ml) and microsomal protein

(0.1 to 1.0 mg/ml). Protein was determined by the method

UM

of Lowry (107). The reaction mixture was incubated 15
minutes and stopped by adding 10% trichloroacetic acid.
The specific enzyme activity was expressed as milli-
micromoles of formaldehyde formed per milligram of pro-
tein per minute.

E. Electron—capture Gas—liquid
Chromatography

 

 

All HEOD analyses employed a Varian Aerograph
152OBM dual column gas-liquid chromatograph equipped with
electron-capture detectors containing tritium (H3) foils.
The recorder was a dual-channel Westronics Model LD 11A5
operating at l mv range and chart speed of 20 in. per hour.
The most frequently used columns were 5' x 1/8" stainless
steel containing 5% QF-l on 100/120 mesh Varaport 30,
or 5% QF—l on 100/120 mesh DMCS-Aw Gas-Chrom Q. Operating
temperatures were: injector, 230°C; column, 195°C; and
detector,195-200°C. Carrier gas was high purity nitrogen
using a flow rate of U0-50 cc per minute through the
detector, with a head pressure of 65 p.s.i. The reten-
tion time of HEOD under these conditions ranged from 3—5
minutes. Recoveries of HEOD were determined by fortifi-
cation of the samples prior to extraction with amounts of
HEOD roughly equivalent to the amount in the sample such
that the final concentration would be twice that of the

unfortified sample. Identification was made by matching

 

u
Varian Associates, Walnut Creek, California.

r
)Westronics, Inc., Fort Worth, Texas.

145

retention times and peak characteristics with pure stan-
dards of HEOD. The minimum measurable amount of HEOD was
10—19 gram (1 picogram) at maximum sensitivity of the
instrument.

HEOD standards were prepared in glassware thoroughly
rinsed with solvents. Concentrations ranged from 0.01
ppm to 2.0 ppm HEOD in hexane. Injections were made with
a Hamilton 701 microsyringe, using 1—5 microliters per
injection. Low-bleed septums were changed at least once

daily. Concentration of HEOD was determined by a standard

curve utilizing a lO—fold range of standards.

CHAPTER IV

RESULTS

During the entire six-week feeding period, there

Luspeared to be no toxic effect by either HEOD or pheno-
luirbital, nor did there appear to be a synergistic effect
maxfifested between the two chemicals on normal mainten-

:ince and production parameters. Period I indicates weeks

1n—3 of treatment; period II, weeks “—6.

The average age and day of lactation on the first

day of the experiment are presented in Table 1. Differ-

(nices were small, with little effect on performance.

A. Feed Intake

Feed intakes expressed on a dry matter basis are

Drwasented in Table 2. Dry matter consumption tended to

(lec:line regardless of treatment. The highest intakes

<>CL:urred in the decontaminated control group (C-D). The
(leczontaminated phenobarbital group (PB-D) had the lowest
iITtakes, expecially for hay. Groups C—D and PB-C con—

SLHned more grain per day, whereas the remaining two groups

DIKBferred hay (Table 3). This may account for the differences

in consumption per unit of body weight (Table U) observed

fOr groups C—D and PB—C. The control group consumed

M6

A7

(TABLE l.—-Average age and day of lactation on first day
of experiment.

 

 

C—C C-D PB-C PB—D

AA/erage age (months) A7 A5 51 AA
Axlerage day of lac-

tation 263 2A8 237 2AA

 

TVXBLE 2.--Dry matter consumption.

 

Vheek C—C C—D PB—C PB—D

 

-------------- kg DM/cow/day—-————-——---—-———

 

 

 

0 16.93 16.97 18.31 15.81
1 15.71 18.02 18.78 16.1A
2 1A.93 18.06 15.36 1A.67
3 15.A3 18.AA 16.88 13.73
A 1A.89 18.15 13.81 13.81
5 lA.77 17.65 15.75 13.38
6 1A.98 l7.Al 15.A9 13.A2
mean

(0—6) 1538:2029a 17.81:o.19 16.3Ai0.66 lA.A2.t0.A3

 

 

a
Standard error of the mean.

A8

TABLE 3.--Feed DM intake as a percentage of total DM intake.

"—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Week 0—0 0—0 PB—C PB-D
Grain
0 58 9 61.8 63.3 59.1
1 55.8 63.6 65.6 61.2
2 53.A 3.6 61.5 55.8
3 55.5 62.6 66.8 58.A
A 53.5 62.5 56.7 56.6
5 53.5 61.8 61.1 55.2
6 56.1 63.2 60.0 55.3
(0—6) 55.2t0.8a 62.7:0.3 62.1:1.3 57.u:0.9
me an
Silage
0 18.1 18.1 16.8 19.“
1 19.5 17.0 16.3 18.7
1 20.6 17.0 18.8 20.6
3 19.8 16.6 16.1 20.7
A 20.6 16.8 21.0 22 2
5 20.8 17.A 19.2 22.8
6 20.2 17.6 19.3 22.6
(0—6) 19.9iO.A 17.2:0.2 18.2:0.7 21.0:0.6
me an
Hay
0 23.0 20.1 19.9 21.5
2A.7 19.u 18.1 20.1
26.0 19.A 19.7 23.6
_ 2A.7 20.8 17.1 20.9
A 25.9 20.7 22.3 21.2
5 25.7 20.8 19.7 22.0
6 23.7 19.2 20.7 22.1
(0—6) 2A.8:0.A 20.110 3 19 6:0.6 21.6:0.A
mean

 

a Standard error of the mean.

A9

 

 

 

 

 

 

TABLE A.-—Dry matter intake as a per cent of body weight.
Week C—C C—D PB—C PB-D
———————————————— % sw-————-———--——-————--——

O 2.87 2.76 3 00 2.72
1 2.63 2.89 3 02 2.71
2 2.A9 2.87 2.39 2.A7
3 2.50 2.8A 2.60 2.25
A 2.37 2.80 2 08 2.23
5 2 37 2.66 2.38 2.18
6 2.3 2.65 _ 2.38 2.15

(0—6 2.51:0.07a 2.78:0.0A 2.55:0.13 2.39:0.09
mean)

a

Standard error of the mean

about 1 kg more dry matter per day than the phenobarbi—
tal group. Also when expressed as a per cent of body
weight, the intakes vary considerably (Table A). Groups
C-0 and PB-D consumed the most and least amount of dry
matter per unit body weight, respectively.

The average per cent dry matter of each feed dur-
ing the control period and weeks 1—3, A-6, (periods I
and II, respectively) is presented in Table 5. A decline
in dry matter consumption occurred over the entire experi—
ment which was primarily attributable to the reduction of
grain offered in response to the decline in milk produc-
tion.

It was anticipated that feed intakes might increase
with phenobarbital feeding. As noted in Table 2 there

appeared to be no effect of phenobarbital on feed intake.

80

 

 

 

 

 

TABLE 5.——Per cent dry matter of feedstuffs consumed.
Period Hay Silage Grain
------------------ % DM------—---—------
Control 85.61 3A.06 86.35
1 85.60 33.2 85.97
11 85.13 32.85 85.71
(Control 11 85.u5:0.16a 33.38:0.36 86.01:0.19
mean)

 

3 .
Standard error of the mean.

0ver the entire experiment each group consumed a
ration which consisted of the following overall per cent
dry matter: C-C, 65.7; C—D, 68.1; PB-C, 66.9; and PB-D,

65.7.

U. Body Weight Changes

 

Changes in body weight from the first period to the
second are shown in Table 6. All animals, regardless of
treatment, gained weight over the entire experiment. The
animals in the control group averaged a 5.0% increase
compared to the phenobarbital-treated group which gained
A.3%.

The expected increase in feed intake by phenobarbi—
tal treatment was also expected to produce a concomitant
increase in body weight. This effect did not materialize.
Rather, the opposite effect was noted.

The ration met all nutrient requirements of main—

tenance and lactation while roughage intake was kept at

51

a minimum in order to superimpose a MgClq infusion experi—
C
ment designed to correct slight depressions in milk fat

percentage.

TABLE 6.——Body weights of experimental animals.

 

 

 

Period C—C C—D PB—C PB—D
——————————————————— kg-—————-———---—--—-—-———
Control 590 615 611 582
I 605 63A 6A0 600
II 628 656 659 619
C. Dosage

 

Contamination of the cows at 0.10 mg HEOD/kg body
weight/day provided each group with between 59 and 65
mg/cow/day (Table 7). Because of differences in body
weight, PB—C received the highest total HEOD intake per
day, PB—D the lowest. Gain in body weight caused an
increase in total daily and period HEOD intake. These
values represent the amount administered to each animal.
However, on the fourth day of the experiment, one cow
in group PB—D regurgitated the bolus for it was found in
the orts the next morning. The data do not include this
lost dose. It is assumed that all other doses were re—
tained by all animals for there is no evidence to the
contrary.

The contamination level in the feed is shown in

Table 8. The total dose administered divided by the

'FATHJS 7.--lH$01)<1osan3e.

 

 

 

 

 

Period C—C C—D PB—C PB—D
-------------- mg HEOD/cow—-——--—-—----—-—--
I 133A.3 132A.1 1A00.3 1252.9
11 1388.3 0 1A50.3 0
Total 2722.6 132A.1 2850.6 1252 9
Per day of
contam-
ination 61.88 63.05 6A.79 59.66
TABLE 8.-—HEOD intake.
C—C C-D PB-C PB-D
Total HEOD, (mg) 2722.6 132A.1 2850.6 1252.9
ppm HEOD in DM A.10 3.A7 A.03 A.02
ppm HEOD "as fed" 2.68 2.36 2.71 2.6A
Total DM intake
during contamin—
ation, (kg) 66A.72 381.6A 703.73 311.78
Feed consumed "as
fed" during con—
tamination, (kg) 1016.07 560.21 1051.27 A7A.7A
% DM of total 7 ,
ration consumed 65.7 68.1 66.9 65.7

 

amount of dry matter consumed during the contamination

period gives the concentration on a dry matter basis.

When corrected for per cent moisture or placed on an "as

fed" basis, group C-D continued to be contaminated at the

lowest level per unit of feed intake. This appears to be

I)?

primarily a function of not only consumption of more hay
and grain relative to silage but also of a lower daily
dose of HEOD.

The cumulative mean total dose given to each group
is enumerated in Table 9. It can be seen that the dose
given up to each succeeding week is essentially the same
for each group. The quantity of HEOD administered rep—
presents the actual amount weighed out to the nearest 0.1
mg, and not the amount calculated for each animal to re—

ceive.

TAULL 9.-—Cumu1ative HEOD dose administered.

 

 

 

Week C—C C—D PB—C PB—D
—————————————————— mg/cow——--—---—-----—-—-—-——
1 A77.6 A98.0 A95.6 A73.A
2 906.9 938.8 9AA.3 890.8
3 133A.3 132A.1 1A00.3 1252.9
A 177A.3 -— 186A.8 ——
5 2277.A —— 2395.2 --
6 2722.6 —- 2850.6 -—
D. Milk Production, and Concentration and

 

Excretion of HEOD in Milk

 

Milk production was quite persistent for each per—
iod, except for a marked decline noted during the second
period for PB-D (Table 10). The daily milk yields de—

clined throughout the experiment for all treatments.

This decline was eXpected since all cows were in the
sixth to tenth month of lactation when contamination be—

gan.

TABLE 10.——Average daily milk production.

 

 

Week C-C C-D PB—C PB—D
———————————————— kg/cow/day—-—-----—---—--———
Control lA.8 19.10 19.A0 16.80
1 13.95 18.00 19.15 16.30
2 l3.A0 18.20 15.20 13.80
3 13.60 16.25 16.20 13.50
A 12.95 16.65 13.70 11.A5
5 12.50 15.25 1A.95 11.65
6 12.15 1A.25 15.25 12.05

 

 

 

(1—6) 13.09:0.28a 16.u3:0.63 15.7A:O.76 13.13:0.75

mean

 

a .
Standard error of the mean.

In Table 10 it can be seen that there was a sub-
stantial drop in milk production in the phenobarbital-
treated groups from week one to week two. This may re-
flect an adjustment to phenobarbital treatment and the
transient decline in dry matter intake. Average daily
production throughout the experiment was highest for C—D,
lowest in C—C and PB-D. Again, these differences prob-
ably are a reflection of energy intake. Milk produc—
tion may have been been affected by treatment with pheno—
barbital (Table 11). The average persistency of milk
yield on phenobarbital treatment was 75% compared to 83%

for the controls. A similar change in milk production

U3
\Jl

was noted in the first period from the production ob-
served prior to the initiation of treatment. Throughout
the experiment the animals receiving the drug appeared
to decline at a faster rate than did the control group.
This may be a reflection of a decrease in dry matter
intake noted for the drug—treated grOUp. The great
decline in daily yield for group PB—D is primarily a
reflection of one cow's production dropping to one—third
the control level while the other cow maintained normal
production. Ho abnormality was noted for the first ani-

mal.

TABLE 11.——Percentage decline in milk production.

 

—:— ———-- .

Period C—C C—D PB—C PB—D

 

--------------- % of control—--—-----—-——-—-—

Control 100.0 100.0 100.0 100.0
1 92.8 91.6 86.9 86.5
11 85.3 81.1 79.6 69.8

 

In order to obtain a different view of milk pro-
duction standardized to eliminate body size and level of
production effects, the daily amount of milk produced per
kg of dry matter consumed is presented in Table 12. These
values represent a measure of efficiency of conversion.
All animals tended to decline in efficiency as the experi-
ment progressed. However, no notable differences appear

between the control and phenobarbital groups. The

56

averages suggest that possibly PB—C and D were more effi—
cient utilizers of feed, which is supported by the rela-
tive differences in preference for grain, i.c., those
that preferred grain produced more milk per unit of
intake.

