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‘vi: . . . vsr
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THESIS

This is to certify that the

} thesis entitled

FACTORS AFFECTING THE STORAGE, EXCRETION,
AND PLACENTAL TRANSFER OF DIELDRIN IN
DAIRY CATTLE

presented by

Darw in G1 1ber t Braund

has been accepted towards fulfillment
of the requirements for

L degree in JEL—

LIRPARY

MlChigJ‘l State
University

 

fir/M

/ Major professor

Date Februar 7 1968

 

0-169

 

 

 

 

 

 

 

 

 

' ABSTRACT

FACTORS AFFECTING THE STORAGE, EXCRETION,
AND PLACENTAL TRANSFER OF DIELDRIN
IN DAIRY CATTLE

by Darwin G. Braund

This study was undertaken to investigate the reten—
tion and excretion of dieldrin when fed in equal dosages
to lactating dairy cows, and to pregnant dairy heifers
during various stages of gestation.

The investigation included analyzing appropriate
samples for dieldrin content with electron—capture gas
chromatography to: (a) establish a normal dieldrin ex—
cretion curve and ascertain whether this excretion pattern
could be altered by dietary changes, (b) determine body
storage of dieldrin when fed prepartum, and the subse—
quent appearance and duration in milk following parturi—
tion, and (c) ascertain the occurrence and degree of
placental transfer, sites of storage in the fetus, and
the time required for dissipation of residues from young
contaminated in utgrg.

Varying the dietary energy level did not signifi—
cantly enhance the rate of dieldrin excretion, although
thyroprotein—feeding did decrease the time required to

reach a mean residue level of 1.0 part per million in

 

 

 

Darwin G. Braund

the milk fat. Residues in whole milk did not change when
the fat content of milk was sharply reduced by feeding
pelleted hay.

Significantly greater dieldrin storage occurred in
the body fat of pregnant dairy heifers contaminated
early rather than late in gestation, even though total
pesticide intake was essentially equal.

Once non—lactating pregnant dairy heifers were con—
taminated with dieldrin, very little was eliminated from
the body until lactation began. The pattern of residue
decline in milk fat was similar for both cows and heifers.
Concentrations dropped to about one—half their initial
levels after three to four weeks, and to one-third by
five to six weeks. At the end of 2A weeks milk still con—
tained detectable levels of dieldrin. Because of plateau—
ing, an exponential decline did not accurately describe
the dieldrin disappearance pattern in milk fat° As the
dieldrin was eliminated from the body via excretion in
the milk, residue levels in shoulder fat of both cows and
heifers declined exponentially.

Approximately 2.5% of the daily intake was excreted
as unchanged dieldrin in the feces during contamination.
Only trace amounts of the original compound were found
in the urine.

Placental transfer of dieldrin occurred in each

dam, resulting in contamination of all newborn calves.

 

   

   
    
  

licldtil ingested k

Viv fat and blood of calves «whiww we'

  
  

‘~L§:wienlined with age and at 52 weeks negligible
.3: as in omental fat were detectable in only one of

female calves.

 

    
 

EEACTORS AFFECTING THE STORAGE, EXCRETION,
AND PLACENTAL TRANSFER OF DIELDRIN
IN DAIRY CATTLE

By

Darwin Gilbert Braund

A THESIS

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

Department of Dairy

DOCTOR OF PHILOSOPHY
1968
l
l

 

 

DEDICATED TO

My wife, Carol, and my parents, whose many I

sacrifices have so meaningfully enriched my life.

ii

 

 

   
 
  

BIOGRAPHICAL SKETCH

of

Darwin G. Braund

Born at Towanda, Pennsylvania, July 22, 193“, Darwin
G. Braund was raised on a dairy farm, and was graduated
from Sayre High School where he was named Outstanding
Senior. In September, 1952, he enrolled at The Pennsyl—
vania State University and was awarded a Sears—Roebuck
Foundation Scholarship and the Allegheny County Boys'
Working Reserve Scholarship.

Following graduation with a Bachelor of Science
degree in Dairy Science, he joined The Pennsylvania State
University Dairy Extension Staff July 1, 1956, and served
four years as Dairy Specialist. In September, 1960, he
enrolled in the Dairy Department of the University of
Wisconsin and received the Master of Science degree in
Dairy Science August, 1961, under the guidance of Dr.

L. H. Schultz.

He Joined Beacon Feeds, Cayuga, New York in
September, 1961, as Dairy and Livestock Specialist, and
was named Assistant Director of Dairy and Livestock

Research January 1, 196A.

 

   
     
 
  
 

:vw~rr 1, 1964, he enrolled in the ?
g"wtchigan State University and completed the
_-3, we» 5 for the Doctor of Philosophy degree in
.Eiiwver, 1967. On December 15, 1967,he assumed the
responsibilities of Project Leader for the'Dairy Exten—
sion Program in the Department of Animal Sciences at

the University of Kentucky.

iv

 

 

ACKNOWLEDGMENTS

Because of the duration and scope of this research
project, the list is long of those whose minds and hands
have contributed to its fruition.

The author wishes to express his gratitude to his
former major professor, Dr. L. D. Brown, for encourage—
ment and counsel during the early phases of his graduate
study. He is grateful to Dr. J. T. Huber, current major
professor, for his guidance and constructive criticism
in the preparation of this thesis.

Sincere appreciation is extended to Drs. R. M.
Cook, J. L. Gill, and H. A. Tucker (Dairy), N. C. Leeling
and M. J. Zabik (Entomology), E. J. Benne (Biochemistry),
and D. A. Reinke (Physiology) for their advice concerning
experimental techniques and preparation of this manu—
script. The contributions of Drs. D. A. Ellis, S. M.
Getty, and G. R. Ruth (Veterinary Surgery and Medicine)
in performing surgery on the experimental animals are
appreciated. Technical assistance was rendered by
George Blank, Mrs. Nancy Millard, and Kim Wilson. The
feeding and management of the cattle by the Dairy Barn
staff and use of the facilities of the Meats Laboratory

is acknowledged.

 

 

  
  
  
 

  
 

s are extended to Dr. C. A. Lassiter,
xgrment of Dairy, for making available the
.. flies and animals, and for financial support through
a Gwehuate Research Assistantship and Research Instructor-
ship.
And last, but not least, deepest appreciation goes
to my wife, Carol, for her ever ready assistance, moral

support, and sacrifice throughout my Ph.D. program.

vi

 

L

DEDICATION

TABLE OF CONTENTS

BIOGRAPHICAL SKETCH

ACKNOWLEDGMENTS
LIST OF TABLES.

LIST OF FIGURES

LIST OF APPENDIX TABLES.

Chapter

I. INTRODUCTION

II. LITERATURE REVIEW

A.

Pesticide Absorption

.1:me

Dermal application
Inhalation

Intravenous administration.
Oral ingestion.

Pesticide Transport in the Mammalian

Body
1.

2.

Solubility and transport in
blood. .
Transfer from the blood.

Factors Affecting Pesticide Storage
in the Body . . . . . .

\IONU‘IJTUUI'UH

Dosage level .

Duration of dosage

Age . . .

Sex

Species

Tissue

Interaction of drugs and other
compounds . . .

Page

ii

\lmUl-l: J‘: .D‘ |—’

 

Chapter

III.

F.

G.

Biotransformation . . . . . .

1. Chemical nature . . . .
2. Identity of metabolites.

3. Relation to species or strain.

Excretion and Storage Loss

1. Respiratory loss

2. Fecal, biliary, and urinary
excretion .

3. Excretion in milk. .

A. Relation of excretion to storage

and storage loss
Placental Transfer.

Experimental Decontamination

EXPERIMENTAL PROCEDURES

A.

Experiment l——Excretion and Storage
of Dieldrin in Dairy Cows Fed Thyro—
protein and Different Levels of
Energy. . . . . . .

1. Experimental animals--Dosage and
duration of contamination

2. Dietary treatments and adjust—
ments. . . . .

3. Sample collection.

Milk .

Body fat and tissues
Feces and urine
Feed.

040693

A. Sample extraction.

a. Milk.
b. Body fat
c. Body tissue
d. Urine
e. Feed and feces
5. Column clean—up procedure for

chlorinated hydrocarbon pesticide
residue extracts

6. Electron— capture gas chromato—
graphic (ECGC) analysis.

viii

Page
21
21
23
25
26

26
28

32
3A
36
39

A9
50

 

 

Chapter

IV.

E.

Experiment 2——Storage, Excretion,
and Placental Transfer of Dieldrin
by Dairy Heifers Contaminated During
Three Stages of Gestation . .

l. Contamination of heifers

a. Contamination during 60 to 0
days prepartum

b. Contamination during 120 to
60 days prepartum

c. Contamination during 180 to
120 days prepartum. . .

2. Dietary treatments imposed during
lactation . . . .

a. Contamination during 60 to 0
days prepartum

b. Contamination during 120 to
60 days prepartum . . .

3. Sample collection

Milk .

Body fat

Blood

Feces and urine.

Feed

Body tissues from newborn
calves

"meJOCTW

A. Sample extraction, clean—up, and
analysis . .

5. Feeding contaminated vs ”clean”
milk to calves contaminated in
utero __

RESULTS

A.

Experiment l——Excretion and Storage of
Dieldrin in Dairy Cows Fed Thyroprotein
and Different Levels of Energy .

Energy intake.

Body weight changes.

Milk production .

Excretion of HEOD in milk fat
HEOD levels in body fat .

HEOD levels in tissues and body
fat of slaughtered cows .

ONU‘I .1:me

ix

Page

51
51

52
55
55

56

6O

61
63

   

 

Chapter Page
B. Experiment 2——Storage, Excretion, and
Placental Transfer of Dieldrin by
Dairy Heifers Contaminated During
Three Stages of Gestation . . . . . 78
1. Experimental treatments and body
weight changes during lactation. . 78
2. Milk and milk fat production. . . 81
3. Body storage of HEOD . . . . 82
a. Pre— and postpartum HEOD con-
centrations in external body
fat. . . . 82
b. Pre— and postpartum HEOD. con-
centration levels in blood . . 87
0. Content of HEOD in body fat
and blood at parturition. . 91
d. Distribution at partirution of
HEOD fed during gestation . . 93
A. Excretion of HEOD . . . . . . 9A
a. HEOD in colostrum milk . . 9A
b. Concentration of HEOD in milk
fat during lactation . . . . 9A
c. Excretion of HEOD during
lactation. . 98
d. Fate of HEOD found in body fat
and blood at parturition. . 102
e. Excretion of HEOD in feces and
urine . . . . . 102
5. Placental transfer of HEOD . . . 10A
a. Tissue residue levels in new—
born male calves . . . 10A
b. Age effect on HEOD concen—
trations in blood and omental
fat of female calves . . . . 108
6. Effect of feeding dams' contami—
nated milk or non— contaminated
milk to calves contaminated in
utero . . . . . . . . . 113
V. DISCUSSION . . . . . . . . . . . 115
VI. SUMMARY AND CONCLUSIONS . . . . . . . 1A2
LITERATURE CITED. . . . . . . . . . . . 1A6
APPENDIX 161

 

Table

1.

10.

11.

LIST OF TABLES

Contamination Data for Groups of Heifers
Fed Dieldrin During. Three Stages of
Gestation . . . . .

TDN Intake of Cows as Percent of the NRC
Requirement . .

Percent of HEOD Intake Excreted in Milk Fat
During the Eight-Week Contamination Period
of All Cows . . . . . . . .

HEOD Excretion Pattern for Cows Contaminated
During Lactation.

Regression Coefficients of ppm HEOD in Body
Fat on Days of Decontamination for Cows
Contaminated During Lactation

HEOD Residues in Tissues and Body Fat of
Cows Fed Thyroprotein or No Thyroprotein
After 18 Weeks Decontamination

Contamination and Ration Schedules for
Heifers.

Total Milk and Milk Fat Production 01'
Heifers During the First 2A Weeks of
Lactation . . . .

HEOD Concentrations in Body Fat of Heifers
Contaminated for 60 Days at Various Stages
of Gestation . . . . . . . . .

Orthogonal Contrast Comparisons of HEOD
Concentrations in Body Fat of Heifers.

Regression Coefficients of ppm HEOD in Body
Fat on Days of Lactation for Heifers Con—
taminated During Various Stages of
Gestation . _ . .

xi

Page

57

63

72

73

76

77

78

81

83

8A

87

 

Table

12.

13.

1A.

15.

16.

17.

18.

19.

20.

2A.

Effect of Stage of Gestation on Blood HEOD
Concentrations of Heifers . . . . . . .

Effect of Stage of Gestation on HEOD Content
in Body Fat and Blood of Heifers at
Parturition. . . . . . . .

Distribution at Parturition of HEOD Fed
to Heifers During Gestation . . . .

HEOD Levels in Colostrum Milk of Heifers
Contaminated During Three Stages of
Gestation . . . . . . . . .

Cumulative Excretion of HEOD in Milk Fat as
Affected by the Stage of Gestation During
Which Heifers Were Contaminated

Cumulative Excretion of HEOD in Milk Fat as
Affected by the Ration

Fate of HEOD Found in Body Fat and Blood of
Heifers at Calving . . . . .

Daily Excretion of HEOD in Feces and Urine of
Non- ~1actating Heifers at Midpoint of 60 Day
Contamination Period.