TABLE l2.——Ratio—milk produced per day vs dry matter
consumed.

 

Week C—C C-D PB—C PB—D

 

-------------- kg milk/kg DM intake----------—

 

 

 

 

Control 0.87 1.13 1.06 1.06
1 0.89 1.00 1.02 1.01
2 0.90 1.01 0.99 0.9A
3 0.88 0.88 0.96 0.98
A 0.87 0.92 0.99 0.83
5 0.85 0.86 0.95 0.87
6 0.81 0.82 0.98 0.90

(1—6) 0.87:0.01d 0.92:0.0u 0.98:0.01 0.92%0.03

me an

 

r0
(.4

Standard error of the mean.

The concentration of HEOD in whole milk is presented
in Table 13. The levels rose rapidly in each group during
the first week, continued to increase in the six-week con-
tamination groups, but declined rapidly when HEOD was
withdrawn at the end of the third week in groups C—D and
PB—D. HEOD concentration declined at a considerably
greater rate in PB-D than in C—D. The variable response
in HEOD concentration among groups during the first three
weeks is almost as great as within—group differences from

one week to the next. The lower HEOD levels in milk

57

observed when contamination ceased appears to be due more to

withdrawal of HEOD rather than to drug treatment. The decline

in the sixth week for group PB—C may be due to one cow

which dried off rapidly.

TABLE l3.-—Concentration of HEOD in whole milk.

 

 

 

 

Week C—C C—D PB-C PB-D r.-
——————————————————— ppb-—-—-—-——-———-—--——-——-———
1 57.6: 6 9a 56.6:7.7 6A. : 7 3 52.3: 3 A
2 110.5113.9 87.717.7 89. i 7.6 135.7il3.3
3 108.71 9.9 129.8i8.2 90.2i 8.3 12A.6i 9.1 _
A 118.9: 8 3 106.9:6.9 122.6: 9 89.7: 7 0 1,.
5 15A.1ill.5 88.AiA.O 1A7. + 3 52.1i 2 9
6 121.6:1A.2 50.0i5.7 92. _ 9 38.61 3 1
a

Standard error of the mean.

In Table 1A, daily excretion of HEOD per cow rose
rapidly and remained elevated as contamination continued.
The most dramatic elevations, in both concentration and
excretion, occurred during the first two weeks. Clear—
ance of HEOD via milk appears to be more a function of
milk production than HEOD concentration. Whereas pheno-
barbital appeared to have a slight effect on milk con-
centration of HEOD, the most pronounced differences due
to phenobarbital were noted for weekly excretion data.
Levels of HEOD in the milk were lower in group PB—D
especially when contamination ceased. This would sug—

gest that phenobarbital both minimizes the amount of

f‘v‘
VV

HHHD excreted via the milk and accelerates excretion upon

withdrawal of HEOD.

TABLE lA.——Daily excretion of HEOD via milk.

 

 

Week C—C C-D PB-C PB-D
--------------- mg/cow/day——-————---——-——-—-——
1 0.80 1.02 1.23 0.85
2 l.A8 1.60 1.36 1.87
3 l.A8 2.11 l.A6 1.68
A 1.5A 1.78 1.68 1.03
5 1.93 1.32 2.20 0.61
6 l.A8 0.71 1.A0 0.A7

 

When the average daily excretion values are com—
bined to obtain period averages the same trends appear
(Table 15). When HEOD was withdrawn (groups C-D and PB—i)
the excretion of HEOD in milk was A0% less when phenobar-
bital was administered. The total HEOD excreted by period
again suggests the above conclusions (Table 16).

The amount of the total HEOD received during the
entire experiment that was excreted in the milk amounted

to one day's dose or about 2.5%.

TABLE l5.——HEOD excreted in milk.

 

Period C—C C-D PB-C PB-D
----------- mg/cow/day-—--——-—------——------—--
1 0.989 1.577 1.350 1.A67

11 1.650 1.270 1.760 0.703

 

 

 

 

 

TABLE l6.——HEHD excreted in milk.
Period C-C C—D PB—C PB—D
—————————————— mg/cow/period——-————---—--—----
I 20.8 33.1 28.A 30.8
II _38.0 29.2 A0.5 16.2
I and II 58.8 62.3 68.9 A7.0
(total)

 

E. Concentration of HEOD in Milk Fat
and Milk Fat Production

The average per cent milk fat of all groups varied
considerably (Table 17). The values at the end of the
contamination period for both decontamination groups were
high, whereas at the end of the eXperiment the percentage
milk fat was lowest. Across all treatments the percentage
milk fat declined, which is possibly a reflection of the
modified restricted—roughage ration imposed on all ani—
mals. The higher value for PB—D in period I was primarily
due to one individual which initially tested very high
throughout lactation, but whose percentage milk fat
declined rapidly after initiation of the experiment.

This depression may have been due to phenobarbital but
data from other cows in this experiment do not suggest
that the drug affected milk fat content. All other ani—
mals, though declining in percentage milk fat, were not
as severely depressed as is usually observed with

restricted-roughage rations. Certainly, there appeared

to

to be no synergistic depression by the use of phenobar—
bital with a restricted roughage diet. The reverse
situation does not seem to exist either. That is, the
data do not support the possibility that phenobarbital
eacted counter to the restricted-roughage ration and min—
iJnized the depressing effect on milk fat caused by the
(iiet. As was illustrated by Braund (15) changes in milk
:fat percentage did not significantly alter total excre-
t;ion of HEOD. His observations seem to be supported by
tJde present eXperiment. During the course of the exper—
iJnent, two official tests by the DHIA supervisor were
ccnqducted. The official values obtained for milk pro—
dliction agreed very closely with the weights recorded
beafore and after the test. Likewise, the results of our
cnvn.Babcock test varied no more than $0.1 percentage
DCDint from that obtained by the DHIA tester. Comparison
Of‘ experimental values with official results not only
sexrved as a check on the conduct of the trial, but also

irujieated that sampling techniques were adequate.

'PAIBLE l7.—-Average percentage milk fat.

 

 

 

h..—

 

‘PeIeied c—c C—D PB—C PB—D
Corrtroi 3.09 2.99 3.73 3.86
I 3.75 3.76 3.36
IL 3,3 2.96 3.38
I and II 3.53 3.36 3.37

mean)

\

61

Maintenance of milk fat production was achieved in
each C group but was markedly reduced in each D group
(Table 18). These differences may be due to the ration,
but probably are due more to the combination of per cent
milk fat decline and decline in milk production seen to

a greater degree in the two D treatments.

TABLE l8.——Milk fat production.

 

 

 

Period C—C C—D PB-C PB-D

------------- kg/cow/period—--——-------—-————
I 10.92 13.02 11.97 12.92
II 10.70 10.2“ 12.19 8.05
I and II 21.62 23.26 2A.16 20.97
(total)

 

The concentration of HEOD in milk fat shows trends
similar to those for whole milk concentrations (Table 19).
Again, phenobarbital—treated animals had a lower HEOD
concentration than the control groups and the rate of
decline appeared to be faster due to phenobarbital treat—
ment. These differences did not appear during the first
week of the trial. Since differences in milk fat percent-
age were small, the expected correction of milk concen—

tration for fat content was also negligible.

F. Concentration of HEOD in the Blood

Samples of blood analyzed for HEOD contained very

low amounts of the pesticide (Table 20). The values are

62

TABLE l9.~-HE00 concentration in milk fat.

————-_.-—_
—--.—.———_

 

 

Week C—C C—D PB-C PB-D
—————————————————— ppm——--—---—-——------—-—--—

1 1.565 1.5U5 1.815 1.32“

2 2.757 2.4u0 2.807 3.U9l

3 3.260 “.080 3.081 3.U97

H 3.569 3.032 3.339 3.026

5 H.512 2.99M H.078 1.866

6 “.691 2.319 3.245 1.298

 

TABLE 20.-—Concentration of HEOD in blood.

 

 

Week C—C C—D PB—C PB-D
————————————————— ppb———————-—--———----—--—--
Control 0 0 0 0
2 3 82 3.U8 3 26 3.07
A 7 2H 3.90 2 88 1.50
5 5 50 1.37 5 37 1 59

 

are highly variable and appear to be related more to the
duration of HEOD dosage than to any other treatment
variable. Analytical problems evolved which caused the
values presented to be estimates rather than precise fig-
ures. The fact that they are all much lower than usually
encountered with the dosage intensity used in this experi-
ment, may be more a function of the large body weight
increases, thus shifting equilibrium toward deposition in
adipose tissue.

The low value for PB—D in the second week would

appear to agree with the high excretion in the feces

63

during this time, but the levels in the other three groups
do not agree with the relationships noted in feces. Cer—
tainly, the effects due to withdrawal of the pesticide
were rapidly seen. As early as seven days after cessa-
tion of the dose blood levels had declined. In the fourth
week it appeared that phenobarbital may have accelerated
the decline of HEOD in blood, but values had increased
again in the fifth week.

0. Concentration of HEOD
in Body Fat

 

 

The levels of HEOD in body fat from the scapular
fat pad are presented in Table 21. Particular care was
taken to minimize trauma and to ensure that newly deposi-
ted adipose was not used in the samplings from each sur-
gical site. The first samples were removed from the
right side of each animal, the second from the left side,
each sample being taken from the upper region of the
incision. The third biopsy was performed on the right
side, the fourth from the left side, both just below the
previous biopsy, on the respective side.

No HEOD was detected in control biopsies. As early
as two weeks there was considerable variation in the HEOD
concentrations. Although the absolute values were dif—
ferent initially, a trend developed by the fourth week,

a week after cessation of HEOD feeding in group C-D and

PB—D. Upon withdrawal of the pesticide, HEOD was depleted

6”

more rapidly from group PB—D than group C—D. The rise
in HEOD concentration was greater throughout the experi-
mental period for group C—C than for group PB—C. In

addition, levels in PB groups were consistently lower

than corresponding controls. All values are somewhat
lower than those observed by Braund (15) using the
same level of contamination as employed in this experi-

ment.

TABLE 21.--Mean HEOD concentration in shoulder fat.

 

 

 

Week C—c C-D PB—C PB—D
----------------- (ppm)-—------———--——-----—-—

Control 0 0 0 0

2 1.21 1.78 1 07 1.53

A 1.56 1.65 1 27 1.10

6 2.96 1.35 2 53 1.18

 

h. Concentration of HEOD in Urine

 

No HEOD was detectable in urine (<0.1 ppb). .8017
vent extraction (ether, petroleum ether, hexane, ethyl
acetate) extracted no HEOD. However, peaks appeared in
the chromatograms which may have been metabolic products
of the parent compound. Alkaline hydrolysis was also
employed on large quantities of urine, in order to des—
troy conjugates of HEOD and improve extractability of
the compound. Acidification to pH 2 did not appear to
enhance HEOD extraction because HEOD is labile in acid

conditions.

65

Daily urine production did not appear to be affec—
ted by treatment or stage of lactation (Table 22). Indi—
vidual variation among animals was considerable, with
average daily outputs ranging from 9—29 kg/cow, but vari—
ation within each animal was low in each period. One
animal in group PB—C had a high urine output throughout
the experiment. There was no trend due to time on treat-
ment, with some animals increasing urine output, others

decreasing during the progress of the experiment.

TABLE 22.—-baily urine production.

 

 

Week C—C C—D PB-C PB-D
—————————————— kg/cow/day—--————---——--——-——-—

2 11.316 73 1u.3:1.8 20.0:3.3 12.1:1.1

11.710.6 12.u:0.9 19.Ui1.6 11.310.8

 

a .
Standard error of the mean.

I. Feces

 

1. Dry Matter Excretion

Dry matter excretion of feces expressed as kg/cow/
day is shown in Table 23. The method used to determine
chromic oxide gave recoveries of 93-98%. The concentra—
tion of chromic oxide ranged from 1—5 mg/kg dry matter,
and was more variable than per cent dry matter. During
the six weeks of the experiment the mean dry matter
excretion for groups C—C, C—D, PB—C, and PB-D was 8.26,

8.83, 6.1“ and 7.78 kg DM/day, respectively. The reason

66

for the higher dry matter excretion in the control than
in the phenobarbital-treated groups is not known. A gen-
eral decline was seen for both phenobarbital groups

whereas the control groups were variable.

 

 

TABLE 23.-— Excretion of dry matter in the feces.
Week C-C C—D PB—C PB-D
—————————————————— kg/cow/day-—-——-—---—---:---——

Control -- -— -- --
1 8.05t0.59a 8.7ut0.u1 7.73t0.u7 8.58:0.71
2 7.33i0.58 9.7910.59 5.3Ai0.29 8.52t0.58
3 8.70il.52 7.07i0.8l 6.86:1.10 8.80:0.63
A 8.95i0.62 7.7510.u1 6.08i0.70 7.8AtO.39
5 8.13i0.A8 10.86il.O8 5.12t0.37 7.15t0.u6
6 8.Ul*0.77 8.75i0.85 5.7210.35 5.77i0.38

 

a. ‘
Standard error of the mean

Percentage Dry Matter

 

The overall average of percentage dry matter was
around 19 with a range of lA—2A. Some variation among
animals was noted, but very little within-animal varia—
tion occurred. There appeared to be no trend or differ—
ences due to treatment. Excellent repeatability was
obtained within samples under the conditions employed and
drying longer than 2“ hours gave no significant change in

the dry matter values.

67
J. Fecal Concentration and Excretion
of HEOD

Extraction efficiency of HEOD from feces was inves—
tigated and a summary of results is presented in Table 2M.
Previous analyses (15) had been performed on feces that
had been oven—dried, ground, subsampled and extracted with
a 3:1 mixture of hexanezisopropyl alcohol. Alkaline hy-
drolysis of dried feces caused interfering peaks to occur
during gas chromatography, and at times appeared to pro—
duce alteration of HEOD. Regardless of the extraction
procedure used, recoveries of HEOD from dried feces were
highly variable. A simple change to the extraction of
wet feces with the same solvent systems and no addition
of water gave improved recoveries. At no time was the
improvement in extraction efficiency between wet and dry
material less than 390%. Water was added to the tubes
during extraction to prevent emulsions from forming and

to permit the isopropyl alcohol to solubilize the feces.

rPABLE 2A.-—Extraction of HEOD from wet and dried feces.

 

 

Dried Normal
Range of HEOD in dry matter
(ppm) 0.670—o.807 2.613—6.019
Relative extraction:
As a percentage of ”dried" 100 390—7A0
As a percentage of "normal" 26—13 100

 

HEOD concentration in feces on a dry matter basis

was highest during the first week of treatment for all

 

.cmoe who no posse esopcmpm

 

 

 

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nun mwa.oamm:.a In: moa.oaom:.a m
-u- mma.oaoom.a --- Ho:.oaeom.H m
w. mmo.oamao.o mem.owemm.m mmo.eafiaa.o wae.oam:o.m :
mmm.oaeem.fi mmm.owemm.a mma.oamam.o o:e.oaome.m m
mm:.oawae.m mmm.oaweo.m swm.oame:.a new.oaems.m m
mam.aaooe.e woa.aamem.m meo.Hamma.m amee.aafiae.z H
o o o o Hopscoo
IIIIIIIIIIIIIIIIIIIIIIIIIIII EaQIIIIIIIIIIIIIIIIIIIIIII
enma sums 6.0 6.0 see:
.aessee see Haves ea aomr as coasaaseeeees--.mm mamae

69

cows, except for those in group PB—D which peaked during
the fourth week (Table 25). The low level in the third
week and the high level in the fourth week for the group
PB-C are difficult to reconcile.

Examination of fecal concentration of HEOD (Table
25) and fecal excretion of HEOD (Table 26) indicates that
fecal dry matter output was much less variable than con-
centration or total elimination of HEOD. The same rela-
tive differences exist regardless of which measure is
used. Phenobarbital—treated animals in the first week
moved in feces the most and least amounts of HEOD. Sim—
ple averages of each treatment indicate that phenobarbi—
tal caused a slightly greater elimination of HEOD in the
first two weeks,but lesser amounts than shown for the
controls were observed during the following four weeks.
Individual variation was sufficient to mask any subtle
effects of phenobarbital treatment. Analysis of variance
failed to indicate differences between groups in any one
week. or primary interest are the extremely high values
observed for each group initially. Usually very little
HEOD is seen in the feces, but concentrations achieved in
this experiment equal and even surpass those seen when
activated carbon is administered.

Tables 27 and 28 present fecal excretion data on
the basis of percentage of the dose excreted per week, and

cumulative, respectively. It can be seen that these

 

70

 

 

 

 

 

TABLE 26.——HE00 excretion via feces.
Week C-C C-D PB-C PB—D
——————————————— mg HEOD/cow/day-------—-—---—-—--—-
Control 0 0 0 0
1 3937311359a 29.3u18.1u 21.72:7.u5 50.90i13.89
2 16.68: 5.75 11.86a1.86 11.61:2.23 22.92: 3.92
3 19.011 n.77 6.3010.7u 6.20:0.78 13.311 1.55
A 17.68: 5.Al 0.82tO.19 19.09t2.71 0.71: 0.23
5 11.21: A.Ol -—- 5.A9:O.7A ---
6 11.231 0.99 --- 7.67:0.76 ---
d Standard error of the mean.
TABLE 27.—-Percentage of weekly HEOD dose excreted in feces
that week.
Week c—c C—D PB—C PB—D
____________________ g__-__--_____-________-__-_
66.5 “7.1 35.1 86.0
2 27.7 18.8 18.1 38.A
3 31.1 11.“ 9.5 25.7
A 28.1 -- 28.8 —-
5 17.8 -- 8.2 --
6 17.7 -- 11.8 --

 

- -'_ at“ "*- '—

 

71

TABLE 28.——Cumulative excretion of HEOD as a percentage
of the total amount administered.

 

 

Week C-C C-D PB—C PB-D
___________________ 7,;__.___....._...__....._......_...__._..
1 66.5 “7.1 35.1 86.0
2 “7.9 33.8 27.0 63.7
3 A2.5 27.3 21.3 52 7
A 39.0 27.8 23.2 53.1
5 3A.3 —- 19.9 ——
6 31 —— 18.6 ——

 

values approximate the relations seen in Tables 25 and
26. The elevated excretion of group PB-D during the
first week represents 86 per cent of the total dose ad-
ministered. Almost two—thirds of the dose was excreted
by C—C in the first week. A precipitous decline in
excretion of HEOD in all groups occurred in the second
week when from one—fifth to two-fifths of the dose given
that week was excreted in the feces. The amount de—
clined as the experiment progressed with no definitive
differences among groups. The decline is reflected in

a marked reduction in fecal HEOD when expressed as a
percentage of the cumulative dose. At no point in the
trial did the amount excreted to date fall below 50%

for group PB-D. Total excretion of HEOD and total
excreted to date (Tables 29 and 30) also indicate trends

similar to the foregoing tables.

 

if

72

TABLE 29.-—Tota1 HEOD excretion in feces.

 

 

Week c—c C—D PB-C PB—D
--------------- mg/cow/week—-——---—--—-——---—-—

1 317.8“ 239.72 173.76 907.20

2 116.76 83.02 81.27 160.4A

3 133.07 UH.10 U3.U0 93.17

M 123.76 5.7“ 133.63 4.97

5 89.68 —- A3.92 —— r1.
6 78.61 -— 53.69 --

 

TABLE 30.~—Cumu1ative excretion of HEOD in feces.

 

 

 

 

Week C—C C-D PB-C PB—D
---------------- mg/cow~--—--------—---—------
l 317.8U 23U.72 173.76 A07.20
2 “34.60 317.7“ 255.03 567.64
3 567.67 361.8u 298.A3 660.81
A 691.A3 367.58 “32.06 665.78
5 781.11 -— “75.98 -—
6 859.72 -— 529.67 --

 

An estimate of the digestibility of the feed con—
sumed is presented in Table 31. The data are calculated
from the weekly dry matter consumption divided into the
weekly dry matter excretion as determined by the chromic
oxide method. Significant differences exist among the
mean per cent intake digested. Each group shows a
marked depression in digestibility for one week, but

these occur at different times for the different groups.

73

TABLE 31.—~Dry matter digestibility.

 

 

 

 

Week C-C C—D PB-C PB—D
___________________ %_______--_-________--__-_
1 48. 51.5 58.8 46.8
2 50. 45.8 65.2 41.9
3 43.6 61.7 59.4 35.9
14 39.9 57.3 56.0 43.2 Mn
3 45.0 38.5 67.5 46.6
6 43.9 49.7 63.1 57.0
mean 45.u:1.6a 50.8:3.u 61.7:1.8 u5.2:2.9
a Standard error of the mean.

 

Point plots of mean daily concentration of HEOD in
fecal dry matter are presented in Figures 2, 3, 4 and 5.
Wide variations exist from day to day, which are primar—
ily a function of variability of the pesticide movement
in the gastro—intestinal tract. Fecal concentration of
HEOD appears to be cyclic for each treatment. A change
in concentration is manifested every 3—6 days. Even
if the days of extremely high concentrations are ignored,
there appears to be no definitive differences among groups
which could be attributed to treatment rather than indi-

vidual variation.

K. Body Burden and Distribution
of HEOD

 

The distribution of the HEOD administered to the

cows is listed in Table 32. A significant amount of the

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82

TABLE 32.——Distribution of the total HEOD dose.

 

Location C—C C—D PB-C PB-D

 

—————————————— % of HEOD dose——--—-----——-----

 

Feces 31.6 27.8 18.6 53.1
Body fat
(2 weeks) 8 0: 11.92“b 7.3g 10.25“"b
(6 weeks) 6 9 —— 5,8 -_ C
Milk 3.1 3.8 3.3 3.1 an.
Body fluids 0 07 0.06 0.04 0.06
Total 42 77 43.56 29.24 66.46

a These values are based on the concentration of HEOD

in the body fat and the total HEOD administered up to

the day of biopsy and used to estimate total burden.

 

These values represent the recovery obtained at the
time of highest HEOD concentration in the fat.

0

The values are not presented because contamination had
ceased three weeks prior to this biopsy.

parent compound was excreted in the feces. The value was
determined by dividing the total amount given to each
animal by the total amount of HEOD excreted for the whole
experiment. Group PB—D had the highest.per cent-of the
dose in the feces, but group PB-C had the lowest. 0n the
average, the drug had very little effect on the fecal
excretion of HEOD. Body fat was assumed to be 10% of

the body weight. Thus, the burden of HEOD in body fat
was determined by multiplying the highest biopsy concen-
-tration of HEOD detected in the fat for each group by

10% of the body weight and dividing by the total dose

administered up to that particular biopsy. Estimations

83

of HEOD in body fat were calculated using the highest
values which occurred at six, two, six and two weeks,
respectively, for groups C-C, C-D, PB-C and PB—D. Also,
estimates were made for all groups using the second
biopsy value in order to examine the effect of a stan-
dard exposure duration for each group. Even though the
body fat levels of HEOD were the greatest at six weeks ,3
for groups C-C and PB-C, a larger per cent of the dose
could be accounted for in body fat at two weeks for
these groups.

In contrast to the high values calculated for per

 

 

cent of the dose in the feces and body fat, considerably
less HEOD was accounted for in the milk. The milk is
not a primary excretory route for HEOD in this experi-
ment. The values for milk were obtained by dividing the
total HEOD excreted in the milk by the total amount of
HEOD administered. A value of 30% body fluids which
were in equilibrium with the blood levels was assumed
for estimation of body fluid burden of HEOD. Extremely low
values were obtained showing that body fluids contribute
little as an HEOD sink.

In summary, the dispostion of the original HEOD
was primarily through feces, body fat and milk. Indi-
vidual cow variation appeared to exert a greater influ-

ence than phenobarbital treatment on HEOD distribution.

84

L. Induction of Hepatic Microsomgl Enzyme
9y Phenobarbital and Metabolism of HEOD

 

 

Since much of the total dose of HEOD in cows was un-
accounted for, and metabolism of dieldrin has been shown
to occur in other species, it is assumed that a large
portion of the remaining dose was converted to metabolites.
Even if complete information on metabolites were available,
a small portion of the dose would still be unaCCounted for
due to errors in estimation and inability to-analyze the
entire animal (e.g. hide and hair, bone marrow, etc.).

In the metabolite fraction, attempts were not-made

 

 

to quantify gas chromatographic responses for two reasons: . r
(1) at present, no metabolite standards were readily avail-
able for identification and measurement of the peaks; and,
(2) the extraction and analytical techniques were not,»
favorable for obtaining all of the original HEOD and still
extract the metabolite(s) quantitatively. Some of the
metabolites are base-labile whereas HEOD is relatively non-
labile in strong bases. Some metabolites are best extracted
at pH 2, with selected solvent systems. It is known_that
a large portion of each metabolite exists as a conjugate
of glucuronic acid; hence it is unextractable if not_
treated first with glucuronidase, then extracted with
solvents of differing polarity.

During the normal analysis of these samples for HEOD,
extra peaks appeared shortly before and after HEOD on the

chromatogram. The responses were sharp and fast, and their

55 ‘9

behavior was similar to metabolic products of DDT, but were
subsequently found different. Even so, they behaved as

‘ polychlorinated compounds which could absorb electrons.
These extraneous responses were especially noted in feces,
which suggests that the primary route of HEOD metabolite
excretion is the same as for HEOD itself. This theory
would be supported by the work of Cole (33) who noted that
50% of an HEOD dose was excreted via the bile in three days
by intact rats. Data published from our own laboratory
(37, 38, 158) also suggest that biliary excretion may
account for a significant part of the excretion of an HEOD
dose.

The use of phenobarbital has shown an increase in
drug metabolizing activity of the liver in previous eXperi-
ments. 1t was expected that it would do so in this trial.
The method employed for assaying the induction of enzyme
activity measured the end product formation of nitrogen—
demethylase on aminOpyrlne. This assay is one of several
which are routinely employed to measure activity of
hepatic microsomal mixed-function oxidases. The results
are tabulated in Table 33. There were no significant
differences among the values and no effect of phenobarbital
was detected. All values were low for untreated animals.
Storage of the tissue prior to assay for nine-ten months
after sampling may partially account for the low levels
of activity but, past experience has shown that the

activity will remain for up to six months.

86

TABLE 33.——Nltrogen demethylase activity of liver micro—

:XUIHHS .

 

 

 

Specific activity

(muM ECHO/mg pro-
tein/min.) 0.60 0.97 0.76 0.70

 

 

CHAPTER V
DISCUSSION

A major effect of phenobarbital on body fat levels
of HEOD was observed in this study. Phenobarbital not 7 g”?
only prevented HEOD concentration from reaching the level
seen in the untreated animals, but it also accelerated ;

the decline in HEOD upon withdrawal of the pesticide.