Daily Excretion of HEOD in Feces, Urine, and
Milk 30 Days Postpartum of Heifers Contami—
nated During the Last 60 Days of Gestation

Mean HEOD Concentrations in Body Tissues of
Male Calves Born to Contaminated Dams

Comparison of HEOD Concentrations in Body
Fat and Blood of Dams at Parturition with
Blood Levels in All Newborn Calves

Effect of Age on HEOD Concentrations in
Blood of Female Calves Born to Contaminated
Dams

Effect of Age on HEOD Concentration in
Omental Fat of Female Calves Born to Con—
taminated Dams.

Page

88

92

93

95

101

102

103

105

106

107

109

112

 

  
 
 

 

vison of HEOD Levels in Omental Fat
, - .ves Contaminated in Utero When Fed
wehs' HEOD-contaminated Milk or Non—

contaminated Milk for 12 Weeks . . . .

 

Contamination Data for Experiments 1 and 2
With Resulting HEOD Levels in Body and
Milk Fat . . . . . . . .
Body Weight Data for Experiments 1 and 2.

Relative Amounts of Estrogen Found in the

Urine of Cows at Various Stages of Pregnancy

Compared with Prepartum Contamination
Periods. . . . . . . . .

HEOD Decline in Milk Fat During Decontami—
nation for All Dietary Treatments

xiii

 
  
  

113

116
117

119

126

 

Figure

10.

LIST OF FIGURES

Experimental Design of Prepartum Contami—
nation . . . . . . . . . . . .

Body Weight Changes During Decontamination
of Cows Fed Thyroprotein and Different
Levels of Energy. . . . . .

Average Daily Milk Production During Con—
tamination and Decontamination of Cows Fed
Thyroprotein and Different Levels of
Energy . . . . . . .

Levels of HEOD in Milk Fat of Cows Contami—
nated During Lactation and Fed Thyroprotein
and Different Levels of Energy . .

Levels of HEOD in External Body Fat During
Decontamination of Cows Fed Thyroprotein
and Different Levels of Energy . .

Body Weight Changes of Heifers Contaminated
at Various Stages of Gestation and Fed
Different Rations During Lactation.

Pre- and Postpartum Levels of HEOD in Ex-
ternal Body Fat of Heifers Contaminated at
Various Stages of Gestation . . .

Pre— and Postpartum Levels of HEOD in
Blood of Heifers Contaminated at Various
Stages of Gestation.

Levels of HEOD in Milk Fat of Heifers Con—
taminated at Various Stages of Gestation.

HEOD Excreted Daily in Milk Fat of Heifers
Contaminated at Various Stages of
Gestation . . . . . .

Page

5A

67

69

71

75

80

86

90

97

100

 

 

I.

II.

III.

IV.

LIST OF APPENDIX TABLES

Data on Individual Cows Contaminated with

Dieldrin During Lactation . . . .

Data on Individual Heifers Contaminated
with Dieldrin During 60 to 0 Days Pre—
partum .

Data on Individual Heifers Contaminated
with Dieldrin During. 120 to 60 Days Pre-
partum

Data on Individual Heifers Contaminated

with Dieldrin During 180 to 120 Days Pre-

partum

Physical Characteristics of Dieldrin

XV

 

Page

162

163

16A

165
166

 

I. INTRODUCTION

Pesticide residues in foods are nothing new, but
interest in the problem by scientific investigators and
the public in general has developed only during the past
20 years (116). The main contributing factor for this
increased attention is the great number of new organic
compounds that have come into general use since 19AO.
Accompanying this use, a demand has arisen for adequate
controls to protect the consumer.

Residues in milk and dairy animals are particularly
troublesome. Presently (December 1967) the tolerance for
any pesticide, other than DDT and its metabolites, in
milk is technically zero. A zero residue level simply
means that a particular product contains less residue
than the analytical chemist is able to detect with avail—
able techniques. However, the more capable the chemist
and the better his technique, the lower the residue level
that can be detected.

In October 1966 the Advisory Committee to the Food
and Drug Administration (FDA) recommended that a toler—
ance level be established with a limit of 0.05 parts

per million (ppm) of DDT, DDD, or DDE, individually or

 

in combination, for whole milk (A0). FDA accepted this
recommendation and established the tolerance indicated
above.

A difficult situation for the dairy industry exists
because of the very widespread use of pesticides on farms,
drift problems, the pooling of milk from many dairy farms
in a milkshed, and the rapidity with which milk has to be
processed and distributed (115). In addition, analytical
techniques which can detect parts per billion (ppb) and
even less of a particular residue are becoming increasingly
available.

The persistence of chlorinated hydrocarbon pesti—
cides in tissues of dairy animals has been well established.
Once an animal is contaminated with a chlorinated hydro—
carbon pesticide, residues are found in the milk long
after intake of the chemical has ceased (8, 12, 15, 16,
20— 22, 3o, 37, 38, 51, 52, 57, 71, 83, 8A, 113, 135).
However, there is a paucity of information relative to
management practices which might effect a more rapid de-
contamination of milking cows. In addition, relatively
few data are available concerning the body storage and
elimination of chlorinated hydrocarbon pesticides fed
prepartum; or of their subsequent appearance and duration
in milk following parturition. An area of additional
interest, which has profound implications for human

health and medicine, is placental transfer of these

 

l.w~ team: and the time required er-"
.i'wesidues from young contaminated 13 gt

‘Awe purpose of the investigations described herein

wqajlo establish a normal dieldrin excretion curve and

'ascertain whether this excretion pattern could be altered

 

by dietary changes. Furthermore, dieldrin storage, ex—
cretion, and placental transfer were studied in prepartum

heifers contaminated during three stages of gestation.

 

 

II. LITERATURE REVIEW

The latest chromatographic methods for residue
analysis with their increased sensitivity give results
which are not always directly comparable with those ob—
tained by older colorimetric methods (A5). During recent
years electron—capture gas—liquid chromatography (GLC)
has emerged as the analytical method of choice for
chlorinated hydrocarbon insecticide residues (68). For
this reason pesticide residue literature obtained since
the development of electron-capture GLC is emphasized in

this thesis.

A. Pesticide Absorption

 

Chlorinated hydrocarbon pesticides can enter the
biological system through the skin, by inhalation, or in—

gestion (69, 1A3).

1. Dermal Application

 

All forms, including the dry powder of dieldrin
and aldrin are absorbed through skin. Absorption of
dieldrin through the skin was as rapid for the crystal—
line as for the solubilized material (62, 63, 1A3).

Presumably the same is true for aldrin. Although the

  

mechanism of the percutaneous absorption of these mater—
ials has not been thoroughly investigated it is probably
related to their lipid solubility (1A3). O‘Brien and
Dannelley (106) examined the penetration through rat
skin of DDT, famphur, carbaryl, malathion, and dieldrin
using radioactive compounds. The penetration rates varied
markedly, increasing in the above order. Moreover, the
rate was not related to the olive oil—water partition
coefficient. However, the vehicles in which the com—
pounds were applied did influence penetration rate, be—
ing slowest for corn oil, then benzene, and fastest for

acetone.

2. Inhalation

Limited data concerning the inhalation of aldrin or
dieldrin are available. Following inhalation Of an
aldrin aerosol, some irritation of mucous membranes was
observed in guinea pigs (56). Baker and associates vapo—
rized 0.50 g of aldrin per 1000 cubic feet of air each
day and did not find any significant effect on mice, ham—
sters, guinea pigs, or parakeets (5). Whitacre and Ware
(13A) exposed chickens, rats, mice, frogs, fish, and
seven species of plants to vaporized lindane for periods
varying from 7 to A6 days. Nearly all animal and plant
tissues showed significant storage of lindane, including
egg yolks of chickens. Usually the residue in an animal

or plant and the duration of exposure were correlated.

 

Witt et al. (138) found that respiratory exposure of
dairy cows to DDT produced a lower level of DDT and its

metabolic products in milk than did alimentary exposures.

3. Intravenous Administration

 

Intravenous administration of DDT to dairy cows was
also studied by Witt et_a1. (138) who theorized that maxi-
mal response would be observed only after the oral dose
was absorbed into the blood. They found that both one—
and six—dose intravenous administrations produced much
larger amounts of DDT and its metabolites in the milk fat
than any other method tested. Levels were 162 to 525%,
respectively, of those obtained with comparable doses of
DDT given by intratracheal infusion or alimentary adminis—
tration in a corn oil solution.

Hayes (60) stated that intravenous administration of
an insecticide may not produce a true maximal response
(death); because, if administered as an emulsion of an
oil solution, the oil droplets may be physically blocked
from passage in small capillaries or taken up by phagocytic
cells. Thus, the fraction of the dose immediately avail—
able for distribution to critical tissues is unknown but
probably small. Witt et_al. (138) were unable to deter—
mine the effect of the vehicle used on absorption of an
intravenous dose of DDT but did show that 33% was ex-
creted in the milk compared to only 3 to 5% appear—
ing in milk from intratracheal infusion or alimentary

administration.

 

 

A. Oral Ingestion

 

Heath and Vandekar (67) found that very little
dieldrin remained for as long as 2A hours in the gut of
rats, but that relatively high proportions of unabsorbed
dieldrin were excreted in the feces. Heath and Vandekar
(67) suggested that absorption of dieldrin is controlled
by the amounts of certain naturally occurring materials
in the gut which enable its passage, and not by the
quantity of dieldrin present. They found that dieldrin
absorption from the gastrointestinal tract varied with
the vehicle used and the absorbed compound appeared in
the portal vein rather than in the thoracic lymph duct.

Witt et_al. (138) concluded that absorption of DDT
by dairy cows from aged-residues on alfalfa was about
twice as efficient as absorption of DDT from an oil solu-
tion. This phenomenon has also been observed by Ely
et_a1. (A6). The increased absorption of DDT from an
aged—residue on alfalfa may be due to a longer rumen ex—
posure time for the alfalfa compared to the oil. It is
not known whether pipetting the acetone solution of the
insecticide on to the grain ration as used in this study
would be more like administration in an oil solution or

as an aged—residue.

 

B. Pesticide Transport in the Mammalian Body
1. Solubility and Transport
in Blood

Moss and Hathway (102) showed that blood serum 3
constituents exerted a powerful solubilizing effect on
dieldrin and Telodrin. Solubilities of these two chlori-
nated hydrocarbon pesticides were A000 times as great in
rabbit serum as in water. These investigators presented
evidence for the binding of dieldrin and Telodrin with
the soluble proteins of the circulating blood. Ultra-
centrifugation demonstrated that radioactivity was
associated with albumin, but not with chylomicra or
lipomicra. In other experiments, DEAE—cellulose—column
chromatography separated from rabbit serum three bands
of dieldrin—lac or Telodrin—1&0 which were associated with
albumin, the a-globulins, and another protein having the
same electrophoretic mobility as albumin, respectively
(65, 102).

The percent of total dieldrin and Telodrin bound
to constituent proteins was not decreased with increased
concentrations of the insecticide. Furthermore, increased
concentrations of pesticide were not accompanied by spill—
over from one protein to another. Dieldrin and Telodrin
were located mainly in the plasma and erythrocytes of
rabbit and rat blood and not in the leucocytes, platelets

or stroma. After treatment of animals with either

 

1”C or Telodrin—14C, the distribution of 1”c

dieldrin-
between the soluble proteins of blood in vivo was identi—
cal with that in vitro. Thus, the radioactivity was due
to unchanged starting materials and not to metabolites.
The absence of metabolites for Telodrin was confirmed by

gas chromatographic studies, in which all of the label

was accounted for as Telodrin.

2. Transfer from the Blood

 

Pesticide levels in blood are difficult to interpret
because it is not known whether the blood levels are a
refleCtion of an immediately prior overt intake or of
an equilibrium with pesticides stored in adipose tissue.
The initial rate of removal of dieldrin and Telodrin from
blood was very rapid, followed by a slow, roughly log—
arithmic rate of removal (102). Witt et_al. (137) showed
that DDT in bovine blood declined rapidly and concluded
that equilibrium was nearly re—established 2A hours after
cessation of pesticide absorption into the blood, al-
though it is felt that their data did not cover a suf—
ficient period of time (2A hr) to firmly establish the
equilibrium point. Cook §£_El: (28) injected dieldrin
into the jugular vein of sheep and goats at a level of
1.2 mg/kg body weight. They found that within 1 hour
after administration dieldrin appeared in rumen fluid
(2-20 ppb), parotid saliva (2—10 ppb), total saliva

(20—80 ppb), and pancreatic juice (30—100 ppb). Dieldrin

 

10

in saliva and pancreatic juice remained rather constant
for A8 hours. This recycling of dieldrin from the blood
to the gut would extend the absorption period and in-
crease the time required for blood to reach equilibrium.
Crosby gt_al. (30) gave two cows single doses of DDT
(13.5 and 15.0 mg/kg body weight, respectively) and
found whole blood DDT increased sharply within the first
two hours after feeding. The levels diminished rapidly
after the peak measured at 17 hours, but did not reach

a plateau until approximately two weeks post—adminis—
tration.

Moss and Hathway (102) proposed that unloading of
dieldrin and Telodrin in the kidney and liver was caused
by breakdown of the carrier proteins, by changes in pH
after penetration into the tissues, and by phagocytosis.
They argued that since all of the plasma proteins enter
phagocytic cells at the same rate (96), a mechanism for
the rapid unloading of bound dieldrin and Telodrin from
blood involving pinocytosis seemed likely, especially
since many water—soluble and water—insoluble substances
are rapidly removed from circulating blood. The with—
drawal of these substances into the sinusoid spaces of
the liver would provide an alternate explanation of

their rapid removal from blood (102).