 

This effect has been noted by other workers using hepta-
barbital and DDT (16, 142). The weight gains noted for
each group could have markedly diluted the pesticide con-
centration. Since most body weight gain is as adipose
tissue a simnificant increase in the adipose pool could
have caused the dilution. The possibility exists that
newly deposited adipose tissue would contain less HEOD
due to the lack of time exposed for incorporation of HEOD
into fat. Although attempts were made to avoid sampling
from fat that had been laid down since the previous biopsy
some of this new tissue may have been removed. Alter-
natively, since HEOD is associated with the lipid frac-
tion of tissues, the combined effect of general adipose
deposition due to body weight gain and the need to re-
place fat removed at a particular site may cause newly

deposited fat to contain greater residue levels than fat

87

88

which becomes contaminated after deposition. Mobiliza—
tion and storage occur continuously, but the equilibrium
is toward storage unless the animal is under a stress
such as lactation. In addition to the above factors,
lactation must have influenced body fat levels of HEOD
in this experiment. The decline in milk production
diminishes the excretion via the milk which would shift —“
the equilibrium toward storage or excretion via the other
routes. The combination of preferential movement of HEOD

with newly deposited adipose as represented by body weight

 

 

increases and the concurrent fall in milk production could

‘Ll' .fl-f'

well have exaggerated the storage levels observed. It
was assumed old tissue was sampled. However, if new
tissue had a lower HEOD level and had been included in
the biopsy then the total HEOD in body fat as it was cal—
culated for Table 21 would be underestimated. Even so,
relative differences would still be expected to remain.
Another factor which can play a role in HEOD storage is
the effect of gestation. Placental transfer can account
for 0.9% of a 60-day dose of HEOD (15). The transfer to
the fetus or fetal fluids of almost 1% of a 60—day dose
is 10-20 times greater than the HEOD estimated to be con—
tained in blood and body fluids (Table 32). In addition,
the data of Braund (15) suggested that as gestation pro—
ceeds, the efficiency of fat uptake of HEOD may have de-

creased. Part of the weight gain noted in all groups must

89

have been due to fetal growth, yet the fetus is of neg-
ligible size until the 6-7th month of gestation. The
major effect of fetal growth would occur during the last
two months of gestation when more than half of the birth
weight is achieved.

The net deposition of fat may not reflect net
storage of a chlorinated insecticide such as dieldrin. :2
A two-compartment model within the fat cell has been pro-
posed which suggests that the primary function of one

compartment is the storage and mobilization of lipids

 

while the other compartment serves as a storage area for g
compounds such as chlorinated hydrocarbons (153). This

may explain why starvation of obese subjects has failed

to produce toxicity symptonm due to released HEOD cir-

culating in the blood at greatly elevated levels. If

the two—compartment situation does exist then the effect

phenobarbital has on decreasing the storage of HEOD in

fat must be substantial because of the difficulty in

moving the pesticide with the mobilized lipids.

The second important finding was that, contrary to
previous reports, HEOD was excreted in significant amounts
in the feces when chronically administered orally over a
period of time. An investigation of extraction tech-
niques in the present study indicated that drying of
samples prior to extraction provided the opportunity for

the pesticides to co—distill with water. Endrin has been

f) 0

shown to decompose on dried soils (4). Heptachlor
‘opoxide and dieldrin require exhaustive ultrasonic ex-
traction from dried soil particles due to the strong
adsorption of the pesticide by the soil (88, 89). The
addition of water to undried soils in order to standardize
all extractions at 20-80% moisture, depending on organic
matter content, has vastly improved recoveries and ex—
traction efficiency (139). A study of several methods

for extracting carbon-14 labelled dieldrin from soil has

been reported (136). The addition of water to the soil

 

prior to extraction improved the recovery of the pesti- L
cide. Saba gt_gl. reported that fortification of air-
dried soil should not be used for measuring the true
recovery rates (138). The addition of water to soil
samples prior to soxhlet extraction has increased the
efficiency of extraction (156). Recent studies have
demonstrated that 85% of the DDT residues in alfalfa hay
could be removed by washing with water or steam (2,3).
Wheeler has stated that extraction is the weakest part
of analysis (154). However, the increased extraction
efficiency in this investigation did not account for the
difference in fecal HEOD excretion from that reported in
past experiments.

Results in the present study support the above ex—
periments. The data in Table 16 emphasized the extensive

improvement that was obtained upon extraction of feces

91

which had not been dried. The feces in this experiment
had an average moisture content of 81%. The process of
drying this material removed “/5 of the original weight.
The fact that HEOD can co-distill with the water phase
must have accounted for a significant portion of the in-
crease in extraction efficiency. The recommended amount
of water for the extraction of soils "high in organic Era
matter” is 80%. The dry matter in feces is almost entirely
organic matter, therefore extraction of the feces in the

same condition as collected from the animal seems to be

 

a reasonable method.

In addition to the loss encountered by co—distillation
of HEOD with water during drying, it would be expected
that HEOD would be tightly bound to organic matter when
dried. in soil, the binding is of such magnitude that it
requires rigorous ultrasonic treatment in order to obtain
adequate recoveries (88, 89). The binding of HEOD to
fecal dry matter has been observed in our laboratory. A
usual method employed to check analytical efficiency is
that of adding known amounts of the compound being ana-
lysed to the unknown sample prior to extraction or at
various other stages in the analytical process. This
procedure is commonly referred to as "fortification" or

' of the sample. An aliquot of the original feces

"spiking'
sample was removed after thorough mixing and split into

five samples. One sample was untreated while another was

92

fortified but not dried. The third sample was dried only
while the fourth was fortified before drying. The fifth
sample was spiked after drying. The results of this study
showed significant differences among the methods used

(Table 3“).

 

 

TABLE 3U.-—Relative differences in extraction efficiency.
Treatment Relative recovery of HEOD
Dried Fortified Endogenous Added
- — lOO -
— + 100 100
+ — 2O -
+ before drying 2O 20
+ after drying 2O 9O

 

The differences above are only relative but they demon-
strate two points. First, the drying of feces markedly
reduces the amount of HEOD detected in the sample. Second,
the fortification of a wet sample which is subsequently
dried and used to measure extraction efficiency would lead
one to falsely assume no loss occurred even though 80%

of the original and added HEOD were not recovered. Thus,
it can be seen that HEOD was lost and/or bound when dried,
but little binding occurs once the sample has been dried.
Drying the sample prior to extraction has been used in
the past in order to provide adequate mixing and sub—

sampling. This process of obtaining a truly representative

 

93

aliquot of the whole sample is desirable in any situation.
However, if representative subsamples are obtained by
drying which substantially reduces the amount of extract-
able residues, then drying negates the accuracy initially
sought. Thus, oven drying of feed or feces samples prior
to extraction of chlorinated hydrocarbons is not recom—
mended. r—

There are factors other than improved efficiency of '
extraction that may have accounted for the excessive fecal

excretion of HEOD. Chromic oxide will bind HEOD in a

 

hexane solution in an amount equivalent to less than 0.1
percent of each daily dose, but it is possible a larger
amount was bound in the gastro—intestinal tract. Binding
of HEOD by chromic oxide may have been enhanced in the
rumen. This idea appears unlikely since fecal excre-
tion of HEOD declined with time while the chromic oxide
dose remained constant. The determination of the chromic
oxide content in feces gave recoveries of less than 100%.
The nature of the calculation of fecal dry matter output
by the chromic oxide technique is such that the lower the
recovery the greater the value will be an overestimation
of dry matter excretion. Total daily dry matter excretion
did not change appreciably during the experiment. Thus,
rate of passage was not affected sufficiently to account
for the decline in daily HEOD elimination in the feces.

Some of the variation in fecal dry matter excretion was

99

due to differences in preference for hay, grain or silage.
The variability of excretion of HEOD in the feces appeared
to be more a function of HEOD concentration than it was

of changes in percent dry matter, dry matter excretion or
rate of passage. All feces samples were analysed in
random order; therefore, any effect of time of analysis
should be distributed over the entire experiment. The

6-week means in Tables 29 and 31 suggest that HEOD excre—

.f'lL~-. II 1'98” L2. F'III

tion was inversely related to digestibility of the ration.

Because many times more HEOD has been accounted for in the

 

feces in this study than has been the case reported pre- ;
viously, the following question arises: if in fact the

dynamic movement of fecal HEOD is associated with the

particulate matter in the gut, why is there a precipitous

decrease in fecal excretion of HEOD beginning the first

day after withdrawal of HEOD from the diet? This rapid

decline would suggest that there are other effects in—

volved.

The first effect which may account for the movement
of HEOD in feces is the action of phenobarbital on biliary
flow (97, 99). Phenobarbital increased biliary flow by
50% in rats. An increase in biliary excretion of several
compounds can occur but to varying degrees. Phenobarbital
can stimulate increases in conjugation of compounds for
excretion. Thus, the combined effects of greater bile

flow and increased transport maxima due to the elevated

rate of hepatic microsomal conjugate formation are syn-
ergistic effects which result in extensive changes in the
rate of elimination of a compound. The substantial saliva
flow in a cow results in a turnover of large quantities
of water. Bile flow may also be great due to the need
for elimination of the waste products of energy utiliza-
tion, emulsification and digestion in the lower gut.
Changes in bile flow noted above can not entirely
account for the decline in HEOD excretion in the feces.
Animals not receiving phenobarbital also eliminated large
quantities of HEOD in the feces. Therefore, if changes
in biliary flow due to drug treatment did occur, this
phenomenon should be evident in the several tables pre-
sented. The cumulative percent of the dose excreted in
the feces declined steadily regardless of treatment.
This suggests that if bile was a major source of HEOD in
the feces, then it may have a saturation point for HEOD
(a transport maximum) which was reached early and was
unable to deal with the continual dosing. But, if there
was a maximum rate of transport, why was there a decline
in fecal excretion of HEOD? Perhaps the hepatic system
was able to adapt to the increased flow rates due to
phenobarbital treatment by decreasing biliary excretion
of HEOD. It is more likely that degradation of HEOD
occurred which caused increasing portions of HEOD-

derived material to be excreted.

 

In}. .I

96

In addition to the changes in bile flow which may
cause general shifts in excretion patterns, individual
variation probably played enough of a role to produce
artificial effects due to phenobarbital. That is, all
four animals in each group were treated alike during the
first three weeks. The large difference between PR-C and
PB-D must have reflected individual responses rather than
a response due to drug treatment. In spite of a lack of
a definitive phenobarbital response for excretion of
HEOD, except during the decontamination period, the indi-
vidual variation was much greater for phenobarbital—
treated cows. It can be seen in Figures 2 through 5 that
the scattering of points was much greater for the animals
receiving the drug. The untreated group had small 3-6
day cycles of excretion of HEOD whereas definitive short-
term changes were difficult to see in the drug-treated
groups. it is difficult to explain this response. Pos-
sibly this was a manifestation of the efforts of the body
to eliminate the compound. Increased stress could have
caused inconsistent responses to that stress. Adapta-
tions or compensations may have varied greatly due to
the drug stimulus. The drug probably affected many
metabolic processes.

In addition to the definitive responses in body fat
storage of HEOD due to phenobarbital and the substantial

fecal excretion regardless of treatment, the concentration

 

97

and excretion of HEOD via milk was unexpected. The pro-
duction or percent milk fat appeared to have no effect on
the relative differences observed for HEOD concentration
and excretion in the whole milk. The great initial in-
creases in concentration were not matched by later rises
in HEOD content. Again, only during decontamination did
phenobarbital have an effect on pesticide clearance. The f‘*
HEOD values for the control groups were much higher during
decontamination than were those on drug treatment. All

concentrations were above the legal tolerance for HEOD.

 

However, at no time did they reach the levels noted by a
Braund (15) in a trial with lactating cows. Perhaps
phenobarbital inhibits excretion of HEOD in the milk.
However, this seems unlikely because there was little
difference in HEOD excretion among groups.

The argument used earlier that previous extraction
techniques were not always able to obtain all the residue
present could be a pitfall for the author in milk analysis.
The method used by Braund for extraction of HEOD from
milk was different from that employed in this study. He
used an oxalatezethanolzether system with a cleanup
procedure prior to injection into the chromatograph (15).
In this study the procedure of Crosby and Archer was used.
This method involved an alkaline hydrolysis of the whole
milk (Al). Recoveries of HEOD were adequate when the

samples were fortified prior to extraction. Values were

98

obtained from milk samples which were from other experi—
ments of herds which were 10—20 times higher than the
levels in this experiment. It is possible that this tech-
nique was not satisfactory for samples obtained in this
experiment. The authors of this method developed it for
the dechlorination of DDT to DDR and it is effective for
the extraction of several chlorinated pesticides from
proteinaceous or fatty tissues. Without the use of radio—

isotopes it is difficult to be certain that the extrac-

.._.' ‘-' Ole‘f-EHLI nil:
.

tion method is entirely effective. The high excretion

 

in the feces of HEOD and its considerable storage in body
fat may explain the low levels seen in the milk. But,

it is difficult to believe that milk is not a better route
for excretion than is seen in this experiment. HEOD
should readily diffuse across the mammary membranes or be
easily transported with lipoidal material in milk.

In considering the possible effects of phenobarbital
on the various tissues no mention has been made of its
effect on the rumen microbial population. Although there
is little evidence that this drug can affect microorgan-
isms, it could be possible that the changes resulting
from drug administration on fecal excretion of HEOD may
be due in part to an alteration of the fermentation
occurring in the rumen. If this did occur it apparently
was not detrimental to the animals since their milk pro—
duction and feed intakes were not different from expected

normal values.

99

Although it is doubtful that there could have been
induction of microbial enzymes which would degrade HEOD,
the evidence exists that the chronic administration of
phenobarbital will induce hepatic enzymes capable of
pesticide degradation. The data in this experiment sug-
gests a similar action on HEOD in lactating cows. The
concentration and excretion patterns in the several tis- is»
sues indicate that either HEOD was assimilated into some
unanalysed tissue or else it was converted to forms which

were undetectable under the conditions employed for

 

analysis. it is unlikely that a substantial portion of L w
the HEOD is located in tissues which were not analyzed,
according to the work of Braund (15) and King et al. (96).
The most logical expectation would be that degradation

of HEOD was by induced hepatic—microsomal enzymes to an
extent commensurate with the unaccounted-for portion of
the dose. The effects of phenobarbital on the microsomal
enzymes of the liver are known to be significant within
two days, but the activity of these enzymes on HEOD may

be slow. These enzymes have also been shown to metabolize
HEOD in vitro (113, 11“). Therefore it is assumed that
the in vivo action of these enzymes would be at least as
great as in vitro. The best measure of this activity was
to have been the nitrogen—demethylase activity assay on
liver microsomes obtained from each of the eight cows.

However, due to an apparent loss of activity in storage,

100

the microsomal preparations showed very little activity
regardless of treatment. In fact, the values were lower
than those normally observed for fresh cow liver. This
fact indicates a Veneral loss of enzyme activity, which
is well documented for the mixed-function oxidases. Had
the activity remained elevated a correlation could have
been established between the decline in HEOD in all tis—
sues except for fat and the increased mixed-function
oxidase activity of the liver.

The low level of HEOD administered has not been
shown to have an inductive effect as noted with pheno-
barbital, but it is possible that the HEOD may have also
caused induction of other enzymes which could have de-
graded it to some extent. it is difficult to determine
the low level effects which could have occurred due to
HEOD and phenobarbital in combination. Phenobarbital may
act in a subtle way on the permeability of HEOD in various
tissues. This effect can be inferred by the body fat
data. Rather than causing the metabolism of HEOD the
phenobarbital could minimize the incorporation of the
pesticide in adipose tissue. Further, it may act to

enhance mobilization from the adipose tissue.

 

 

 

[WIAIWWSR \II

\

SUHVAEY AND CONCLUSTONS

Two groups of four cows each were contaminated for
three weeks with Hhflb at the level 0.1 mg/kg body weight
per day orally. Two of the cows in each group were con-
taminated for three more weeks, while the remaining two

animals received no HEOD. Phenobarbital was superimposed

 

on (UK) errnui<lf foui'innus for'. ix wrmdas. Uuadxrfi cormmuni-
nation and decontamination samples of feces, milk, body
fat, blood, urine and liver were taken in order to study
the metabolic fate of the pesticide. All cows were fed

a ration of hay, grain and corn silage which was balanced
to meet the requirements of maintenance and lactation.

A major finding was that from 18-53% Of the HEOD
was excreted in the feces. Phenobarbital treatment ap-
peared to have its most pronounced effect during decon-
tamination when it was able to accelerate the excretion
of HEOD over that of the untreated group. Phenobarbital
had no effect on dry matter intakes or excretion com—
pared to the control group. the concentration of the
pesticide in the fecal dry matter was highly variable
both among cows and from one day to the next. The re-

Spon e to withdrawal of the pesticide was a rapid decline

lOl

102

in fecal HHHH UHHuwHtPHtiHH and total HEOD excretion.
The decline appeared to be as much a function of with—
drawal of HHOM as of phenobarbital treatment. Pheno-
barbital—treated cows had a much greater variability of
HEOD concentration in the feces than did untreated con-

trols. independent of drug administration was the peak

 

fecal excretion of HEOD in the first week of the experi— r:
ment. Mean daily excretion ranged from 22—51 mg/cow/day
during the first week, which declined to 12-73 mg/cow/
day in the second week.
The reason that substantial fecal elimination of L‘

HEOD had not been detected previously is attributed to
the use of extraction procedures in this experiment that
maximized the recovery of the endogenous pesticide.
Former methods used as the first step a drying of the
feces which has been shown to not only cause losses via
co—distillation of the pesticide with the water being
removed but also through inextractable binding of the
compound to organic matter in the drying process.

The concentration of orally administered HEOD in
body fat was lower in phenobarbital-treated animals during
contamination than in controls. It was also lower in
those animals which were decontaminated and receiving
the drug. Maximum levels of HEOD in the body fat were
approximately the same as the level in the diet as fed,

ranging from 1.53—2.96 ppm at the highest point.

103

The level of HHOh in the milk was approximately
l/20th that in the diet as fed. A general increase in
concentration and daily excretion of HEOD was noted
during contamination. Upon decontamination, the decline
in these parameters appeared to be a function of with-

drawal of Hhflh as well as phenobarbital treatment, as

was indicated for feces. Milk fat percentage and pro- f'
duction declined slightly, presumably due to the slightly :
restricted—roughage diet, but this alteration did not L
affect the overall movement of the pesticide in the milk. é

Very low and highly variable levels of HEOD were ;

 

found in the blood. Ho HEOD was detected in the urine

of any of the experimental animals. it is assumed that
the degradation products of the pesticide will be found
in the urine as conjugates of glucuronic acid.

The formation of metabolites of HEOD was maximized
by the use of phenobarbital to induce hepatic microsomal
drug metabolizing enzymes. Although liver microsome
preparations had low enzyme activity, it is concluded
that activity was lost upon extended storage. The ap-
pearance of unidentified compounds having a behavior in
the gas chromatograph similar to chlorinated hydro-
carbon pesticides, suggests that metabolites may have
been formed in this experiment. These additional peaks
occurred primarily in the feces. They must have been
conjugated in the bile and subsequently excreted in the

feCOS.

10“

It i; numgemted that phenobarbital may be used with
(liscretion to aid in the decontamination of animals con-
t:aining chlorinated hydrocarbon pesticide residues above
‘the legal tolerance. The drug appears to be particularly

eidkvitive when ineestirw1<yf the pesticidr:iur3 ceased.

 

LITERATURE

105

CITED

 

8.

10.

ll.

LITERATURE CITED

Archer, T. h. and D. G. Crosby. 1967. Extraction
and location of selective chlorinated hydro—
carbon residues in alfalfa hay. Bull. Environ.
Contam. Toxicol. 2:191.

Archer, T. E. and D. G. Crosby. 1968. Removal of
DDT and related chlorinated hydrocarbon resi-
dues from alfalfa hay. J. Agr. Food Chem.
16:623.

Archer, T. E. and D. G. Crosby. 1969. The decon—
tamination of animal feeds. Residue Rev.
29:13.

 

Asai, R. 1.; N. w. Westlake; and F. A. Gunther. 1969.
Endrin decomposition on air—dried soils. Bull.
Environ. Contam. Toxicol. “:278.

Baldwin, M. K. and J. Robinson. 1969. Metabolism
in the rat of the photo-isomerization product
of dieldrin. Mature 22U:283.

hann, J. M.; T. J. DeCino; N. w. Earle; and Y. Sun.
1956. The fate of aldrin and dieldrin in the
animal body. J. Agr. Food Chem. 4:937.

3enson, W. H. 1969. Note on nomenclature of dieldrin
and related compounds. J. Ass. Offic. Anal.
Chem. 52:1109.

Bishop, J. L. and J. T. Huber. 1964. Secretion of
Telodrin in the milk of cows fed varying levels
of Telodrin. J. Dairy Sci. “7:552.

howden, D. M.; M. E. Clarke; and W. D. Sargent. 1968.
Influence of ration on the excretion of dieldrin
in milk of dairy cows. Pesticide Progress 6:1.

Boyd, E. M. and C. P. Chen. 1968. Lindane toxicity
and protein-deficient diet. Arch. Environ.
Health 17:156.

Boyd, E. M. and E. S. DeCastro. 1968. Protein—

deficient diet and DDT toxicity. Bull. Wld.
Hlth. Org. 38:191.

106

13.

1“.

17.

18.

107

Boyd, E. M. and C. J. Krijnen. 1969. Dietary pro-
tein and DDT toxicity. lull. Environ. Contam.
Toxicol. 9:256.

Boyd, E. M. and J. Stefec. 1969. Dietary protein
and pesticide toxicity: With particular
reference to endrin. Canad. Med. Ass.cl.
101:335.

Boyd, E. M. and F. 1. Taylor. 1969. The acute oral
toxicity of chlordane in albino rats fed for
28 days from weaning on a protein-deficient
diet. Industr. Med. 38:92. E'

Braund, D. G. 1968. Factors Affecting the Storage,
Excretion and Placental Transfer of Dieldrin
in Dairy Cattle. Ph. D. Thesis, Michigan State
University.

Braund, D. G.; L. D. Brown; and J. T. Huber. 1968.
Effect of heptabarbital on storage of dieldrin i
in rats. J. Dairy Sci. 51:982 (Abstr.).

 

Braund, D. 0.; L. D. Brown; J. T. Huber; N. C.
Leeling; and M. J. Zabik. 1968. Placental
transfer of dieldrin in dairy heifers Con-
taminated during three stages of gestation.
J. Dairy Sci. 51:116.

Braund, D. 6.; L. D. Brown; J. T. Huber; N. C.
Leeling; and M. J. Zabik. 1969. Excretion
and storage of dieldrin in dairy cows fed
thyroprotein and different levels of energy.
J. Dairy Sci. 52:172.

hraund, D. d.; R. R. Langlois; O. J. Connor; and
E. E. Moore. 1970. Feeding phenobarbital
and activated carbon to accelerate dieldrin
residue removal in a contaminated dairy herd.
J. Dairy Sci. in press.

Brooks, G. T. 1969. The metabolism of diene-
organochlorine (cyclodiene) insecticides.
Residue Rev. 27:81.

Brooks, G. T. and A. Harrison. 1963. Relation be—
tween structure, metabolism and toxicity of
the cyclodiene insecticides. Nature 198:1169.

26.

29.

30.

L2.)

3?.

108

Brooks, d. T. and A. Harrison. 1966. Metabolism
of aldrin and dihydroaldrin by houseflies
(M. Domestica L.) in vivo and by housefly
and pig liver microsomes. Life Sci.
512315.

 

Brooks, 0. T. and A. Harrison. 1969. Hydration of
HEOD (dieldrin) and the heptachlor epoxides
by microsomes from the livers of pigs and
flabbits. Bull. Environ. Contam. Toxicol.

:352.

brooks, d. T.; A. Harrison; and S. E. Lewis. 1969. -
Cyclodiene epoxide hydration by microsomes
from mammalian liver and houseflies. Biochem.
Pharmacol. 18:557.

Brown, V. K. H.; C. 0. Hunter; and A. Richardson.

 

 

1969. A blood test diagnostic of exposure to }
aldrin and dieldrin. Brit. J. lndustr. Med. i
21:283. 2

E

Brown, V. K. H.; J. Robinson; and A. Richardson.
1967. Preliminary studies on the acute and
subacute toxicities of a photoisomerization
product of HEOD. Fd. Cosmet. Toxicol. 5:771.

Bruce, W. N.; R. P. Link; and G. C. Decker. 1965.
Storage of heptachlor epoxide in the body fat
and its excretion in milk of dairy cows fed
heptachlor in their diets. J. Agr. Food Chem.
13:63.

Buck, W. R. and W. van Note. 1968. Aldrin poisoning
resulting in dieldrin residues in meat and
milk. J. Am. Vet. Med. Assoc. 15321972.

Burns, J. J. 1969. Interaction of environmental
agents and drugs. Environ. Res. 2:352.

Casida, J. E. 1969. Radiotracer studies on metab-
olism, degradation and mode of action of
insecticide chemicals. Residue Rev. 25 lug.

Chadwick, R. W. 1967. The Acceleration of Dieldrin
Metabolism by DDT and Other Hepatic Microsomal
Enzyme Inducing Agents. Ph. D. Thesis, Utah
State University.

Christensen, H. E. 196“. The distribution of aldrin
and dieldrin in the tissues and body fluids of
experimental animals. D.S.I.H. Thesis, Uni—
versity of Cincinnati.

\"x—
L‘.
a

3M.

Mb.

91.

NH.

109

Colii, J. I“. 11168. lhidriji arki Dielihain: A (hnnpard;u)n
of Hepatic Excretion in the Hat. D.S.E.H. Thesis,
University of Cincinnati.

Conney, A. H. 1965. Enzyme induction and drug
toxicity. Symposium on drugs and enzymes,
Second lnternat. Pharmacol. Meeting, Prague,
fergamon Press, New York. 9:277.

Conney, A. H. 1967. Pharmacological implications
of microsomal enzyme induction. Pharmacol. Rev.
19:317.

Conney, A. H.; R. M. Welch; R. Kuntzman; and J. J.
Rurns. 1967. Effects of pesticides on drug
and steroid metabolism. Clin. Pharmacol.
Ther. 8:2.

Cook, R. M. 1970. Metabolism of Xenobiotics in
Ruminants. Dieldrin recycling from the blood
to the gastrointestinal tract. J. Agr. Food
Chem. in press.

Cook, R. M; J. W. Thomas; R. S. Emery; and J. T.
Huber. 1967. Dieldrin recycling from the
blood to the gut in ruminants. J. Animal Sci.
26:1“86 (Abstr.)

Cook, R. M. and K. A. Wilson. 1970. Metabolism of
Xenobiotics in Ruminants. Phenobarbital in—
duction of liver microsomal nitrogen—demethylase.
J. Agr. Food Chem. in press.