 

 

11

C. Factors Affecting Pesticide Storage

in the Body

Most of the chlorinated hydrocarbon insecticides

 

are retained to a greater or lesser extent and for vary—
ing periods of time in the body fat. The fact that
chlorinated hydrocarbon materials are stored in adipose
tissue is not surprising in View of the known solubility
of these compounds in fats and fat solvents and their

relative insolubility in aqueous solvents (21).

l. Dosage Level

In several species, less DDT was stored at higher
than at lower dosage levels (59, 87). Lehman (87) showed
that the same was generally true for aldrin, dieldrin,
endrin, and isodrin; in contrast, lindane was stored with
the same efficiency at all dosages studied. Methoxychlor
showed a threshold and was stored only at the higher
dosage levels.

Ivey e£_al. (72) found that 0.25 ppm of aldrin in
the diet of steers for 12 weeks resulted in 0.99 ppm of
dieldrin in the body fat, and 10 ppm aldrin in the diet
increased the dieldrin content of fat to 39.2 ppm. Thus,
in this study the storage of dieldrin was almost exactly
proportional to the amount of aldrin fed. Not all
species show the same storage when maintained on similar

dosage levels (53).

 

 

12

2. Duration of Dosage

 

Soon after a single dose of any chlorinated hydro—
carbon insecticide, distribution is relatively equal in
most tissues. However, there is a gradual redistribution
leading to higher concentrations in fat than in other
tissues (3A, 67).

Barnes and Heath (7) showed that two equal doses
of dieldrin given to rats within three weeks of each
other were more toxic than the sum of the two given as
a single dose. Rats that survived one dose of dieldrin
remained much more sensitive than normal rats to a second
dose given up to three weeks later. Heath and Vandekar
(67) theorized that after an original high dose of
dieldrin, a concentration barrier develops between the
blood and fat, so that the dieldrin from the new dose is
diverted away from the fatty tissues, and much more
dieldrin reaches the central nervous system.

At a constant daily dose, the storage of DDT in
fat increased steadily for a period, but eventually
reached a maximum and plateaued in the rat (82), monkey
(AA), cattle (108), dog (111% and man (61, 6A).

It had been claimed (22) that cattle and sheep
achieve storage equilibrium for a wide range of in—
secticides in four to eight weeks, but more recent work
shows that a steady state is not achieved in cattle fed

DDT until 18 to 21 weeks of administration (8A). There

 

 
  

‘17? steady state of stu

       
  
 
    

stem conditions. Therefore, few if

9‘

imates seem completely reliable (60).

  
  

‘The time necessary for the rat to reach a steady

state in the storage of DDT was estimated to be as little

  

as seven weeks (1A0) and as much as 19 to 23 weeks (82).
The most likely value was considered to be 17 weeks (AA).
In the monkey, the time is unknown but it was less than
26 weeks (AA). In man, the time was somewhat greater
than 52 weeks (61).

Some authors have assumed a steady state when their
earliest measurements were made. Others have accepted
the highest observed storage level as the steady state.
Hayes (60) emphasized that the latter assumption would
be erroneous if storage were really incomplete or if a
steady state had already been reached and the high value
observed represented part of the random variation of a
lower mean, which would be ascertained eventually by a

longer—term study.

3. Age

Nothing is known about the relation of age to sus—
ceptibility of contamination by chlorinated hydrocarbon
pesticides. What might have been interpreted as an age

effect in previous studies can be explained by the

 

1A

intensity and duration of dosage. No age is completely
immune to contamination. To illustrate this, placental
transfer of chlorinated hydrocarbon pesticides has been
reported in dogs (A8), rabbits (65, 66), rats (13, 65,

66), dairy calves (12, 1A, 16, 2A, 75, 83),and man (39).

A. Sex

At any substantial level of intake in excess of
about 0.05 mg/kg per day, female rats store more DDT and
derived compounds than do males (A3). Similar results
have occurred with benzene hexachloride (36), chlordane
(l, 2), aldrin, endrin and isodrin (80), and heptachlor
epoxide (110). Female rats did not store significantly
more methoxychlor than males (81).

The greater storage of most chlorinated hydro—
carbon insecticides in the female, as compared with the
male rat, is probably characteristic of most tissues and
not of fat only. This is documented by findings with
DDT (33), aldrin, dieldrin, endrin and isodrin (80).

The difference in the storage of DDT and DDE be—
tween male and female rats which had been fed DDT was
reduced greatly but not reversed by hormones of the
opposite sex, gonadectomy, or a combination of these
treatments (A3). The difference in DDT storage between
the sexes is small or absent in the dog (1A1), the hog

(58), and the monkey (AA).

   

 

15

5. Species

Gannon §t_al. (53) reported species differences in
the storage of dieldrin. They found that steers stored
more dieldrin than cows or hogs while sheep stored the
least for any given level in the diet. In a related
experiment (5A) dairy cows showed slightly less dieldrin
in tissues than did steers. Losses through lactation
most likely accounted for this difference. Dieldrin was
fed at levels of 0.1 to 2.25 ppm in the diet of steers,
hogs, 1ambs,and dairy cows for 12 weeks and residues were
found in renal fat, body fat, heart fat, udder fat, steak,
and roast. Six weeks after feeding of the insecticide
had ceased levels in most of these tissues had decreased
by 50% or more (52, 53).

Bovard et_al. (10) fed apple pomace contaminated
with 103 ppm DDT to non-lactating beef heifers for 10A
days. Average residues of 75.7 ppm DDT were detected in
fat samples taken 79 days after pomace feeding began.
Presumably some additional residue was deposited during
the 25 days of pomace feeding which followed these fat
analyses, but no other fat samples were obtained until
80 days after pomace feeding ceased. At this time DDT
residues averaged A9.7 ppm. Based on limited data they
concluded that DDT residues dissipated most rapidly
immediately after pomace feeding was stopped, but the
animals still contained 8.6 ppm DDT 566 days after last

receiving contaminated feed.

 

16

It has been well established that the decline in
residue concentration of chlorinated hydrocarbon pesticides
in lactating dairy cows following cessation of intake is
due primarily to elimination via the milk (12, 1A, 16, 21,
30, 52, 5A). There is no obvious reason for the faster
rate of decline in residue concentration for the non-
lactating animals (steers, hogs, lambs) used by Gannon
§£_al. (53) and by Bovard et_al. (10) (beef heifers),
than occurred in the present studies (12, 16) with non—

lactating dairy heifers.

6. Tissue

As previously mentioned the chlorinated hydrocarbon
pesticides tend to be stored more extensively in adipose
than in other tissues. There is some indication that the
occurrence of DDT (85) and dieldrin (53) in tissues other
than adipose is determined by their lipid content. The
high DDT content of yellow bone marrow (108) is almost
certainly explained by the high fat content of the tissue.
The rather high levels of DDT reported for the adrenal
(89) and ovary (126) may reflect the high lipid concen—
tration in these organs.

Radeleff and Polen (109) fed heptachlor to beef
cattle for 16 weeks at levels varying from 1 to 60 ppm.
They found that complete conversion of heptachlor to
heptachlor epoxide occurred in the animal body and that

higher concentrations of the epoxide were noted in the

 

 

17

renal than in subcutaneous fat. Long §t_al. (88) found
slightly more endrin in internal than in external fat of
lambs.

Gannon gt_a1. (52, 53) investigated dieldrin
accumulation in tissue of steers and cows after feeding
the insecticide (0.1 to 2.25 ppm) for 12 weeks. At 12
weeks, brain and kidney were free from residues regard-
less of dosage level. Highest concentrations attained
were 0.6 ppm in the heart, 0.7 ppm in the liver, 6.2 ppm
in renal fat, A.8 ppm in body fat, 7.0 ppm in heart fat,
5.6 ppm in udder fat, 1.3 ppm in steak and 1.2 ppm in
roast. Claborn et_al. (23) found highest levels of
dieldrin in the body and renal fat of steers while less
was detected in muscle tissue. Fries and Kane (50) con—
ducted a total balance study of DDT and DDE fed to male
calves. They found similar residue levels in subcutaneous
and perirenal fat; whereas, lower concentrations were
noted in the lipids of liver and brain.

Heptachlor epoxide in the tissues of a male calf
and a lactating cow were reported by King gt_gl. (75).
Concentrations in 11 different tissues of the newborn
calf averaged about 1 ppm (range 0.AA to 2.00) while the
residue was 3 to A times higher in the hair and hide.
Residues in extracted fat of 18 tissue samples removed
from a lactating cow were very uniform. The overall

average was 0.53 ppm with lowest concentrations in the

 

l8

brain and highest in liver, hide and hair. Matsumura
and Hayashi (93) found that in the German cockroach
(Blattella germanica L.) dieldrin formed a complex with
nerve components which was inextractable with organic
solvents. They suggested that such a binding phenomenon
involved a complex formation with nerve components other
than simple lipids.

Chlorinated hydrocarbon pesticides accumulate at
much higher concentrations in adipose than in other
tissues. This raises the question regarding the exact 1
nature of the storage process. The concept has existed
for some time that adipose tissue functions primarily
in fat accumulation (131). However, studies in recent
decades indicate adipose tissue may be part of the
reticulo—endothelial system (RES). Under certain conditions
adipose tissue has the capacity to develop a hematopoetic
function and synthesize antibodies (128). The possibility
that adipose tissue is part of the RES suggests that it
could play a role in immune processes (130). These con-
siderations caused Wasserman and Wasserman (128) to pro—
pose that adipose storage of chlorinated insecticides is
an active process in the sense of phagocytosis.

7. Interaction of Drugs
and Other Compounds

 

 

Durham (A2) has recently published the first review

on the subject of pesticide interactions with a variety

  

19

of factors. In this comprehensive report he discussed
the relationship of pesticide contamination to factors
such as physiologic state (species, sex, age, nutri—
tional status, and hereditary defects in enzyme systems)
disease, environment (temperature and light), route of
exposure, formulation of the pesticide and interaction
with other chemical agents.

Street (122, 123) demonstrated a reduction in
dieldrin storage in fatty tissues of the rat when DDT was
administered simultaneously. In further work Street et_al.
(125) tested selected drugs for their effectiveness in
reducing dieldrin retention by rats. They found that
oral administration of heptabarbital, aminopyrine,
tolbutamide, and phenylbutazone reduced tissue concen-
tration of dieldrin. Heptabarbital was the most effective
drug used and resulted in an 80% decrease in tissue
dieldrin when fed at a rate of 225 mg/kg rat/day. In
comparison, DDT at A mg/kg rat/day effected a 72% re—
duction. Braund (13) obtained similar results with rats
fed heptabarbital and dieldrin concurrently. At the end
of two weeks contamination, dieldrin storage in rats re—
ceiving the drug was reduced 75%. The drug was also ef—
fective in eliminating the pesticide already stored in
the body. Six weeks after cessation of dieldrin feeding,
residues had been reduced 96% in animals receiving the
drug during decontamination, while much higher concen—

trations were detected in groups receiving no drug.

 

 

20

In additional studies Street and co—workers (12A)
found that interactions among certain organochlorine
insecticides influenced their storage in rat adipose
tissue. DDT administration markedly depressed storage
of dieldrin and heptachlor when the three pesticides were
fed simultaneously. DDT also promoted a rapid depletion
of pre—existing dieldrin stores. The DDT effect on
cyclodiene storage was postulated to result from stimu—
lation of detoxifying enzymes. Supporting evidence for
the postulate included the depressing effect on dieldrin
storage by several enzyme-inducing drugs, and the stimu—
lation of ascorbic acid biosynthesis by DDT. However, this
theory was challenged by the failure of inhibitors of pro—
tein synthesis to stop the effect of DDT on dieldrin
storage. DDT interaction with dieldrin metabolism was
also demonstrated in swine and sheep, but not in chickens
(12A).

Nutritional interactions in dieldrin toxicity have
recently been investigated by Tinsley (127). At a dietary
level of 20 ppm he found dieldrin accentuated an essential
fatty acid stress in rats due to the action of dieldrin
in the metabolism of polyunsaturated fatty acids. A
riboflavin deficiency resulted in a depressed growth rate
in female rats subjected to dieldrin stress. The vitamin
inadequacy accentuated dieldrin toxicity and increased

the level of dieldrin in liver. The hypothesis was

 

21

proposed that a riboflavin deficiency could limit the
detoxification process.

Phillips (107) reported that, in the rat, DDT de—
creased the utilization of vitamin A and carotene and
that rations low in protein accentuated dieldrin toxicity.
The combination of the low protein and dieldrin stresses

reduced the levels of vitamin A in liver (86).

D. Biotransformation

 

1. Chemical Nature

 

Processes concerned with the metabolism and decompo—
sition of pesticides are important for several reasons.
First, the duration of toxic action can be related to the
rate and manner in which the compound is metabolized.
Second, the rate of elimination from the body is dependent
upon the physical and chemical properties of the meta—
bolic products. Third, the ability of a compound to
reach a site of action may be limited by the rate at which
it is metabolized and the character of the metabolic pro—
ducts. Fourth, the toxicity of a compound can be de—
creased or intensified upon conversion to a metabolite.
These factors emphasize the need for knowing the pathways
of metabolism as well as the degree of accumulation of
metabolic products in tissues.

Menzie (99) has classified metabolic pathways into
seven groups that cover the majority of biotransformations

which pesticides undergo.

 

ion (in the sense that oxygen, as

hydroxyl, takes part or is postulated to

take part in one or more Of the steps).