Crosby, D. C. 1969. The non-metabolic decomposition
of pesticides. Ann. N. Y. Acad. Sci. 160:82.

Crosby, D. C. and T. E. Archer. 1966. A rapid
analytical method for persistent pesticides in
proteinaceous samples. Bull. Environ. Contam.
Toxicol. 1:16.

Crosby, D. H.; T. E. Archer; and R. C. Laben. 1967.
DDT contamination in milk following a single
feeding exposure. J. Dairy Sci. 50:90.

Cueto, C.,Jr. and W. J. Hayes, Jr. 1962. The detec-
tion of dieldrin metabolites in human urine.
J. Agr. Food Chem. 10:366.

Curley, A. and R. Kimbrough. 1969. Chlorinated
hydrocarbon insecticides in plasma and milk of
pregnant and lactating women. Arch. Environ.
Health 18:156.

91.

96.

97.

98.

110

(Izmii'nciclai , .J. ; I . Ii. {lil)brilxl; ZIH(1 IQ. \/. ldvzirut. 1‘)(il.
'fh(\riet(unnirviticni of‘ chimunic (inth> irn sanniles
of feed and excreta by acid digestion and
spectrophotometry. Canad. J. Anim. Sci.
91:167.

Damico, J. N.; J. T. Chen; C. E. Costello, and E. O.
Haenni. 1968. Structure of Klein's metabolites
of aldrin and dieldrin. J. Ass. offic. Anal.
Chem. 51:98.

Datta, P. H.; E. P. Laug; J. 0. Watts; A. K. Klein;
and M. J. Nelson. 1965. Metabolites in urine
of rats on diets containing aldrin or dieldrin.
Nature 208:289.

Davidow, R. and J. L. Radomski. 1953. Isolation of
an epoxide metabolite from fat tissues of dogs
fed heptachlor. J. Pharmacol. Exp. Ther.
107:259.

Davies, J. E.; W. F. Edmundson; C. H. Carter; and
A. Rarquet. 1969. Effect of anticonvulsant
drugs on dicophane (DDT) residues in man.
The Lancet, ii. p.7.

Deichmann, W. H.; i. Dressler; M. Keplinger; and
W. E. MacDonald. 1968. Retention of dieldrin
in blood, liver and fat of rats fed dieldrin
for six months. Indus tr. Med. Surg. 37:837.

DeMott, H. J.; J. T. Miles; S. A. Hinton; and L. J.
Hardin. 1966. Secretion of heptachlor
epoxide in milk. J. Dairy Sci. 99:1995.

borough, H. W.; R. B. Davis; and G. W. Ivie. 1970.
Fate of Temik-Carbon—19 in lactating cows
during a 19-day feeding period. J. Agr. Food
Chem. 18:135.

Durham, W. F. 1967. The interaction of pesticides
with other factors. Residue Rev. 18:21.

Feil, V. J.; R. D. Hedde; . . My Hkie and C. H.
Zachrison. 1970. Dieldrin- C Metabolie m in
Clheep. Identification of trans-6 ,7- dihy—
droxydihydroaldrin and 9- (_y_—epoxy) hydroxy-

1,2,3, ,10, 10— hexachloro— 6 ,7— —epoxy— —1 ,9, 3
6, 7 8,8a- octahydro- l ,9- endo- 5, 8- -exo— -dimethano-
naphthalene. J. Agr. Food Chem. 18: 120.

 

5‘.) .

61.

63.

69.

C\
C7

111

Wouts, J. R. 1963. Factors influencing the metabo—
lism of drugs in liver microsomes. Ann. N. Y.
Acad. Sci. 109:875.

Fries, G. F. 1970. Organochlorine pesticides and
the dairy industry. J. Dairy Sci. 53:367.

Erie , G. F.; A. M. Hartman; and L. P. Dryden. 1969.
The effect of hexobarbital on the retention
of DDT analogues by the rat. Bull. Environ.
Contam. Toxicol. 9:320.

Storage of dieldrin in tissues and its excre—
tion in milk of dairy cows fed dieldrin in 1
their diets. J. Agr. Food Chem. 7:829. '

Gannon, N.; R. P. Link; and G. C. Decker. 1959. W

Gannon, N.; R. P. Link; and G. C. Decker. 1959.
Storage of dieldrin in tissues of steers, hogs, :
lambs, and poultry fed dieldrin in their diets. ;
J. Agr. Food Chem. 7:826. '

 

Gannon, N.; R. P. Link; and G. C. Decker. 1959.
Insecticide residues in the milk of dairy cows
fed insecticides in their daily ration. J.
Agr. Food Chem. 7:829.

Gillett, J. W. 1968. "No effect" level of DDT in
induction of microsomal epoxidation. J. Agr.
Food Chem. 16:295.

Gillett, J. W. 1969. Microsomal epoxidation: Effect
of age and duration of exposure to dietary
DDT on induction. Bull. Environ. Contam.
Toxicol. 9:159.

Gillett, J. W. and T. M. Chan. 1968. Cyclodiene
insecticides as inducers, substrates, and in—
hibitors of microsomal epoxidation. J. Agr.
Food Chem. 16:590.

Gillett, J. W.; T. M. Chan; and L. C. Terriere.
1966. interactions between DDT analogs and
microsomal epoxidase systems. J. Agr. Food
Chem. 19:590.

Gutenmann, W. H. and D. J. Lisk. 1967. Metabolism
of TDK herbicide in the dairy cow. J. Dairy
Sci. 5021516.

66.

67.

70.

71.

75-

7M.

75-

76.

112

Gutenmann, W. H. and D. J. Lisk. 1970. Metabolism
and excretion of Bromacil in milk of dairy
cows. J. Agr. Food Chem. 18:128.

Hart, L. H. and J. R. Fouts. 1963. Effects of acute
and chronic DDT administration on hepatic
microsomal drug metabolism in the rat. Proc.
Soc. Exper. Biol. Med. 11H:388.

Hathway, D. E. 1965. The biochemistry of dieldrin
and Telodrin. Arch. Environ. Health 11:380.

Hathway, D. E.; J. A. Moss; J. A. Rose; and D. J. M.
Williams. 1967. Transport of dieldrin from
mother to blastocyst and from mother to foetus
in pregnant rabbits. Eur. J. Pharmacol. 1:167.

Hayes, W. J., Jr. 1965. Review of the metabolism
of chlorinated hydrocarbon insecticides espe-
cially in mammals. Ann. Rev. Pharmacol. 5:27.

Hayes, W. J., Jr. and A. Curley. 1968. Storage and
excretion of dieldrin and related compounds.
Effect of occupational exposure. Arch. Environ.
Health. 16:155.

Hayes, W. J., Jr.; F. F. Ferguson; and J. S. Cass.
1851. The toxicology of dieldrin and its
bearing on field use of the compound. Am. J.
Trop. Med. 31:519.

Hayes, W. J., Jr.; G. E. Quinby; K. C. Walker; J. W.
Elliott; and W. M. Upholt. 1958. Storage of
DDT and DDE in people with different degrees
of eXposure to DDT. Arch. 1nd. Health 18:398.

Heath, D. F. and M. Vandekar. 196A. Toxicity and
metabolism of dieldrin in rats. Brit. J.
Industr. Med. 21:269.

Hedde, R. D.; K. L. Davison; and J. D. Robbins. 1970.
Dieldrin-luC Metabolism in Sheep. Distribution
and isolation of urinary metabolites. J. Agr.
Food Chem. 18:116.

Henderson, G. L. and D. G. Crosby. 1966. The photo-
decomposition of cyclodiene insecticides. ACS
Second Western Regional Meeting. San Francisco,
October 16.

77..

78.

79.

80.

83.

8A.

85.

86.

87.

113

Henderson, 0. L. and D. G. Crosby. 1967. Photo-
decomposition of dieldrin and aldrin. J. Agr.
Focxi Ctmnn. lf3z888.

Henderson, J. L. 1965. Insecticide residues in milk
and dairy products. Residue Rev. 8:74.

Hironaka, R. 1968. Elimination of dieldrin from
beef cattle. Can. Vet. J. 9:167.

Hoffman, W. S.; H. Adler; W. I. Fishbein; and F. C.
Bauer. 1967. Relation of pesticide concentra—
tions in fat to pathological changes in tissues.
Arch. Environ. Health 15:758.

Huber, J. T. and J. L. Bishop. 1962. Secretion of
heptachlor epoxide in the milk of cows fed
field—cured hay from soils treated with hepta-
chlor. J. Dairy Sci. A5:79.

Hunter, C. G. and J. Robinson. 1967. Pharmaco—
dynamics of dieldrin (HEOD). I. Ingestion by
human subjects for 18 months. Arch. Environ.
Health 15:614.

Hunter, C. C. and J. Robinson. 1968. Aldrin,
dieldrin and man. Fd. Cosmet. Toxicol. 6:253.

Hunter, C. C.; J. Robinson; and K. W. Jager. 1967.
Aldrin and dieldrin-—the safety of present ex-
posures of the general population of the United
Kingdom and the United States. Fd. Cosmet.
Toxicol. 5:781.

Hunter, C. C.; J. Robinson; and M. Roberts. 1969.
Ingestion by human subjects for 18 to 2A
months, and postexposure for eight months.
Arch. Environ. Health 18:12.

Hyams, D. E.; S. M. Sabesin; N. J. Greenberger; and
K. J. Isselbacher. 1966. Inhibitor of intes-
tinal protein synthesis and lipid transport by
ethionine. Biochim. Biophys. Acta 125:166.

Ivey, M. C.; H. V. Claborn; H. D. Mann; R. D. Radeleff;
and G. T. Woodard. 1961. Aldrin and dieldrin
content of body tissues of livestock receiving
aldrin in their diet. J. Agr. Food Chem. 9:37U.

88.

()(’

91.

93.

UN.

()6.

‘18.

11A

Johnsen, R. E. and R. 1. Starr. 1967. Ultrasonic

JODHJG

Kruii:s,

Keanie,

Keane,

Kearus,

extraction of insecticides in soil. 1. Com—

parison of extraction methods and solvent sys-
tems over three time intervals. J. Econ. Ent.
60:1679.

n, R. E. and R. 1. Starr. 1970. Ultrasonic
extraction of insecticides in soil. II. Re-
finement of the technique. J. Econ. Ent.
63:165.

V. W. and O. J. Jonasson. 1965. The detec-
tion and persistence of chlorinated insecti—
cides in human and animal blood. Canad. J.
Public Health 56:U33.

W. T. and M. R. Zavon. 1969. Validity of a
critical blood level for prevention of dieldrin
intoxication. Arch. Environ. Health 19:36.

W. T. and M. R. Zavon. 1969. The total body
burden of dieldrin. Bull. Environ. Contam.
Toxicol. “:1.

W. T.; M. R. Zavon; and S. H. Withrup. 1969.
Dieldrin poisoning in dogs: relation to
obesity and treatment. Brit. J. Industr.

Med. 26:338.

Kimbrough, R. D.; T. B. Gaines; and W. J. Hayes.

1968. Combined effect of DDT, pyrethrum and
piperonyl butoxide on rat liver. Arch. Environ.
Health 16:333.

Kimura, F. T. and V. L. Miller. 1957. Improved deter-

K i ”If. a

Klaass

Klaass

mination of chromic oxide in cow feed and feces.
J. Agr. Food Chem. 5:216.

R. L.; A. Weisshaar; R. W. Hemken; J. W. Lester;
and E. Heinle. 1967. Intake and retention of
heptachlor epoxide by dairy animals during non—
lactating periods. J. Dairy Sci. 50:991
(Abstr.)

en, C. D. 1969. Biliary flow after microsomal
enzyme induction. J. Pharmacol. Exp. Ther.
168:218.

en, C. D. 1970. Effects of phenobarbital (PB)
on the plasma disappearance (PD) and biliary
excretion (BE) of drugs in rats. Fed. Proc.
29(2):5u3 (Abstr.)

99.

100.

101.

102.

1.06 .

107.

115

Klaassen, C. D. and G. L. Plaa. 1968. Studies on
the mechanism of phenobarbital-enhanced sulfo-
bromophthalein disappearance. J. Pharmacol.
Exper. Ther. 161:361.

Klein, A. K.; J. D. Link; and N. F. Ives. 1968.
Isolation and purification of metabolites
found in the urine of male rats fed aldrin and
dieldrin. J. Ass. offic. Anal. Chem. 51:895.

Korte, F. and H. Arent. 1965. Metabolism of In-
secticides. IX. (1) Isolation and identifi-
cation of dieldrin metabolites from urine of
rabbits after oral administration of dieldrin-
luc. Life Sci. “:2017.

Kunze, F. M. and E. P. Laug. 1953. Toxicants in
tissues of rats on diets containing dieldrin,
aldrin, endrin and isodrin. Fed. Proc.
122339 (Abstr.)

Laben, R. C. 1968. DDT contamination of feed and
residues in milk. J. Animal Sci. 27:16U3.

Laben, R. C.; T. E. Archer; D. G. Crosby; and S. A.
Peoples. 1965. Lactational output of DDT
fed prepartum to dairy cattle. J. Dairy Sci.
A8z701.

Lichtenstein, E. P.; K. R. Schulz; T. W. Fuhremann;
and T. T. Liang. 1970. Degradation of aldrin
and heptachlor in field soils during a ten-
year period. Translocation into crops. J.
Agr. Food Chem. 18:100.

Liska, B. J. and W. J. Stadelman. 1969. Acceler—
ated removal of pesticides from domestic
animals. Residue Rev. 29:51.