 

a. Hydroxylation of aromatic rings
b. Oxidation of side chains to alcohols,
ketones, or carboxyl groups
c. Dealkylation from oxygen or sulfur
(ether cleavage)
d. Sulfoxide formation
e. N-oxide formation
2. Dehydrogenation and dehydrohalogenation
3. Reduction
A. Conjugation
a. Amide formation
b. Metal complex
0. Glucoside or Glucuronic acid
d. Sulfate
5. Hydrolytic reactions
a. Cleavage of esters
b. Cleavage of amides
6. Exchange reactions
7. Isomerization
Illustrative examples of substances known to under—

go each of the biotransformations were also indicated.

   

23

2. Identity of Metabolites

 

Metabolites of chlorinated hydrocarbon insecticides
which have been identified were summarized by Hayes (60).
He made no attempt to separate the metabolites according
to their certainty of identification. Many metabolites
of chlorinated hydrocarbon insecticides have not been
identified although they have been clearly recognized by
solubility or chromatographic properties. Even when a
new metabolite is indicated, further study is usually re—
quired to determine whether it is one of the previously
unidentified metabolites of the compound.

3. Relation to Species
or Strain

 

Bann et_al. (6) found that aldrin was readily con—
verted to dieldrin in the body of beef and dairy cattle,
pigs, sheep, rats, and poultry. This change was indepen-
dent of the mode of entry, since it occurred following
oral ingestion or subcutaneous injection.

When aldrin—lac was fed to male rats (25, 91) or
administered orally to male and female rats, the active
material excreted in feces and urine consisted of aldrin,
dieldrin, and considerable amounts (up to 75% of the
label appearing in feces and up to 95% in urine) of a
mixture of unidentified hydrophilic metabolites. At a
feeding level of A.3 ug per day, a saturation level was

reached after about eight weeks and daily excretion of

 

 

2A

the active material approximated the entire activity
administered daily. When injected intravenously, 16.2%

4C and 13.0% of dieldrin—lac was excreted within

of aldrin—l
four hours, via the bile, into the intestinal canal,
mainly as hydrophilic compounds (78, 101). Recent

studies with rat livers showed that this epoxidation

was performed by the microsomes (139). Jensen (73)

showed that biliary excretion was responsible for almost
all of the DDT metabolites in feces of rats.

Paper chromatography of extracts of feces and urine
showed a high percentage of aldrin—luC initially. The
percentage of unchanged aldrin decreased while that of
hydrophilic metabolites increased for about 12 days.
Thereafter, the distribution of aldrin and metabolites
remained constant as long as aldrin was administered.
After aldrin administration was discontinued, the relative
percentage of aldrin in feces decreased and dieldrin in—
creased (91). Paper and thinvlayer chromatography showed
that two additional compounds appeared in the feces and
in the urine, and that one of the compounds in both feces
and urine behaved similarly. Hydrolysis of the main com-
pound from urine with alcoholic KOH resulted in a new
acidic compound (91). When rabbits were given aldrin
intravenously, hydrolysis of the metabolites gave the

aldrin diol (78).

 

~in was excreted. Of the dieldrin administered,

FIRES excreted within 1A days in the feces. Datta

1' et a1. (35) showed that dieldrin was metabolized in rats

to two compounds more polar than dieldrin and unstable
to alcoholic KOH.

When labeled dieldrin was administered to a rabbit
via stomach tube, six metabolites were isolated and puri-
fied via thin—layer chromatography. The acute oral toxi—
city of the main metabolite, which accounted for about
86% of the total activity in the urine, was only one—
twelfth to one-sixteenth that of dieldrin (77).

Urine of men with occupational exposure to dieldrin
contained at least two neutral, polar, chlorinated meta-

bolites, which are thus far unidentified (31).

E. Excretion and Storage Loss
True excretion, i.e. elimination of previously
absorbed material, of chlorinated hydrocarbon insecti-
cides may occur by way of expired air, urine, feces,
milk, dermal secretions (136), eggs,and fetus (12, 1A,
16, A8, 65, 66, 75, 83).

 

 

 

26

1. Respiratory Loss

 

Respiratory excretion may be important for certain
fumigants. McCollister (95) found that although monkeys
eliminated 20% of absorbed radioactive carbon tetra—
chloride by exhalation during the first 18 hours follow—
ing exposure, ll}C continued to be measureable in samples
of respired air for a period of four weeks. Material
derived from the carbon tetrachloride was also found in
. the urine and feces.

2. Fecal, Biliary, and
Urinary Excretion

 

 

For a wide range of compounds, urinary excretion
has been studied more thoroughly than fecal excretion,
yet fecal excretion is more important for certain com—
pounds. Studies by Hayes (60) showed that in the rat
fecal excretion of DDT exceeded urinary excretion ir-
respective of the route of administration. The same was
true of Perthane (9), methoxychlor (129), aldrin (9A),
and dieldrin (67). In fact, 90% of the excretion of
dieldrin—derived material was via the feces (67). It
has been suggested that most residual chlorinated hydro—
carbon insecticides are excreted chiefly in the feces
(60).

Biliary excretion has been shown for DDT (73, 112),
Perthane (9), methoxychlor (129), aldrin (101), and

dieldrin (67). The rate of biliary excretion may be

 

ates of chlorinated hydrocarbon insecticides

~s certain. Bleiberg and Larson (9) collected bile

 

from two rats given radioactive Perthane intravenously
and in an eight hour period recovered 25 and 55% of the

1”C. When the bile samples were combined and given to

a third rat, about 70% of the 114C appeared in the feces
and 13% in the urine over a three day period. This
experiment also showed the relatively rapid excretion of
the metabolites compared with the parent compound.
Enterohepatic circulation of dieldrin was demonstrated
by Heath and Vandekar (67) who found that the proportion
of unchanged dieldrin excreted could be increased from
3% to 10% of the total excretion by cannulation of the
bile duct.

The bile appears to be the principle route through
which DDT metabolites enter the feces. The bile duct
was cannulated in a rat before intravenous injection of
radioactive DDT and 65% of the labelled dose was re—
covered in the bile, 2% in the urine, and only 0.3% in
the feces (73). Furthermore, the possibility of some

contamination of feces by urine could not be excluded.

 

28

Biliary excretion of metabolites suggests, but
does not prove, that the liver is an important site for
their production. For aldrin and dieldrin, the high
concentrations of both the parent compound and its
hydrophilic metabolite in the liver support this view
(101).

However, Mendel and Walton (98) have presented evi—
dence that the normal flora of the gastrointestinal tract
may be more important than the liver in conversion of
p,p'—DDT to p,p'—DDD. They showed that coliform bacteria 7
isolated from feces of control animals could effect re—
ductive dechlorination of p,p'—DDT to p,p'—DDD.

The urine is a more important route for excretion
of several chlorinated hydrocarbon insecticides than for
DDT. Stohlman, Thorpe, and Smith (121) recovered about
18% of the chlordane that had been fed from the urine of
rabbits. Approximately 80% of the radioactivity derived
from both a and y benzene hexachloride was found in the

urine and only about 20% in the feces (76).

3. Excretion in Milk

 

Gannon et_a1. (51, 52) found that the first
dieldrin appearing in milk of cows which grazed treated
pastures was directly proportional to dieldrin intake.
Later concentrations were determined by the combined
effect of daily intake and amount released from the

body fat. Finally, when dieldrin had been removed from

 

29

the diet the slowly declining residues that persisted in
the milk apparently were associated with dieldrin stored
in the body fat.

As would be expected, while pesticides are being
ingested, excretion rates in the milk are proportional
to the dosage for DDT (22, 1AA), dieldrin (22, 33),
aldrin, heptachlor, methoxychlor (22, 26), heptachlor
epoxide (A, 71), and endrin (79). Moreover, the slopes
of the lines relating concentration in milk to that in
feed were identical for most of the compounds mentioned
(22).

Once an animal has been contaminated with chlori-
nated hydrocarbon pesticides, residue excretion in milk
may continue for many months. Bruce et_al. (21) fed
various heptachlor levels (0.5 to 50 ppm) to cows for
12 weeks. The maximum concentration of heptachlor epoxide
appearing in milk fat was A60 ppm. This level occurred
after 12 weeks of feeding the pesticide. Detectable
residues were still present in both body and milk fat 23
months later. Other studies have shown that after
hepachlor epoxide (22, 37, 38, 51, 57, 71, 113, 135),
dieldrin (12, 1A, 16, 22, 51, 52, 135), Telodrin (8, 57),
and DDT (20, 22, 30, 51, 83, 8A, 135) are stored in body
fat, they continue to contaminate the milk long after
chemical intake has been discontinued. Laben et_a1.

(83, 8A) calculated that DDT in milk declined at 9 to

 

3O

11% per week after maximum concentrations of DDT in milk
fat were 19, 231 and 812 ppm for three treatment groups.
Rate of decline was 6.6% per week for their control group
which had a maximum milk fat DDT concentration of about

2 ppm. However, Brown et_a1. (20) studied DDT excretion

in eight cows during the first 59 days of lactation and
found no marked change in the level of DDT or its metabolic
products in milk. The level of DDT and its degradation
products in this study ranged from 0.58 to 0.79 ppm in

milk fat.

It has been shown (117, 119) that 10% or more of
the DDT derived from residues on forage was excreted subse—
quently in the milk. When daily DDT intake was approxi—
mately 126 mg/cow (7 to 8 ppm on the hay) 15 to 23% was
secreted. On some days certain cows secreted up to 32.5%
of their DDT intake in their milk (119). Shepherd et_a1.
(117) found the output of DDT in milk varied from 5 to
30% when daily intake varied between 100 and 700 mg per
cow. However, these results must be viewed with caution,
because they depend not only on analysis of DDT in milk,
but also on analysis of DDT residues in hay. Any syste—
matic failure to detect all of the residue in the hay
would lead to an over—estimation of the proportion of the
dose recovered in the milk. Ely et_a1. (A6) were able
to recover in the milk of cows only 1.8 to 7.0% of the

DDT administered as an oil solution.

 

31'

Huber and Bishop (71) reported that cows fed
heptachlor and heptachlor epoxide residues on hay secreted
20 to 29% of the insecticide in their milk in the form of
heptachlor epoxide. Again, the accuracy of the analysis
of the residues in hay has bearing on the credibility
of these data. Recent evidence (103, 133) has indicated
that routine extraction techniques are inefficient when
applied to crops containing solely an internal accumu—
lation of dieldrin. Wheeler §t_§l. (133) emphasized that
the extraction of insecticides from plant materials,
whether surface or internal residues, is often the weak—
est link in the entire analytical procedure.

Demott gt_al. (38) found that the percentage of
heptachlor secreted into the milk as the epoxide ranged
from A to 22, with an overall average of 12. Those cows
on alfalfa hay that contained 0.29 ppm heptachlor or
heptachlor epoxide secreted an average of 9% of the in-
take in their milk, whereas those on alfalfa with 0.08 ppm
secreted 17% in their milk. This difference in secretion
rate as influenced by concentration in the feed was also
observed by others (52).

Zweig et_al. (1AA) reported finding about 0.3 to
2.2% of the daily dose of DDT in the milk while Laben
et_al. (83) calculated that 3% of the dose was excreted
in the milk. By estimating urinary output of DDA they

calculated that urine and milk would account for 17 to

 

32

19% of the DDT fed. Witt _e_t_a1_. (138) investigated
contamination of milk from different routes of animal
exposure to DDT. The amount of administered insecticide
which they accounted for in the milk varied from 0.78%
to 32.2%. The percent of the dose recovered in the milk
did not seem to be correlated with milk production,
lactation number, stage of lactation, or weight of the
cow.

A. Relation of Excretion to
Storage and Storage Loss

 

 

High storage is generally correlated with slow
excretion; thus, when excretion is relatively inefficient,
storage tends to be high and storage loss slow.

Some of the earliest studies on residues of chlori—
nated insecticides indicated that the rate of storage loss
was not constant after dosage was discontinued. In 19A9,
Kunze gt_al. (82) reported that following small doses of
DDT about one—half remained in storage a month after
dosage was discontinued and one-fourth after three months.
Male rats, which had stored chlordane in their fat at a
concentration of about A0 ppm, reduced stores by about
25% in five days and about 50% in 20 days (2). When
these values for storage of DDT and chlordane are plotted
on semilog paper the resulting curves are not strictly
logarithmic. Similar curves were published by Cannon

and Decker (51) in connection with the concentration of

 

33

dieldrin in milk following reduction or termination of
dosage. Hayes (60) found similar curves for the storage
of DDT and the excretion of DDA after dosage was stopped
in man.

Greater efficiency in excretion at higher dosage
levels accounted for, at least in part, the lesser amounts
of DDT stored at high compared with low dosages (59, 87).
Similarly, greater excretion efficiency may partially
explain the reduced storage of DDT and DDE in males than
female rats (60).

Hayes (60) pointed out that although the term ”half—
life" is frequently used to refer to storage loss, the
term "percent per day" is preferable when accounting for
both storage loss and excretion. The latter term is far
more appropriate for describing changes in rates pro—
duced by starvation or some other temporary factor. More—
over, the rate of loss of some residual chlorinated in—
secticides decreases as storage decreases. The term
half—life is completely valid only when stores decrease
according to a logarithmic pattern. Hayes (60) cited the
data of Heath and Vandekar (67) as an example of the
rapid change in loss that might occur. In this study
about 5% of a single intravenous infusion of tagged
dieldrin was excreted daily in the bile, but the amount
excreted daily increased to more than 10% in rats that

had been starved for a few days.

 

3H

F. Placental Transfer

Relatively few data are available on placental
transfer of chlorinated hydrocarbon pesticides to the
fetus or on the dissipation of residues from young con—
taminated in utero.