Lowry, 0. H.; N. J. Rosebrough; A. L. Farr; and R. J.
Randall. 1951. Protein measurement with the
Folin—phenol reagent. J. Biol. Chem. 193:265.

Ludwig, G. and F. Korte. 1965. Metabolism of
Insecticides. X.(l) Detection of dieldrin
metabolite by GLC—analysis. Life Sci. A:2027.

Ludwig, C.; J. Weis; and F. Korte. 196“. Excretion
and distribution of aldrin—18C and its metabo—
lites after oral administration for a long
period of time. Life Sci. 3:123.

 

110.

111.

115.

116.

117.

118.

119.

120.

116

Lykken, L. and J. E. Casida. 1969. Metabolism of
organic insecticide chemicals. Canad. Med.
Ass. J. 100:1U5.

Matsumura, F. and G. M. Boush. 1967. Dieldrin:
degradation by soil microorganisms. Science
156:959.

Matsumura, F.; G. M. Boush; and A. Tai. 1968.
Breakdown of dieldrin in the soil by a micro-
organism. Nature 219:965.

Matthews, H. 6., Jr. 1968. Dieldrin Metabolism in
Mammals. Ph. D. Thesis, University of Wisconsin.

Matthews, H. B., Jr. and F. Matsumura. 1969.
Metabolic fate of dieldrin in the rat. J.
Agr. Food Chem. 17:8“5.

Miller, D. D. 1967. Effect of thyroprotein and a
low—energy ration on removal of DDT from
lactating dairy cows. J. Dairy Sci. 50:1UUU.

Morgan, D. F. and C. C. Roan. 1970. Chlorinated
hydrocarbon pesticide residue in human tissues.
Arch. Environ. Health 20:A52.

Morsdorf, K.; G. Ludwig; J. Vogel; and F. Korte.
1963. The excretion of aldrin-C1 and dieldrin-

C1“ and their metabolites through the bile.
Med. Exp. 8:90.

Moss, J. A. and D. E. Hathway. 196A. Transport of
organic compounds in the mammal. Partition of
dieldrin and Telodrin between the cellular
components and soluble proteins of blood.
Biochem. J. 91:38A.

Moubry, R. J.; G. R. Myrdahl; and H. E. Halliday.
1968. Dieldrin residue in an orchard—dairy
area of Wisconsin. Festic. Monit. J. 2:51.

Moubry, R. J.; G. R. Myrdahl; and W. E. Lyle. 1968.
Investigation to determine the respective
residue amounts of DDT and its analogues in
the milk and back fat of selected dairy animals.
Festic. Monit. J. 2:“7.

Moubry, R. J.; G. R. Myrdahl; and A. Sturges. 1968.
Rate of decline of chlorinated hydrocarbon
pesticides in dairy milk. Pestic. Monit. J.
2:72.

12A.

197.

13].

132.

117

Nash, T. 1953. The colorimetric estimation of
formaldehyde by means of the Hantzsch reaction.
biochem. J. 55:U16.

O'Brien, R. D. 1967. Insecticides. Action and
Metabolism. Academic Press, New York.

 

 

O'brien, R. D. 1967. Effects of induction by pheno-
barbital upon susceptibility of mice to insecti—
cides. Bull. Environ. Contam. Toxicol. 2:163.

O'Brien, R. D. and C. E. Dannelley. 1965. Penetra-
tion of insecticides through rat skin. J. Agr.
Fo<nl Ginsu. 13:2?“5.

Park, K. S. and W. N. Bruce. 1968. The determina—
tion of the water solubility of aldrin, diel—
drin, heptachlor and heptachlor epoxide. J.
Econ. Ent. 61:770.

Peakall, D. H. 1970. Pesticides and the reproduc-
tion rf birds. Scientific American, April,
rp- 73—78.

Richardson, A.; M. K. Baldwin; and J. Robinson. 1968.
Metabolites of dieldrin (HEOD) in the urine
and faeces of rats. Chem. Ind. 588-587.

Richardson, A.; M. Baldwin; and J. Robinson. 1968.
Identification of metabolites of dieldrin (HEOD)
in the faeces and urine of rats. J. Sci. Fd.
Agric. 19:52A.

Richarson, L. A.; J. R. Lane; W. S. Gardner; J. T.
Teeler; and J. E. Campbell. 1967. Relation—
ship of dietary intake to concentration of
dieldrin and endrin in dogs. Bull. Environ.
Contam. Toxicol. 2:207.

Robinson, J. 1969. The burden of chlorinated hydro-

carbon pesticides in man. Canad. Med. ass.
{-10 lO(—):1800

Robinson, J. 1970. Persistent pesticides. Ann.
Rev. Fharmacol. in press.

Robinson, J.; A. Richardson; B. Bush; and K. E.
Elgar. 1966. A photo-isomerization product
of dieldrin. bull. Environ. Contam. Toxicol.
1:127.

 

138.

1110.

181.

182.

118

Robinson, J.; M. loberts; M. Baldwin; and A. I. T.
Walker. 1969. The pharmacokinetics of HEOD
(dieldrin) in the rat. Fd. Cosmet. Toxicol.
7:317-

Rosen, J. D. and D. J. Sutherland. 1967. The nature
and toxicity of the photoconversion products of
aldrin. Bull. Environ. Contam. Toxicol. 2:1.

Saha, J. G. 1968. Comparison of several methods for
extracting dieldrin-C18 from soil. Bull.
Environ. Contam. Toxicol. 3:26.

Hana, J. G. 1969. Significance of organochlorine
insecticide residues in fresh plants as pos-
sible contaminants of milk and beef products.
Residue Rev. 26:89.

Saba, J. G.; B. Bhavaraju; and Y. W. Lee. 1969.
Validity of using soil fortification with
dieldrin to measure solvent extraction effi-
ciency. J. Agr. Food Chem. 17:87U.

 

 

Saba, J. C.; H. Bhavaraju; Y. W. Lee; and R. L.
Randell. 1969. Factors affecting extraction
of dieldrin-18C from soil. J. Agr. Food Chem.
17:877.

Schafer, M. L. 1968. Pesticides in blood. Residue
Rev. 2A:19.

Soto, A. R. and W. B. Deichmann. 1967. Major metab—
olism and acute toxicity of aldrin, dieldrin
and endrin. Environ. Res. 1:307.

Street, J. C. 196A. DDT antagonism to dieldrin
storage in adipose tissue of rats. Science
1A6:1580.

Street, J. C. 1969. Organochlorine insecticides
and the stimulation of liver microsomal enzymes.
Ann. N. Y. Acad. Sci. 160:274.

Street, J. C. 1969. Methods of removal of pesticide
residues. Canad. Med. Ass. J. 100:15A.

Street, J. C. and A. D. Blau. 1966. Insecticide
interactions affecting residue accumulation in
animal tissues. Toxicol. Appl. Pharmacol. 8:“97.

ll”).

18”.

1‘“).

119

Street, J. C.; F. L. Mayer; and D. J. Wafistaff.
inJ. Ecological significance of pesticide
interiutions. lndustr. Med. 38:91.

Stuil, J. W.; W. H. Brown; F. M. Whiting; L. M.
Sullivan; M. Mulbroth; and J. M. Witt. 1968.
Secretion of DDT by lactating cows fed thyro-
protein. J. Dairy Sci. 51:56.

Stull, J. W.; W. H. Brown; F. M. Whiting; and J. F.
Witt. 1968. An epidemiological study of
pesticide contamination in milk on an Operating
dairy farm. iull. Environ. Contam. Toxicol.
3:135.

Tinsley, I. J. and R. R. Claeys. 1970. Influence
of dietary methionine on metabolism of DDT in
the rat. J. Agr. Food Chem. 18:107.

Unpublished information.

U. S. Department of Health, Education and Welfare.
1969. Report of the Secretary's Commission on
Pesticides and Their Relationship to Environ-
mental Health. farts I and II.

Ware, 0. W.; B. J. Estersen; and W. P. Cahill. 1968.
An ecological study of DDT residues in Arizona
soils and alfalfa. Pestic. Monit. J. 2:129.

Wertheimer, H. E. 1965. Handbook of Physiology.
Section 5. Adipose Tissue. American Physio—
logical Society, Washington, D. C.

 

Wheeler, W. H.; D. E. H. Frear; R. O. Mumma; R. H.
Hamilton; and R. C. Cotner. 1967. Quantita-
tive extraction of root-absorbed dieldrin from
the aerial parts of forage crops. J. Agr.
Food Chem. 15:227.

Wiheelagr', i]. i%.; D. E. H. Frwear°; ii. 0. Mtunnui; R. H.
Hamilton; and R. C. Cotner. 1967. Absorption
and translocation of dieldrin by forage crOps.
J. Agr. Food Chem. 15:231.

Williams, 1. H. 1968. Note on the effect of water
on soxhlet extraction of some organochlorine
insecticides from soil and comparison of this
method with three others. J. Ass. offic. Anal.

Chem. 51:715.

157.

l60.

161.

1 if) L" .

lei.

120

Williams, 8.; f. A. Mills; and R. E. McDowell. 196A.
Residues in milk or cows fed rations containing
low concentrations of five chlorinated hydro-
carbon pesticides. J. Ass. offic. Anal. Chem.
“7:112“.

Wilson, K. A. and R. M. Cook. 1970. Metabolism of
Xenobiotics in Ruminants. Use of activated
carbon as an antidote for pesticide poisoning
in ruminants. J. Agr. Food Chem. in press.

Wilson, K. A.; R. M. Cook; and R. S. Emery. 1968.
Effects of charcoal feeding on dieldrin excre—
tion in ruminants. Fed. Proc. 27:558 (Abstr.)

Winterinvham, H. P. W. and J. M. Barnes. 1955.
Comparative response of insects and mammals
to certain halogenated hydrocarbons used as
insecticides. Physiol. Rev. 35:701.

Witt, J. M.; W. H. Brown; G. 1. Shaw; L. S. Maynard;
L. H. Sullivan; R. M. Whiting; and J. W. Stull.
1966. Rate of transfer of DDT from the blood
compartment. Bull. Environ. Contam. Toxicol.
1:1H7.

‘—(

Witt, t. M.; R. M. Whiting; W. H. Brown; and J. W.
Stull. lfiflb. Contamination of milk from dif—
ferent rwutes of animal exposure to DDT. J.
Dairy Sci. U9:370.

Rook, B. J. anl R. M. Cook. 1968. Comparative drug
metabolism in five species. J. Dairy Sci.
51:()81 (I/F\‘LL.’L3tro )0

APPENDIX

121

122

TABLE A1-—Daily milk production.

 

 

 

 

 

 

c—c C-D PB—C PB—D
Day
771 833 820 832 799 991 819 837
1 6.49 23.15 15 35 22 93 23.38 15.44 14.67 18.84
2 4.77 19.98 13 62 18.84 2 .34 14.53 15.21 19.52
3 6.81 22 02 14.76 20.66 22.47 15.44 14.53 19.75
4 7.04 22.02 13.85 21.11 22.25 16.80 12.49 22.02
5 6.36 22.25 14.98 21.34 22.93 16.12 13.17 20.20
6 5.90 21.34 14.98 22.47 20.88 17.93 11.58 19.07
7 6.13 22.93 13.85 23.84 21.57 17.25 9.31 21.57
8 5.90 22.25 13.62 23.61 20.20 14.07 9.76 19.75
9 5 90 22.70 13.62 23.61 19.07 14.30 9.53 18.84
10 5.90 22.47 13.85 24.06 18.84 12.26 8.85 19.07
11 5.90 23.15 14.07 22.93 16.57 12.71 9.31 17.71
12 6.13 21.11 13.62 22 70 15.89 12.49 9.76 16.80
13 5.22 20.43 13.17 22.47 17.48 13.39 9.76 19.07
14 6.34 19.30 14.30 22.25 16.12 13.17 8.63 19.30
15 5.22 17.71 2 71 21.34 16.34 13 17 8.85 17.25
16 5.68 20.20 12.26 19.75 15.66 13.17 8.63 17.93
17 5.45 22. 2 12 71 21.11 17.25 14.53 6.58 19.30
18 5.90 21.57 10 67 19.98 17.48 14.30 9.99 18.84
19 4.77 22.47 12 49 2 .20 19.52 14.53 7.04 18.61
20 5.68 23 15 11.80 21.11 19.52 14.98 9.08 20.20
:1 5.2: 22.02 11.35 22.47 19.75 14.07 6.81 18.61
s 4.54 21.79 2.49 18.84 18.39 13.17 8.85 18.84
23 4.77 22.25 11.58 22.93 17.48 12.71 6.36 13.93
24 4.99 22.25 11.35 22.70 18.16 11 58 5.68 17.93
25 4.54 22.93 11.58 21.57 18.84 10.22 7.04 14.30
26 3.41 22.25 11.35 23.61 19.30 7.04 6.36 15.89
27 4.31 2 .34 10.22 21.11 19.30 4.99 7.04 17.03
28 4. 7 21.79 10.22 22.70 21.34 6.58 7.95 18.16
29 4.09 17 71 10.22 22.02 18.39 5.45 6.36 16.57
30 4.54 21.11 9.53 22.25 18.84 7.72 6.13 17.93
31 4.09 19.75 9.99 21.57 19.52 9.53 6.13 17.03
32 4.09 18.61 9.76 22.25 19.07 10.67 5.22 17.71
33 3.86 21.57 8.85 21.34 19.98 10.90 6.13 18.16
34 4.54 20.20 9.31 19.07 18.61 11.35 4.99 17.48
35 3.86 21.57 9.53 20.66 18.61 12.94 5.68 18.61
36 4.09 21.34 8.63 20.66 19.30 11.35 5.90 17.03
37 4.33 20.88 8.85 21.34 19.07 11.58 4.99 17.71
38 3.41 20.43 8.85 22.02 19.30 13.17 5.22 17.71
39 3.41 20.66 8.63 22.02 19.07 13.39 5.90 18.61
40 4.31 20.88 8.17 21.11 18.61 13.17 4.77 18.39
41 3.18 19 98 8.17 19.75 18.16 12.94 4.54 17.93
42 4.09 20.88 8.40 19.98 16.80 13.62 5.45 19.75
43 3.86 20.66 8.17 18.39 15.21 13.62 4.99 19.52
44 3.41 21.11 7.72 19.52 14.30 13.85 4.99 19.52
45 3.18 20.66 7.95 19.98 14.76 14.53 ‘ 5.45 19.30

 

123

TABLE A2-—Percentage milk fat.

 

PB—D

PB-C

771 833 820 832 799 991 819 837

Day

 

percentage milk fat -

.7

.2

2

.4

.O

3.3 2

3

2

23:4. 56 78

997090.“.

85206085

22221212

711q.7832

3.“ 333333

81868 32.4

4.“. 333333

39.“. 78 558

33333333

20766267

33221222

273.114.2510

33333233

11191079

uuu3uu33

02209537

33332222

30
31
32
33
34
35
36
37

5114. 35976

2 3333333

147712928

3u3uu233

2223013“.

33333333

80039171

«JUfiuh93fiH394

56217523

21221222

25111927

33333232

71986968

3“. 333333

15728829

 

129

TABLE A3——Concentration of HEOD in whole milk.

 

 

 

 

 

 

c-c C—D PB—C PB—D

Day 771 833 820 832 799 991 819 837
-------------------- Dnm - - - - ~ - - - - - — - - - - - -

1 0 0 0 0 0 0 0 0

2 0.0191 0.0630 0.0392 0 0993 0 0571 0.0669 0 0319 0.0908

3 0.0991 0.0871 0.0396 0 1126 0 0792 0.0988 0 0690 0.0552

9 0.0539 0.1199 0.0569 0 0999 0 1095 0.0856 0 0792 0.0980

5 0.0989 0.0918 0.0531 0 0359 0 0999 0.0312 0 0967 --

6 0.0807 0.0509 —— 9 0 0989 0 0591 0.0990 0 0972 0.0595

7 0.0962 0.0935 0.0508 0 0373 0 0667 0 0352 0 0973 0.0567

8 0.0611 0.0576 0.0659 0 0988 0 0678 0 0581 0 0525 0.0507

9 0.0935 0.1189 0.0990 0 0897 0 0958 0 0830 0 1093 0.1219
10 0.0998 0.1213 0.0926 0.0825 0 0892 0 0896 0 1995 0.1671
11 0.0830 0.1000. 0.0906 0 0596 0 0915 0 1050 0 1120 0.0733
12 0.0790 0.2300 0.0998 0 1990 0 0890 0 0880 0 1390 0.1780
13 0.0720 0.1360, 0.0860 0 0623 0 1000 0 1150 0 1090 0.1920
19 0.0680 0.1910 0.0970 0 0615 0 0992 0 0539 0 1352 0.1660
15 0.0956 0.1139 0.1012 0 0619 0 0707 0 1399 0 1762 0.1357
16 0.0766 0.1667 0.1262 0 0820 0 0895 0 1262 0 1131 0.1381
17 0.0629 0.1109 0.1936 0 1080 0 0525 0 1158 0 0739 0.1267
18 0.0560 0.1193 0.1053 0 1306 0 0756 0 0693 0 1225 0.1361
19 0.0802 0.1996 0.1330 0 1376 0 0802 0 0567 0 1130 0.1520
20 0.0979 0.1688 0.1277 0 1016 0 0839 0 0902 0 1599 0.1112
21 0.1012 0.1690 0.1900 0 2096 0 0680 0 1230 0 1683 0.1239
22 0.0775 0.2015 0.1390 0 1800 0 0928 0 1262 0 1015 0.1111
23 0.0760 0.1180 0.1100 0 1615 0 0830 0 0995 0 1030 0.0920
29 0.0850 0.1130 0.0870 0 1080 0 1290 0 0800 0 1990 0.0990
25 0.0910 0.1370 0.1200 0 0990 0 1990 0 1090 0 0700 0.1260
26 0.0780 0.1383 0.1020 0 0990 0 1100 0 1260 0 0760 0.0760
27 0.0960 0.2000 0.1070 0 0910 0 0860 0.1350 0 0820 0.0720
28 0.0990 —- -- 0.0770 0 0990 0 1200 0 0997 0.0699
29 0.1290 0.1320 0.0960 0.0830 0 1228 0 2820 0 0893 0.0628
30 0.1050 0.1950 0.0910 0 0870 0 0970 0 3360 0.0850 0.0699
31 0.1210 ,0.1590 0.1050 0 0770 0 1230 0 2100 0 0590 0.0552
32 0.1030 0.1630 0.0980 0 0856 0 1150 0 2099 0 0520 0.0506
33 0.1980 0.1750 0.0872 0.0770 0 1270 0 1370 0 0590 0.0550
39 0.1370 0.1860 0.1080 0 0700 0 1080 0 0970 0 0990 0.0390
35 0.1060 0.2960 0.0790 0.0796 0 1800 0 1099 0.0560 0.0960
36 0 1600 0.1798 0.1260 0 0700 0 1216 0 1132 0 0980 0.0919
37 0 1058 0.1968 0.0810 0 0708 0 1308 0 1972 0 0386 0.0396
38 0 0965 0.1705 0.0612 0 0999 0 1525 0.0785 0 0298 0.0372
39 0 0835 0.2010 0 0790 0 0396 0 1535 0.0590 0 0530 0.0932
90 0 0970 0.2155 0.0792 0 0592 0 1925 0.0965 0 0918 0.0952
91 0 1016 0.1918 0.0360 0 0690 0 1221 0.0623 0 0980 0.0280
92 0 1306 0.1379 0.0593 0 0222 0 0689 0.0622 0 0932 0.0396
93 0 1572 0.0796 0.0393 0 0295 0 0662 0.0800 0 0297 0.0390
99 0 0662 0.1105 0.0978 0 0298 0 0712 0.0662 0 0209 0.0598
95 0 1126 0.1327 0.0910 0 0289 0 0921 0.0659 0 0375 0.0385
96 0 0585 0.1310 0.0960 0 0960 0 1300 0.0880 0 0300 0.0360

 

O = no HEOD detected

—— = sample was not analysed for HEOD

125

TABLE A9--Fecal dry matter excretion.

 

 

 

 

 

 

C-C C—D PB—C PB-D
Day 771 833 820 832 799 991 819 837
_________________ kg/day — — - - — - - - - - - - - -
l __ .. __ _. __ _- __ --
2 -- -- -- -- 9.69 9.36 -- --
3 11.26 6.15 8.11 10.09 6.05 9.56 11.01 5.00
9 9.67 6.95 9.21 8.19 3.65 8.83 6.97 6.61
5 10.16 5.79 7 52 6.91 6.22 6.92 10.91 12.39
6 5.69 8.60 10 33 9.98 8.09 8.53 10.77 7.89
7 6.29 6.66 7.20 7 99 6.88 7.00 7.92 9.81
8 8.92 10.65 9.32 11.17 9.50 8.95 9.99 5.29
9 7.11 9.31 19.51 8.52 5.69 6.11 6.21 7.29
10 6.90 11.25 10.33 5.69 6.96 6.93 7.06 9.51
11 8.96 2.66 11.90 7.58 3.57 9.72 8.08 9.79
12 6.21 8.99 10.88 11.39 3.98 5.79 9.83 5.90
3 6.25 9.89 11.18 8.56 9.76 6.36 7.77 8.93
19 5.53 5.85 9.98 8.06 9.69 6.91 8.95 7.10
15 6.91 7.88 11 27 8.29 3.68 6.28 8.30 19.68
16 27.39 9.57 7.99 3.26 3.56 16.51 5.39 11.95
17 6.58 8.20 11.95 3.11 6.85 9.62 6.28 9.39
16 5.63 8.71 7 95 9.97 6.52 5.33 10.12 6.98
19 9.15 3.98 8.99 12.65 3.93 6.90 10.99 7.70
20 8.29 8.56 5.85 9.35 3.39 19.31 12.89 9.37
21 6.76 8.70 10.90 6.27 5.97 5.73 10.39 5.39
22 3.85 6.92 7.39 9.96 6.69 2.21 9.65 7.85
23 9.62 6.98 7.29 6.33 9.89 8.69 8.15 5.65
29 10.09 9.87 8.62 5.83 10.03 3.71 7.27 --
25 6.52 12.65 10.11 5.89 3.37 8.01 9.99 8.99
26 6.37 8.69 7.99 6.03 5.92 3.70 8.56 6.95
27 7.52 13.20 8.87 7.65 5.25 9.56 7.38 9.93
99 -- 8.99 7.92 8.93 7.99 2,95 6,29 8,63
29 6.91 10.09 10.83 7.89 6.07 —- -- 7.90
30 5.08 8.78 11.90 6.69 5.29 3.05 5.99 9.79
31 5.97 6.91 11.67 8.67 3.39 —- 5.09 8.99
32 10.25 11.08 23.55 19.96 5.33 7.09 8.15 6.52
33 7.77 10.91 19.92 5.15 5.12 9.65 6.90 7.97
39 5.82 7.31 11.75 6.56 6.98 5.68 9.82 11.32
35 7.79 8.98 8.90 12.38 3.81 9.79 7.22 6.23
36 7.98 11.37 8.31 9.20 9.93_ 3.99 8.86 8.07
37 8.17 7.91 10.82 10.92 6.23 8.10 9.25 5.37
38 10.85 10.62 9.97 12.81 6.65 7.30 9.98 5.72
39 7.35 11.76 7.99 8.28 5.58 5.97 5.71 8.52
90 9.25 6.99 11.75 13.05 5.88 7.83 6.32 6.31
91 7.33 10.92 7.92 9.26 6.21 9.90 5.60 9.72
92 5.83 8.11 6.33 6.19 5.87 6.57 5.19 6.39
93 12.30 5.62 8.20 -- 3.38 6.03 5.83 9.50

 

126

TABLE A5--Concentration of HEOD in feces.

 

 

 

 

 

 

 

c—c 0—0 PB-C PB-D
Day 771 833 820 832 799 991 819 837
---------------- ppm in dry matter - - - - — - - - — - -
1 0 0 0 0 0 0 0 0
2 -— -- -- -- 2.002 0.386 -- --
3 0.893 1.809 0.709 0.609 9.999 1.577 5.729 1.319
9 1.232 9.093 3.899 0.908 6.139 0.805 2.259 1.682
5 11.128 1.880 0.650 5.088 2.299 1.157 5.280 0.737
6 9.133 9.079 2.672 3.285 2.091 2.961 1.369 20.197
7 1.389 1.779 3.875 1.006 3.100 16.639 3.621 9.822
8 20.730 2.205 3.398 5.565 0.969 1.716 10.220 19.585
9 2.319 0.539 0.506 0.705 1.258 9.598 3.971 2.981
10 0.550 1.952 1.809 3.828 1.969 2.296 1.996 1.363
11 9.337 10.182 1.078 3.680 1.967 0.591 9.235 0.289
12 0.666 9.553 1.261 0.998 1.030 0.361 1.389 1.175
13 0.722 1.799 0.559 1.686 1.615 3.106 5.266 0.785
19 0.973 0.762 1.536 1.939 2.066 9.369 6.539 2.630
15 0.712 0.293 0.982 0.686 2.055 1.909 3.979 1.626
16 0.182 —— 0.639 0.990 2.019 0.292 3.082 0.506
17 2.933 1.616 0.329 1.351 0.229 0.807 2.529 0.862
18 7.329 7.610 0.985 1.929 1.980 0.592 1.290 1.129
19 1.329 3.279 0.929 0.599 3.050 0.978 2.202 1.993
20 3.191 0.736 1.752 1.138 1.597 0.929 0.551 2.261
21 1.193 1.919 0.387 1.002 1.929 0.599 0.598 9.263
22 2.096 3.057 1.667 1.298 1.59 3.715 1.301 1.375
23 1.693 2.666 0.172 0.213 3.351 2.155 0.095 0.186
29 1.705 0.790 0.150 0.099 1.986 9.250 0.071 --
25 1.706 0.923 0.090 0.033 9.765 2.598 0.161 0.016
26 1.601 9.065 0.026 0 9.920 6.897 0 0.022
27 0.351 2.053 0 1.861 9.320 0
28 -- 1.991 0.661 1.392
29 2.089 0.587 2.295 --
3“ 0.715 0.955 0.572 1.602
31 0.169 0.281 1.770 --
32 6.982 0.166 1.122 0.157
33 0.902 0.185 0.672 1.009
39 1.010 2.308 0.895 0.899
35 3.163 0.926 2.805 1.089
36 1.995 1.121 2.098 1.901
37 1.297 0.713 1.791 0.223
38 1.022 1.620 2.011 0.639
39 1.989 0.801 2.279 0.807
90 1.981 1.703 1.002 1.092
91 0.863 1.260 -- 1.586
92 2.789 1.338 1.231 1.316
93 -- 1.526 2.621 0.995
O = no HEOD detected

-- = sample was not analysed for HEOD

 

TA

 

””1991119[11911191991ES