Finnegan et_al. (48) showed that both DDT and DDD
were transferred across the placenta of the dog. Pla—
cental transfer of dieldrin and Telodrin have been demon—
strated in the rat and rabbit (65, 66). Clark (2“) re—
ported dieldrin in calves born to cows fed contaminated
corn silage and cull potatoes. Laben §t_al. (83) found
DDT in three stillborn calves from dams fed DDT prepartum.
Heptachlor epoxide was distributed throughout the tissues,
but was three to four times higher in the hide and hair
than in other tissues, of a calf whose dam had consumed
heptachlor—treated alfalfa for 6 to 12 weeks prepartum
(75).

Hathway (65) investigated transplacental passage of
dieldrin and Telodrin in rabbits. When a single injec—
tion of Telodrin-1&0 was made into an ear vein of 24—day
pregnant rabbits, the absence of 14C in the amnionic
fluid eliminated the possibility of Telodrin—lac trans—
port occurring from the uterine cavity at this stage of
gestation through the vascularized yolk~sac or splan—
chnopleure. Guinea pig and rabbit antibodies are
selectively transferred to the fetus by way of the yolk—

sac mechanism (ll).

 

35

During a 40—minute period, Telodrin concentration
in fetal blood did not decline, whereas the concentration
of Telodrin in maternal blood decreased. Hathway (65)
concluded that Telodrin—14C administered to the mother
concentrated on the fetal side of the placenta. The
passage of insecticide from mother to the fetus continued
while the external to internal concentration gradient was
favorable. Conversely, continued uptake of 1”C by the
fetal blood against an adverse concentration gradient was
not indicated. Moreover, maternal blood was constantly
unloading insecticide, which was partially taken up by
maternal fat depots.

Two—way placental transfer of dieldrin in rabbits
was demonstrated by Hathway (65). Seven minutes after a

1M

single intravenous injection of dieldrin— C into 2&—

day pregnant rabbits, the concentration of 1MC in fetal
blood was 93% as high as in maternal blood. This re—
lationship between fetal and maternal blood concentrations
remained roughly constant throughout the experimental
period (H0 minutes). As evidence for two—way trans—
placental passage, Hathway et_al. (66) intravenously in—
jected dieldrin—luc into the umbilical vein of a single
fetus of a 24—day pregnant rabbit. Radioactivity was
found seven minutes later in the blood of the injected

fetus, and also in maternal blood and in the blood of a

non—injected fetus. It was found that after a single

 

36

u

intravenous injection of dieldrin—l C in pregnant rabbits
a very small uptake of insecticide by the free blasto—
cysts rapidly occurred. Following their implantation,
the rate of dieldrin—lac uptake by blastocysts from
maternal blood was significantly slower. Dieldrin was
secreted from the endometria of treated animals ir—
respective of pregnancy. During the second half of
pregnancy allantoic and amnionic fluids were free from
140.

The transfer of compounds across the placenta is
regulated by the same physico—chemical mechanisms which
control the transport across other cell walls (66). In
this regard, the method of passage of dieldrin across
the blood—brain barrier (67), the erythrocyte stroma (102),
the wall of the gut (65),and the blastocyst membrane (66)
seems related. Non—ionized compounds with high lipid
solubility, like dieldrin, can rapidly cross the placenta
.in either direction, provided that the concentration
gradient is favorable. Other compounds of low lipid
solubility are only slowly transferred across the placenta,
and many larger molecules also soluble in lipids are in—

capable of crossing the placental barrier.

G. Experimental Decontamination

 

It has been documented in this review that chlori—
nated hydrocarbons are stored largely in the body fat

and exhibit a relatively long retention time in the

 

37

animal. There is a paucity of information relative to
management practices which might result in a more rapid
decontamination. The possibility of increasing the rate
of decontamination via an increased rate of body fat
depletion has been suggested. Brown §£_al. (20) in—
vestigated this approach but their results were incon—
clusive because fat mobilization was not actually mea—
sured and the cows studied lost an average of only 17 kg
body weight during the first month postpartum.

Fries et_al. (#9) studied the rate of depletion of
DDT and its metabolites when body fat loss was estimated
by complete energy balance trials. They concluded that
DDT and DDE were depleted from the animal at rates in—
dependent of fat mobilization. In contrast, DDD de-
pletion appeared to be closely associated with the fat
losses. However, the practical importance of clearing
DDD through fat loss is minimal because it is stored to
a much lesser degree than DDE and DDT.

Starvation diets in poultry have decreased the
level of DDT found in eggs (132). Miller (100) investi—
gated the effect of thyroprotein and a low-energy ration
on removal of DDT from lactating dairy cows. He found
no appreciable difference in rate of decline of DDT
between cows that received normal rations and those fed
thyroprotein. However, cows on a low-energy ration

showed an appreciable increase in rate of decline.

 

38

iz'uded that cows and heifers contgmawaxés

u wiuh DDT were more difficult to decontaminate than

 

‘ irgssting the same amount of pesticide postpartum.
I'Prepartum contamination resulted in milk fat containing
7 to 12 ppm DDT compared with five to seven ppm DDT in

the milk fat for animals contaminated postpartum.

 

III. EXPERIMENTAL PROCEDURES

A. Experiment l-—Excretion apd Storage of
Dieldrin in Dairy Cows Fed Thyroprotein
and Different Levels of Energy

 

 

 

1. Experimental Animals——
Dosage and Duration of
Contamination

 

 

Sixteen lactating Holstein cows, averaging 39 months
of age and 115 days postpartum were selected from the
Michigan State University dairy research herd. Cows were
assigned to one of four outcome groups based on daily
milk production and stage of lactation.

For 56 days each animal was contaminated with techni—
cal dieldrin in grain at the daily rate of 0.11 mg/kg body
weight. The technical dieldrin contained 88.4% active
ingredient [1,2,3,A,10,10—hexachloro—6,7—epoxy-l,
U,Aa,5,6,7,8,8a-octahydro—l,A—engo—exo—S,8—dimethano-
naphthalene (HEOD)]. Because the analytical procedure
measured HEOD, not dieldrin, all intake and analytical
data reported hereafter in this thesis will be expressed
as HEOD equivalent. Intake of HEOD was equivalent to
1.9“ ppm in the total diet or 3.25 ppm in the dry matter

(DM) consumed.

 

A0

Intake of dieldrin was based on body weights of
cows obtained two consecutive days just prior to con—
tamination. Once each week during treatment the total
weekly intake of dieldrin for each cow was thoroughly
mixed in 6.35 kg of grain by delivering the necessary
volume of an acetone solution containing 5 mg/ml dieldrin
onto the grain from a buret. Thorough mixing was ef-
fected by tumbling the grain in a six—gallon covered
polyethylene container. Grain was then divided into
seven—0.907 kg (2 lb) units and placed in individual
paper bags identified with the appropriate cow number.

A bag of dieldrin—containing grain was fed to the appro—
priate cow in a clean manger at 8:00 A.M. daily, immedi—
ately following weighback of unconsumed feed. Only after
the contaminated grain had been completely consumed was
the remaining portion of the morning grain allotment
offered.

During contamination, TDN was furnished to provide
100% of the NRC (105) requirement for maintenance and milk
production. The requirement for growth instead of
maintenance was used to calculate TDN needed by first
lactation heifers.

All cows received a ration of A.5 kg of alfalfa—
grass hay, 13.6 kg of corn silage and sufficient concen—
trate to balance the TDN requirement. Adjustments in

concentrate level were made every two weeks based on

 

Al

the average milk production for the preceding seven days.

Cows remained in a stanchion barn at all times except for

two hours each morning when they exercised in a paved lot.

Body weights were obtained on two consecutive days at
two—week intervals throughout the contamination and de—
contamination period. Milk yields were recorded each
milking and amounts of feed offered and unconsumed were
weighed daily.

2. Dietary Treatments and
Adjustments

 

At the end of the eight—week contamination period
the four groups of four cows each were randomly assigned
to one of the following dietary treatments:

a. 75% of the NRC requirement for TDN

b. 150% of the NRC requirement for TDN

c. 150% of the NRC requirement for TDN plus 22 mg

of iodinated casein (Protamone)l daily per kg
of body weight

d. 100% of the NRC requirement for TDN (Control)

Grain intake was adjusted every two weeks as pre—
viously described. Hay and corn silage levels remained
constant throughout the experiment except for the low—
energy ration in which each was reduced proportionately

to achieve 75% of requirement.

 

1Supplied by Agri—Tech, Inc., Kansas City, Missouri.

 

A2

Thyroprotein was fed by placing the required daily
dose in gelatin capsules which were injected directly into
the rumen with a balling gun. During the first five days
thyroprotein was divided daily into two equal portions
which were administered at the A.M. and P.M. feedings.
Thereafter, the total daily dose was given at the 8:00 A.M.
feeding only. At biweekly intervals thyroprotein dosage

was adjusted for changes in body weight.

3. Sample Collection

 

a. Milk.——Aliquot milk samples were collected and
composited daily from each cow just prior to contamination,
on the third and sixth day following the start of con—
tamination, and at weekly intervals thereafter until the
eighteenth week of decontamination. The milking machine
buckets were rinsed with water after each cow was milked
to reduce cross contamination. Milk fat was determined
according to the Babcock method on each composite sample
following the P.M. milking. Milk samples were refriger—
ated overnight and extracted the following day for HEOD
analysis.

b. Body Fat and Tissues.—-Biopsies of external

 

body fat weighing 5 to 10 g were removed from the shoulder
area of two cows in each treatment group at the end of
contamination and during the eighth and sixteenth week of
decontamination. A 2% Procaine solution was used as

local anesthetic. Fat samples were placed in plastic

bags and stored frozen until extracted for HEOD analysis.

 

 

A3

Following termination of the decontamination period
six cows were sacrificed for various reasons not related
to the experiment. Samples of external shoulder fat,
omental fat, kidney fat, liver, kidney, and udder tissues
were removed, frozen, and stored at —15°C until extracted
for HEOD analysis.

0. Feces and Urine.——During the third week of de—

 

contamination total feces and urine were collected for

five days from two cows in each treatment group. Feces
were collected in metal pans placed at the rear of each
cow and urethral catheters were used for shunting urine
to tenrgallon cans.

After thorough mixing, two percent of the feces ex—
creted daily was placed in a plastic bag and frozen. The
five daily samples from each cow were thawed, composited,
and dried at 80°C for 72 hours. After weighing, the dried
samples were ground in a Wiley mill through a 1 mm screen.

Daily urine collections from each cow were stirred
and an aliquot sample placed in a plastic jar and frozen.
Each subsequent day's sample was added to the original
container such that at the end of a collection period
approximately two liters of urine had been saved from
each cow.

d. Feed.——Prior to and at monthly intervals during
the entire experiment, hay (1 kg), corn silage (2 kg),

and grain (1 kg) were sampled for dieldrin analysis.

 

AA
1

For hay, a Penn State Forage Sampler was used to obtain
cores from about 25 bales which were representative of
each sampling period. All samples were placed in plastic'

bags and refrigerated until extracted for HEOD analysis.

A. Sample Extraction

 

Extraction and clean—up procedures were developed
in the Pesticide Analytical Laboratory of Michigan State
University (1A2). All solvents were of reagent grade and
were checked for interferring impurities by gas chroma—
tography before use. Extraction proceeded as follows:

a. Milk.——

(i) After warming to room temperature milk was

thoroughly mixed by shaking and 30 m1 of whole

milk were placed in a 500 ml flask, to which was
added one gram of sodium or potassium oxalate,

25 m1 ethanol, and 100 m1 diethyl ether.

(ii) The flask was then shaken for 10 minutes on

a Burrell Wrist—Action Shaker, Model CC2 set at

6 1/2.

(iii) The diethyl ether was decanted and saved.

(iv) A 100 m1 equivolume mixture of diethyl ether

and n—hexane was added to the original sample two

separate times. After shaking for 10 minutes

each time at a setting of 2,the diethyl ether was

decanted and saved.

 

2Burrell Corporation, Pittsburg, Pennsylvania.

 

A5

The three extracts were combined and dried by the
addition of about 5 g anhydrous Nagsou. After
standing for 30 minutes, the combined extract was
filtered into a graduated cylinder. The amount

of extract was then recorded and the Na2SOu was
rinsed with about 20 ml of diethyl ether.

(v) Next, the extract was concentrated to approxi—
mately 10 ml in a Kuderna—Danish Concentrator in
preparation for column chromatographic clean-up.

b. Body Fat.——

(i) The frozen sample (5 to 10 g) was weighed and
then blended with Dry Ice in a Waring Blendor3 until
very finely powdered. The powdered sample was then
poured through a powder funnel pre—cooled with Dry
Ice into a 500 ml flask.

(ii) The Blendor and funnel were slowly rinsed with
250 ml of nfhexane which was allowed to drain

into the flask. The flask was then shaken at a
setting of 5 on the wrist-action shaker for approxi—
mately 30 minutes. Prior to shaking flasks had
been covered with Saran—Wrap squares secured by a
rubber band. A few pin holes were made in the
center of the squares to allow for escape of

residual CO2.

 

3The Blendor had been pre—cooled with Dry Ice be-
fore the fat sample was added; thus preventing the powder
sample from adhering to the sides of the container.

 

A6

(iii) The extract was then filtered through
anhydrous Na2SOu into a graduated cylinder; the
flask was rinsed with an additional 50 ml of
nehexane, and the volume of extract recorded.
All n—hexane was evaporated from a 25 ml aliquot
of extract for determination of percent lipid.
(iv) A volume of extract containing the equi—
valent of 2 g of fat was then introduced into a
250 ml separatory funnel containing 50 ml of
acetonitrile saturated with n—hexane. The mix—
ture was shaken vigorously for two minutes and
acetonitrile layer was drained into a 500 ml
separatory funnel. The last step was repeated
with two successive washings of acetonitrile
which were accumulated in the same 500 ml
separatory funnel.

(v) One hundred ml of n—hexane and 200 ml of a
10% aqueous NaCl solution were added to the
acetonitrile in the 500 ml separatory funnel and
the mixture was shaken for two minutes. The lower
layer was discarded. Another 200 m1 portion of
NaCl solution was added, the shaking repeated,
and the lower layer again discarded.

(vi) The n—hexane layer was transferred to a 250
ml flask, dried with anhydrous Na2SOu, and then
introduced into a FlorisifD:Celité9* (5:1) column

for clean—up.

 

*
Standardized Copyright symbol.

 

A7

0. Body Tissue.--

(i) Approximately 10 g of frozen tissue were
weighed and steps (1), (ii), and (iii) for body
fat were performed except that all n—hexane was
evaporated from a 50 ml aliquot to determine the
percent lipid in the extract.

(ii) The extract was concentrated to about 10 ml
in a Kuderna—Danish Concentrator and was then
introduced into a Florisif®:Celite@r(5:l) column
for clean—up.

d. Urine.——

(i) One liter of urine was filtered through a 5 u
millipore filter to remove particulate matter
without loss of pesticide. The filtrate was
divided into three l—liter separatory funnels and
each portion was extracted in order (left to
right) once with the same 100 ml of n—hexane.

This "progressive—extraction” was repeated, ex—
tracting each portion in reverse order (right to
left) with another 100 ml of nfhexane. This pro—
cedure was repeated for the third time, adding
another 100 ml of n—hexane to the center separatory
funnel and then extracting the other two fractions.
(ii) An additional 100 ml of n—hexane was used for
quantitative rinsing of the separatory funnels.

The four 100 m1 n—hexane extracts were combined

 

*Standardized Copyright symbol.

 

A8

and concentrated to a 5 ml volume. Two pl were
then injected into the gas chromatograph for HEOD
quantification.

(iii) The millipore filters were dried in a vacuum
oven for 10 minutes at 60°C to 70°C. Next, the
filters were placed in a l6 x 150 mm culture tube
with a Teflon—lined cap and 5 ml of n—hexane
added. The tube was shaken for 10 minutes and

2 ul of the hexane extract were injected into the
gas chromatograph.

e. Feed and Feces.-—

 

(i) A 100 g sample of feed or feces was blended
for two minutes in a Waring Blendor with A00 ml of
a 3:1 mixture of n—hexane and isopropyl alcohol
(IPA). The extract was then filtered through
glass wool into a l—liter separatory funnel.

(ii) Next, the IPA was removed with five or six
washes of water, or until no odor of IPA could

be detected. The extract was then dried for at
least 30 minutes over anhydrous Na2SOu.

(iii) The extract was then filtered into a gradu—
ated cylinder, the volume recorded and the Nagsou
rinsed with an additional 20 ml n—hexane. Next,
the extract was concentrated in a Kuderna—Danish
Concentrator to 10 ml for introduction into a

163) ®*

Florisi :Celite (5:1) clean—up column.

 

*
Standardized Copyright symbol.

 

A9

5. Column Clean—up Procedure
for Chlorinated Hydrocarbon
Pesticide Residue Extracts

 

 

 

Chromatographic clean-up columns were 20 mm OD by
600 mm Pyregxglass tubes equipped with a coarse fritted
glass disc and Teflon stopcock. A clean—up column con—
sisted of one—half inch of anhydrous Na280u, 10 g of
Florisif®:Celit;®*(5:l), and one—half inch of anhydrous
Na2SOH on top. Sufficient nehexane was used to wet the
column, then an additional 10 ml were added. The n—hexane
was allowed to flow through the column until the top
layer of anhydrous Na2SOu was just covered with n—hexane.
The concentrated sample and the rinse of the sample con—
tainer were then poured into the column. The sample was
allowed to drain into a 500 ml volumetric flask until it
just covered the top layer of anhydrous Na2SOu at which
time n—hexane was added until 500 ml had been eluted.
At higher levels of dieldrin an aliquot of the 500 ml
sample was saved for injection into the gas chromato—
graph, but at lower levels this amount was concentrated
in a Kuderna—Danish Concentrator to 100 ml or 10 ml.

The Florisi1®z Celite

(5:1) mixture for column
clean—up was prepared as follows: 2000 g of Florisil“
and 250 ml of distilled water were thoroughly mixed in
a large plastic bag. This deactivated Florisil and

A00 g of acid-washed Hyflo Super Cel Celite5 were poured

 

uFloridin Company, Hancock, West Virginia.

5Johns Manville C0,, Manville, New Jersey.

*Standardized Copyright symbol.

 

50

into a A—liter Erlenmeyer flask and shaken for approxi—
mately one hour or until all lumps had disintegrated.
After standing overnight the mixture was shaken for an
additional hour.

For calibration, three columns were prepared with
new Florisif§:0elitgyemixture as previously described.
Instead of adding the concentrated sample, 25 ml of 100
ppm HEOD were added. The HEOD was then eluted with 500
ml of nehexane and the eluate collected in 50 ml aliquots.
These aliquots were analyzed by gas chromatography to
ascertain that no HEOD appeared before the third or
after the ninth 50 ml aliquot. If either of these con—
ditions was not met, more water or Florisif§:Celit§gewas
added to the mixture and re—calibration performed.

6. Electron—capture Gas

Chromatographic (ECGC)
Analysis

 

 

Analytical columns (one—eighth inch i.d. by 6

x .
ft Pyreia glass) were packed with 5% silicone DC—ll6 on

60/80—mesh Gas-Chrom 0.6 The instrument used for analysis
was a Beckman GC—A gas chromatograph7 equipped with an

8

electron-capture detector and a Bristol 1 mV recorder.

 

6Applied Science Laboratories, State College,
Pennsylvania.

7

Beckman Instruments, Inc., Fullerton, California.

8Bristol Instruments, Waterbury, Connecticut.

*
Standardized Copyright symbol.

 

51

Operating temperature parameters were: injection port —
250°C, column — 200°C, and detector — 350°C. Ultra—pure
helium9 was used as a carrier gas at a flow rate of

A0 ml per minute. HEOD residue recoveries were deter—
mined by comparison of retention time (about 2 1/2 min)
and peak height with known standard samples injected
into the gas chromatograph. Sensitivity was such that

12 g) of HEOD gave a measurable

two picograms (2 x 10—
peak.

Recovery of HEOD for various types of materials was
calculated by fortifying samples with known amounts of
HEOD and then extracting. Percent HEOD recovered for
different materials were: milk, 93%; body fat, 96%;
body tissue, 90%; urine, 98%, feed and feces, 10A%.

B. Experiment 2——Storage, Excretion,
and Placental Transfer of Dieldrin
by Dairy Heifers Contaminated

During Three Stages of
Gestation

 

 

 

 

l. Contamination of
Heifers

 

Thirty—one pregnant heifers utilized in this experi—
ment were fed technical dieldrin in grain at the daily
dose of 0.11 mg/kg body weight (5 mg/cwt) for 60 days.

The dieldrin was mixed in the grain and contamination

 

9

Matheson Co., Joliet, Illinois.

 

   

52

accomplished as described for Experiment 1. Based on
total feed consumption during the contamination period,
HEOD intake was equivalent to about 5.A ppm in the
total diet. The contamination scheme is shown in
Figure 1. During contamination and until parturition,
all heifers were fed hay at a daily rate of 1.5 kg/lOO
kg of body weight. Grain was fed in sufficient amounts
to bring TDN intake up to 115% of the NRC requirement
(122).

a. Contamination During 60 to 0 Days Prepartum.-—

 

Eighteen heifers were contaminated for 60 days starting
60 days prepartum. Problems were encountered with three
of the animals which necessitated that they be replaced
in the experiment. The specific reasons for removal
were: one animal calved to an earlier breeding date and
had only 3A days of contamination; a second heifer had

a non—functional udder after calving; and the third
heifer suffered from calving paralysis a week after
parturition.

The calving span for the 18 heifers used on the
experiment extended from November 3, 1965, to April 15,
1966. Based on actual calving order, heifers were
assigned at parturition to one of three dietary groups.

Some variation occurred in the duration of
dieldrin feeding. Feeding commenced on the 60th day

preceding the projected calving date. If calving

 

 

 

 

Figure l.——Experimental design of prepartum
contamination.

 

Plan m Pesticide Contamination

 

 

 

 

(GROUP I
BRED CALVED
L DIELDRIN
L
r ,
283 CO 0
GROUP U
BRED CALVED
', ,
L m
283 IZO 60 O
GROUP HI
BRED CALVED

L —-V//////////

 

 

55

occurred before 60 days of contamination had elapsed,
the feeding of dieldrin was discontinued. If, however,
calving did not occur by the expected date, dieldrin was
fed until the animal calved. If parturition had not
occurred by 8:00 A.M. the dieldrin for that day was
fed. Thus, the contamination period for individual
animals ranged from 52 to 7A days with an average of
58.7 days.

b. Contamination During 120 to 60 Days Prepartum.——
Seven heifers were contaminated for 60 days at the pre—
viously described level (0.11 mg/kg) commencing 120 days
prior to the expected calving date. The extra animal
included in this group was originally scheduled for the
l80—day contamination group but calved two months early.
The calving span ran from March 31, 1966, to May 10, 1966.
The mean number of days at which contamination commenced
was 112.1 days prepartum, and the time between end of
contamination and parturition avergged 52.1 days (Table l).

c. Contamination During 180 to 120 Days Prepartum.——
An additional group of six heifers was contaminated at
the same rate as the other two groups for 60 days begin—
ning 180 days before expected parturition. For this
group, contamination began an average of 177.5 days pre—
partum resulting in a mean time of 117.5 days between
end of contamination and calving. The calving span for

these animals extended from June 9, 1966 through October

 

56

12, 1966. Data on each individual animal are provided in
Tables 11, III, and IV of the Appendix. Mean group
values for the three stages of contamination are shown

in Table l.

2. Dietary Treatments
Impbsed During Lactation

 

 

The following dietary treatments were employed to
evaluate specific feeding practices which might affect
the rate of residue persistence and excretion during
lactation:

a. Contamination During 60 to 0 Days Prepartum.——

 

(i) Group A (6 animals——medium energy)——Alfalfa
hay was fed at 2.0 kg/lOO kg body weight and grain
at 1.0 kg/3.5 kg milk produced.

(ii) Group B (6 animals——high energy)——Alfalfa hay
was fed at 1.5 kg/100 kg body weight and grain at
1.0 kg/2.0 kg milk produced.

(iii) Group C (6 animals-—depressed milk fat)——
These heifers were fed the same as Group B except
that dehydrated alfalfa pellets replaced long hay
for the purpose of depressing percent milk fat.

b. Contamination During 120 to 60 and 180 to 120

 

Days Prepartum.——The 120-day (7 animals) and l80—day
(6 animals) groups were fed similarly to Group A pre—
viously described. All heifers were fed 9.1 kg of grain

daily for two weeks postpartum. After calving, grain

 

 
 

 

m.ssH

 

 

 

m.saa smo.o mmmm o.oo mom a oma-oma
H.NHH H.mm mmo.o mqflm o.oo sqm s omnoma
m” s.mm o :mo.o aaam s.mm mam ma ones
w\w: we mx wasp
.pz mama .3.m
asses . .

EWWMMMMmm mcfi>fimo UQm mpom oz w><
cospmsfiampsoo QmwmwmwwMmeo pom Qomm msomflom Expsmgomm
. when mass .02 no .02 was aomm

oz Hm590< poanom COHmeHEMono

 

mo mowmpm

.mowpmpmow

omnnp mcflssc QHMUHmHU pom whoaflon mo majosm son sump cospmsfiSmpsooll.H mqm<e

 

58

was fed in accordance with specific treatment regimes.
Biweekly adjustments were made based on milk production
during the preceding week. All heifers were weighed
twice within A8 hours following parturition, and on two

consecutive days at biweekly intervals during lactation.

3. ‘Sample Collection

 

a. Milk.-—For most animals aliquot samples of
colostrum milk were collected from each of the first
eight consecutive milkings following parturition. This
procedure was initiated after five heifers had calved,
thus the data on colostrum are not complete. Colostrum
samples were frozen and stored in pint cardboard con—
tainers of cylindrical shape. Once each week samples
were analyzed for milk fat and extracted for HEOD analysis.
After the colostrum period, daily milk composites were
collected from each cow at weekly intervals for the first
six weeks, and thereafter at 8, 10, 12, 16, 20, 2A, and
30 weeks of lactation. The milking machine buckets were
rinsed with water after each animal was milked to re—
duce cross contamination. Percent milk fat was deter-
mined on each composite sample following the evening
milking as previously described.

b. Body Fat.-—Approximately 5 to 10 g of external
body fat were taken from each heifer prior to contami—
nation, at the end of contamination, at partuirition,

and at 8, 16, and 2A weeks postpartum. These samples

   

 

59

were removed, handled, and extracted as previously
described.

c. Blood.——About 10 ml of blood was collected from
the tail vein of each animal at the same time that the
biopsy operation was performed. Ethylenediaminetetraacetic
acid (EDTA) was used to prevent clotting and the samples
were frozen until extracted for HEOD analysis.

d. Feces and Urine.—-Tota1 collections of urine

 

and feces were made during two 5—day periods, one at the
midpoint of contamination for the l80-day group and the
second at 30 days postpartum for two animals in each of
the 60—day dietary groups. Collections were conducted
in digestion stalls equipped to automatically separate
urine and feces. Sampling procedures were identical to
those described earlier.

e. Feed.-—Throughout the duration of the experi—
ment, samples of grain, hay, and alfalfa pellets were
taken at monthly intervals for HEOD analysis. Procedures
used were the same as discussed previously.

f. Body Tissues from Newborn Calves.——Seventeen

 

newborn male calves were prevented from nursing, trucked
to the Michigan State University Meats Laboratory, and
slaughtered soon after birth. Each calf was stunned with
a captive—bolt gun and immediately exsanguinated. The
carcasses were skinned, eviscerated, and placed in large

plastic bags. The bags were then sealed and frozen.

 

60

The whole frozen carcasses were sliced into one—
eighth inch slices on a band saw and then ground through
a heavy—duty meat grinder with a one—half inch die.

After mixing the ground tissue in a large plastic tub,
the tissue was reground through the one—half inch die
five consecutive times with thorough mixing after each
grinding. Then the tissue was twice ground through a
one—fourth inch die with mixing as before. It was then
ground through a five—sixty—fourth inch die, and after
thoroughly kneading this finely ground material about 10%
was placed in a plastic bag as a subsample. From this
amount, 100 g were analyzed for HEOD content as previously
described. The liver, kidney, renal fat, and brain were
also weighed, ground, and frozen for HEOD analysis.

Omental fat samples were removed by surgical biopsy
from all female calves at birth prior to ingestion of
colostrum. Both omental and shoulder fat biopsies were
taken at 8, 16, 2A, and 52 weeks of age. Blood samples
were taken concurrently with fat biopsies similar to the
manner previously discussed for the heifers. Methods for
fat, tissue, and blood extractions and analyses were the
same as described for the heifers.

A. Sample Extraction,
Clean—up, and Analysis

 

 

Procedures for extraction, column clean—up, and

analysis of samples taken during Experiment 2 were

 

61

identical with those described earlier under Experiment 1
for milk, body fat, body tissue, feces, urine, and feed.
Blood samples were extracted according to Crosby
and Archer (12A). All solvents were of Nanograde‘g’56
quality. The gas chromatograph used was an Aerograph
Model 152010 equipped with an electron—capture detector
and a Westronics Model LDllA recorder.11 The most satis—
factory chromatographic column used was a 5' x 1/8" o.d.
stainless steel tube packed with 8% Silicone DC-ll and
A% QF—l on 60/80 mesh Gas—Chrom Q. Carrier gas was
ultra—pure nitrogenl2 (A0 p.s.i.) with a flow of 30—A0
ml/minute. Operating temperatures were: injection port,
190°C; column, 175°C; and detector, 190°C. HEOD residue
recoveries were determined by comparisons of retention
time and peak height with known standard samples in-
jected into the gas chromatograph. Sensitivity was such
that 2 picograms (2 x lO-lZg) of HEOD gave a measurable
peak.
5. Feeding Contaminated vs
”Clean" Milk to Calves
Contaminated in Utero
To ascertain the effect of feeding milk from con—

taminated animals on residue levels in growing calves,

 

10Varian Aerograph, Walnut Creek, California.

llWestronics, Inc., Fort Worth, Texas.

l2Liquid Carbonic, Division of General Dynamics,
Detroit, Michigan.

*
Standardized Copyright symbol.

 

62

female calves born to dams contaminated during the last
60 days of gestation were alternately assigned to either
a contaminated or non—contaminated milk diet. The calves
receiving contaminated milk were allowed to nurse their
dams for 2A hours and were then removed and fed contami—
nated colostrum from their dams for an additional 2A
hours. Starting A8 hours after birth these calves were
fed the dieldrin—containing milk from their own dams
until weaned at 12 weeks of age.

Calves fed "clean" milk were removed from their
dams immediately after birth without being allowed to
nurse. Dieldrin-free colostrum, frozen from non—contami—
nated cows, was fed for the first A8 hours. Thereafter,
dieldrin—free milk was fed until the calves were weaned
at 12 weeks of age.

A sample of the milk fed to each calf was taken
five days per week and composited for HEOD analysis. Hay
and grain fed to these calves were sampled at monthly
intervals for HEOD analysis. All calves were weighed on
two consecutive days at four-week intervals. Milk, hay,
and grain feeding were done according to the regular

Michigan State University calf rearing program.

 

 

IV. RESULTS

A. Experiment l——Excretion and Storage of Dieldrin
in Dairy Cows Fed Thyroprotein and
Different Levels of Energylj

 

 

 

1. Energy Intake

Energy intakes for each treatment during contami—
nation and decontamination are calculated as percent of
the 1966 NRC requirement for TDN and shown in Table 2.

TABLE 2.——TDN intake of cows as percent of the NRC
requirement.a

 

 

 

 

Contami- Decontamination
Tre t ent nation
a m First Second Third Fourth Last
8 wks A wks A wks A wks A wks 2 wks
%
150% NRC 97.7 110.5 115.A 115.2 125.2 130.3
75% NRC 99.6 80.2 79.1 76.A 73.9 73.9
100% NRC 101.0 10A.9 100.9 100.8 98.1 96.8
150% NRC +
thyroprotein lOl.A 98.7 lO6.A 127.1 l38.A 138.0

 

aNational Academy of Sciences-—National Research
Council (NRC). 1966. Nutrient Requirements of Dairy Cattle,
Publication 13A9.

 

13Presented in part at the Sixty—First Annual Meeting
Of tflie American Dairy Science Association, Oregon State
Lhr1versity, Corvallis, June, 1966 (15).

63

 

1 1 1111111 11.11

6A

All cows were offered 100% of the NRC requirement during
the eight—week contamination period. During decontami—
nation it was intended to feed two groups 150%; but, be-
‘cause of appetite limitations during the first eight
'weeks, intake in proportion to requirement increased
gonly slightly. This was especially true for the cows
fed thyroprotein because an increased response in milk
production resulted in a correspondingly higher require—
ment. During the latter weeks of treatment, when lower
production resulted in decreased energy requirements,
appetite appeared less limiting and TDN intake was con—
siderably above requirements for both the thyroprotein
and high energy groups, but it never reached the desired

150%.

2. Body Weight Changes

 

Thyroprotein administration caused a rapid loss of
body weight of cows averaging 57 kg four weeks after
initiation of feeding. Thereafter, body weight plateaued
until the twelfth week of decontamination and small gains
were noted between 12 and 18 weeks. Body weight remained
constant for the control cows until late in the treatment
period after which slight increases were noted, while
the 150% group exhibited an early and sustained increase.
Weights of the 75% group decreased slowly during the en—
tire decontamination period. Larger weight losses for
this treatment were anticipated but they did not material—

ize. Body weight data are shown in Figure 2.

 

 

   

3. Milk Production

 

The average daily milk production is presented in
Figure 3. A sharp initial increase was obtained for the
thyroprotein treatment with a smaller increase for the
150% group. The definite drop in production during the
fourth week of decontamination and subsequent recovery
was due to several days of extremely hot weather which
adversely affected most animals in the herd. As a re—
sult of decreased energy intake, the 75% group responded
with a larger production decline than the other groups.
These decreases preceded body weight loss. The precip—
itous decline in production for the 150% group during
weeks 9, 10, and 11 occurred because the highest pro—
ducing cow in the group suffered an udder injury and
acute mastitis.

A. Excretion of HEOD in
Milk Fat

 

The percent of the HEOD intake excreted during the
contamination period is presented in Table 3. By the
end of contamination approximately 19% of the total HEOD
fed had been excreted in the milk fat.

HEOD residues in the milk fat of weekly samples
fluctuated considerably and as indicated in Figure A,
dropped rapidly for all cows, regardless of treatment,
to about one—third the initial levels after five weeks
of decontamination. Thereafter, the rate of decline

was slower with apparent plateaus in concentrations

 

 

 

   

 

 

 

66

Figure 2.——Body weight changes during decontami—
nation of cows fed thyroprotein and different levels of
energy.

 

 

 

2952332083 85m;

 

 

 

 

 

9 w. v. N_ O. m w ¢ N .
_1 . a a _ . _ q _ - 4 a _ . _ _ q 00.
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000...? I, 1: on. '.000000 a o
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noon»... ..... -\ 0,.‘0 10¢
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\.\.‘o 1 om
-Om

zo_._.<z_2<._.zoowo 02550 wez<Io FIG—m3

>oom

ml 'N/VQ lHQ/JM

 

 

 

 

 

 

 

 

Figure 3.--Average daily milk production during
contamination and decontamination of cows fed thyro—
protein and different levels of energy.

 

AVE. DAILY MILK kg.

   
 

 

 

 

 

AVERAGE DAILY MILK PRODUCTION

3O -' O,

D \.

T. ..\.a..

. , r |50°/o NRC + THYROPROTEIN

I- 0‘ “so \‘

\ .s

25’ wines. 6“- "“7.

7 ' . ‘0 .equso‘y. NRC

- ‘:.\‘...... /l O:. .

.- \‘b::‘., Q / ."

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L L I I 1 I 1 I I I 1 I 1 I u I n I I l l l n l 1 J

| 2 4 6 8 2 4 6 8 IO [2 I4 I6 I8
CONTAMINATION DECONTAMINATION

 

 

 

   

 

70

 

Figure A.—-Levels of HEOD in milk fat of cows
contaminated during lactation and fed thyroprotein
and different levels of energy.

   

zo_.r<z_2<._.zoomo

w v. m m 3
zo_._.<z_2<.rzoo

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‘\ I

 

— - — A — q — u — _ a — . _ u — -

 

 

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@2350 ._.<.._ v3.2, 2. com:

 

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n—

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b/fm’ ‘lVJ mm NI 0034

 

 

 

 

72

TABLE 3.——Percent of HEOD intake excreted in milk fat
during the eight-week contamination period of all cows.

 

,—

Weeks of Contamination

 

1 2 3 A 5 6 7 8

 

% of intake
excreted
weekly 11.7 19.6 15.2 l5.A 16.8 21.5 23.9 2A.A

% of intake
excreted to
date 11.7 15.6 15.5 l5.A 15.7 16.7 17.7 18.6

 

occurring between weeks 5 to 10 and 12 to 18 of decontami-
nation. Eighteen weeks after dieldrin feeding stopped,
the milk from twelve cows still contained measurable levels
of HEOD (0.01 ppm). Calculation of regression coefficients
for the HEOD levels in milk fat, with transformation to
log Y + 1, and statistical tests for goodness—of—fit,
showed that the HEOD residues in milk fat did not decline
at a straight line, exponential rate.

During the 18 weeks of decontamination an average
of about 19% of the total HEOD intake was excreted in the
milk fat (Table A). There was no significant treatment
difference in the percent of HEOD intake excreted. The
mean percent excretion of HEOD was almost equally divided
between the eight weeks of contamination and 18 weeks of
decontamination. Therefore, 26 weeks after initiation of
contamination about 37% of the total HEOD ingested had

been accounted for in the milk fat (Table A).

 

 

 

 

73

TABLE 4.-—HEOD excretion pattern for cows contaminated
during lactation.

 

Percent of Intake Excreted During:

 

 

 

Treatment HEOD

Group Intake 8 wk 18 wk 26 wk

Cont. Decont. Total
mg , %

150% NRC 2970 22.5 19.0 41.5
75% NRC 2692 19.4 19.2 38.6
100% NRC 2939 14.7 16.6 31.3
150% NRC +
thyroprotein 2939 16.9 21.0 37.9
S. E. of
Mean 160 3.5 2.8 5.4
Overall
Mean 2885 i 80 18.6 i 1.7 19.0 i 1.4 37.4 i 2.7

 

5. HEOD Levels in
Body Fat

 

During decontamination HEOD residues were determined
in samples of external shoulder fat removed from two cows
in each group at eight—week intervals. These data are
plotted in Figure 5. Initial values averaged 10 ppm, but
declined exponentially to approximately 5 ppm at eight
weeks and 1 ppm at 16 weeks. Regression coefficients of
ppm HEOD in body fat on days of decontamination are
shown in Table 5. Although the cows fed thyroprotein
declined at the fastest rate, numbers were probably in—

sufficient to detect a significant difference.

 

 

   

 

74

 

Figure 5.——Levels of HEOD in external body fat
during decontamination of cows fed thyroprotein and
different levels of energy.

 

 

 

zo_._.<z:2<._.zoowo wxmm?
m

_

._.<u_ >oom .._<zmm._.xm 2. Dow:

 

 

N_

b/fm’lva A008 NI 003H

 

 

 

 

76

TABLE 5.——Regression coefficients of ppm HEOD in body fat
on days of decontamination for cows contaminated during

 

 

lactation.
Treatment Regression ' Standard Correlation
% of NRC Coefficient Error R2
150 —.OO677 .00126 .88
75 —.OO6A8 .00188 .75
100 —.00590 .00161 .77
150 +
thyro—
protein —.00705 .00261 .65

 

6. HEOD Levels in Tissues
and Body Fat of Slaughtered
Cows

 

After 18 weeks of decontamination six cows were
slaughtered for various reasons. By coincidence three
animals were from the thyroprotein—fed group and one
from each of the other three treatments. Residue levels
in fat at slaughter indicated a definite difference be—
tween thyroprotein~fed cows compared with the three
others; therefore, the data for cows which did not re-
ceive thyroprotein were pooled and mean values are pre—
sented in Table 6.

Residues in kidney, liver, and udder tissues
showed little difference between the two groups. How-
ever, residues in shoulder, kidney, and omental fat were

three to five times higher for the thyroprotein—fed

77

cows. The differences for concentrations in omental

fat were highly significant-(P < .01) and levels in the

kidney fat approached significance (P < .10).

TABLE 6.——HEOD residues in tissues and body fat of cows
fed thyroprotein or no thyroprotein after 18 weeks of

 

 

 

 

 

decontamination.
PPM in Tissues PPM in Fat
. . Shoulder Kidney Omental
Kidney Liver Udder Fat Fat Fat

Mean for
3 non—
thyro—
protein a c
cows .03 .13 .07 1.24 .57 .72
Mean for
3 thyro—
protein b d
cows .03 .15 .10 3.69 2.80 2.42
S. E. of
Mean .01 .03 .02 1.2 .8 .2
Ratio:
Thyro—
protein
non-thyro—
protein 1.0 1.2 1.4 3.0 A.9 3.4

a’bDifference approaches significance (P < .10).

C’dMeans are significantly different (P < .01).

 

 

 

78

B. Experiment 2——Storage, Excretion, and
Placental Transfer of Dieldrin by Dairy
Heifers Contaminated During Three
Stgggs of Gestationi”

 

 

 

 

l. Experimgntal Treatments
and Body Weight Changes

During Lactation

Lactation rations, mean HEOD intake, and stage of

 

gestation during which contamination occurred are sum-
marized in Table 7. Body weight data obtained for each

heifer on two consecutive days at biweekly intervals

TABLE 7.——Contamination and ration schedules for heifers.

 

 

HEOD Fed Total Conc. Gest. Stage Lactation
Prepartum Intake in Feed at Contam. Ration
days mg mg/kg months
60—0 A 316A 5.6 8—9 Normal
60—0 B 2982 5.4 8—9 High
Grain
60—0 C 3189 5.A 8—9 Pelleted
Hay
120—60 3145 5.3 6—7 Normal
180—120 2939 5.4 4—5 Normal

 

during lactation are plotted in Figure 6. The 60—day B
group, receiving the highest level of energy intake

reached minimum body weight at six weeks postpartum,

 

luPresented in part at the Sixty-Second Annual
Meeting of the American Dairy Science Association,
Cornell University, Ithaca, New York, June, 1967 (16).

 

 

Figure 6.——Body weight changes of heifers con—
taminated at various stages of gestation and fed
different rations during lactation.

 

 

 

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81

but regained post-calving weight between 22 and 24 weeks.
Losses in weight for other groups were of a greater
magnitude and more persistent.
2. Milk and Milk Fat
Production

Table 8 shows the total yield of milk, milk fat,
and percent milk fat during the first 24 weeks of
lactation. Although milk production was highest in
those groups fed the diet of high grain and pelleted
hay, mean production was not significantly different
among the five treatment groups.

TABLE 8.——Total milk and milk fat production of heifers
during the first 24 weeks of lactation.

 

 

 

HEOD Fed . Milk
Prepartum Milk Fat Fat
days kg % ks
60-0 A 3718 3.27a 120ab
60—0 B 3880 2.78b ioobC
60-0 0 A149 2.21C 92C
120—60 3908 3.65a 142a
180—120 3A9A 3.52a 123ab
S. E. of Mean 285 0.16 8
a,b,c

Means sharing a common superscript are not
significantly different (P < .05).

 

 

82

Milk fat percentages for the three groups on the
normal rations (60—A, 120—, and 180-day) were signifi-
cantly higher than for 60—B or 60-C heifers (P < .05).
Milk of the 60—0 group contained less fat than that of
the 60—B animals (P < .05). Milk fat yields for the
groups on the normal ration did not differ, but were
significantly higher (P < .05) than for the group re—
ceiving pellets; moreover, fat yield for the l20—day
group was significantly higher (P < .05) than for the
high—grain group.

3. Body Storage of
HEOD

 

a.——Pre— and Postpartum HEOD Concentrations in

 

External Body Fat.——HEOD levels in external body fat

 

samples removed from the shoulder area of each experi—
mental animal at different pre- and postpartum stages
are presented in Table 9 and Figure 7. Although total
intake was very similar, the stage of gestation during
which the pesticide was ingested exerted a highly
significant effect upon storage levels in body fat

(P < .005). Mean HEOD levels at the end of contami—
nation were 10, 18, and 34 ppm, respectively, for the
three stages of gestation. During the two— and four—
month period between end of contamination and partur-
ition, concentrations for the 120— and 180—day animals

changed only slightly.

 

 

 

 

83

 

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84

Following parturition, body fat residues declined
exponentially as the pesticide was excreted in the milk.
At eight weeks postpartum HEOD concentrations were
approximately one-half their level at parturition with
the relative differences highly significant as indicated
in Table 10. By 16 weeks postpartum no significant
differences existed in body fat residues among treatments.

TABLE lO.——Orthogonal contrast comparisons of HEOD concen-
trations in body fat of heifers.

 

During Lactation

 

 

Treatment End At

Contrast Contamination caIVing 8 wk. 16 wk. 2A wk.
60 A, B, C a

VS 120, 180 9696* *tHe *th N.S. N.S.
60 A vs b

60 B, C N.S. N.S. N.S. N.S. N.S.
60 B vs

60 C N.S. N.S. *** N.S. N.S.
120 vs 180 *** *** *** NS NS

 

aTreatment ccmpa:i*ons significantly different
(P < .005).

bTreatment comparisons not significantly different
(P < .05).

After 24 weeks of lactaticn the average HEOD concentration
in body fat of all heifers was approximately 1 ppm With

no significant differences among groups Three of 31
animals had less than .001 ppm in body fat at 24 weeks

postpartum.

 

 

 

 

Figure 7.-—Pre— and postpartum levels of HEOD in
external body fat of heifers contaminated at various
I stages of gestation.

 

 

     

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87

Regression coefficients of ppm HEOD in body fat on
days of lactation are shown in Table 11. Residues in
heifers contaminated during 180— to 120—days prepartum
declined at a significantly faster rate (P < .01) than
(those in any other group.

TABLE ll.—-Regression coefficients of ppm HEOD in body fat

on days of lactation for heifers contaminated during various
stages of gestation.

 

 

 

HEOD Fed Regression Standard Correlation
Prepartum Coefficient Error R2
days

60—0 A —.00A98a .00046 .85
60-0 B —.00A1Aa .00034 .88
60—0 0 —.00A8ia .00053 .80

120 — 60 -.00514a .00041 .88

180 — 120 —.00723b .00051 .90

a,b

Regression coefficients sharing a common super—
script are not significantly different (P < .01).

b.—-Pre— and Postpartum HEOD Concentrations in

 

Blood.——Blood samples obtained simultaneously with fat
biopsy samples were analyzed for HEOD and the results are
presented in Table 12 and in Figure 8.

In contrast to the highly significant differences
in body fat residue levels found at the end of contami—
nation, blood HEOD concentrations were not significantly

different. Mean blood levels were approximately l5, l8,

 

 

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. all. ..

89

Figure 8.——Pre— and postpartum levels of HEOD in
blood of heifers contaminated at various stages of
gestation.

 

 

 

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91

and 16 ppb (parts per billion) at the end of contami—
nation for the groups contaminated at the respective
stages of gestation (60-, 120—, and l80—days prepartum).
During the two— and four—month period between cessation
of HEOD intake and parturition, blood levels dropped
from 18.4 to 6,9 ppb for the l20—day group and from
16.2 to 7.“ ppb for the l80—day heifers.

At parturition, blood HEOD concentrations were
significantly higher (P < .01) for the 60—day heifers
than for either the 120— or l80—day groups as indicated
in Table 12. Stages of contamination had no significant
effect on blood HEOD levels at 8, 16, or 24 weeks post—
partum and by 2M weeks postpartum concentrations for all
groups averaged about 1 ppb.

0. Content of HEOD in Body Fat and Blood at

 

Parturition.——Presented in Table 13 are the calculated
amounts of HEOD contained in the body fat and blood at
parturition based on concentrations determined for each
tissue. To determine the total body burden of pesticide,
body fat content of heifers was estimated at 10%, where—
as 57.4 ml/kg body weight was assumed for blood volume.
Based on these calculations, approximately 17, 30,
and 59% of the pesticide consumed was accounted for at
parturition in the blood and body fat of the 60—, 120—,
and l80eday groups, respectively. These differences are

highly significant (P < .01).

 
 

 

 

92

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93

d. Distribution at Parturition of HEOD Fed During

 

Gestation.—-Calculated in Table 14 is the distribution at
parturition of the HEOD fed during gestation. Based on
these values about 34% of the total intake was accounted
for at parturition. The major amount was found in body
fat and blood, with approximately 2.5% excreted in the
feces during contamination and about 1% recovered in the
calf carcasses.

TABLE lU.——Distribution at parturition of HEOD fed to
heifers during gestation.

 

HEOD Fed Excreted in In Blood and Recovered Total % of
Prepartum Feces During Body Fat in Calf Intake Ac—
Intakea At Calvingb Carcasses counted for

at Calving

 

 

days %
60-0 A 2.5 iu.8C 1.0 18.3C
’60—0 B 2.5 19.3C 1.0 22.8C
60—0 c 2.5 16 [C 1,0 20 2C
120—60 2.5 29 6d 1.0 33 1d
180—120 2.5 59 he 1.0 62 96
Mean 2 5 50 l l.O 33 6

 

aCalculated from data obtained in total collection
trial at midpoint of Contamination period for l80—day group
(see Table l9).

bBody fat compOSItion estimated at 10%; whole blood
volume calculate at 57.” ml/kg body weight, Biology Data
Book, 196M.

C’d’eMe-ans sharing a common superscript are not
significantly different (P < .01)

 

‘ rum Milk.—-It is a

  

1% present in the first milk available at‘

for all animals as summarized in Table 15. JMean
concentrations in milk fat of the first eight milkings
were 17.6, 23.7, and 30.9 ppm for the respective stages

of contamination. These values reflect the body fat
levels of HEOD at parturition (9.9, 16.8, and 31.7 PPm,
respectively).

b. Concentrations of HEOD in Milk Fat During
Lactation.-—Residue levels in milk fat during lactation
are presented in Figure 9. Concentrations dropped rapidly
for all heifers with similar, but less definite patterns
than noted for Experiment 2. As with Experiment 2, the
regression of HEOD level in milk fat on days of lactation
did not decline at a straight line, exponential rate.
Concentrations in milk fat for heifers in groups 60-A and
60—B were not significantly different; whereas, grOup
60—C animals, which averaged 2.2% fat for the first 24
weeks (compared with 3.2% for the control group), ex-
hibited higher HEOD concentrations (P < .01).

Residue concentrations for the 120— and l80—day
heifers were Significantly greater than for any of the
60—day treatments (P < .01). Moreover, HEOD levels for
the l80—day animals were higher than for the lEO—day

heifers, again reflecting a higher (P < .05) prepartum

 

 

 

95

 

 

 

 

 

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Figure 9.—-Levels of HEOD in milk fat of heifers
contaminated at various stages of gestation.

 

   

c2883 .0 9.33

 

 

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98

storage of the pesticide in the body. At 30 weeks post—
partum, milk fat residues had declined to less than 1
ppm for nearly all groups.

c. Excretion of HEOD During Lactation.——The mean

 

daily excretion of HEOD is shown in Figure 10. Of the
three 60—day groups, the amount of pesticide excreted
daily was slightly higher for the group fed pellets be—
cause of the increased concentrations in milk (Figure 9).
Although the total amount of pesticide excreted during

24 weeks of lactation was about 43% greater for the
pellet—fed than the other 60—day groups, differences were
not significant. Both the 120— and l80—day groups ex—
creted significantly larger amounts of pesticide during
lactation than the 60—day groups (P < .01).

Stage of gestation during contamination had a
significant effect on the cumulative percent of total
pesticide ingested which was excreted during lactation
(Table l6). After 24 weeks of lactation, the 120— and
l80-day heifers had excreted over “0% of their total in—
take compared with about l8% for the 60—day groups. The
means for the 60—day heifers were significantly less than
for the 120— and l80—day groups at each six week interval
(P < .01).

In Table 17 are shown ration effects on pesticide
excretion. Animals fed pelleted hay had excreted a
higher percentage (P < .05) of their pesticide intake in
milk fat by l8 and 24 weeks than those on normal or high

grain diets.

 

 

 

Figure lO.--HEOD excreted daily in milk fat of
heifers contaminated at various stages of gestation.

 

 

   

Mg /cow/day

0')

UI

 

Dafly HEOD Exmefian
in Milk Fat During Lactation

18x0 day
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102

TABLE l7.—-Cumulative excretion of HEOD in milk fat as
affected by the ration.

 

 

 

Ration HEOD Fed 6 wks 12 wks 18 wks 24 wks
mg —————————————— % of intake —————————————

Normal 3164 7.6 12.2 114.5a 15.11a

High a a

grain 2982 8.2 11.9 14.5 15.8
Pelleted b b

hay 3189 10.2 16.4 20.0 21.5

S.E. of

Mean 2.9 2.9 2.9 2.9

a b

’ Means sharing a common superscript are not
significantly different (P < .05).

d. Fate of HEOD Found in Body Fat and Blood at
Parturition.——Presented in Table 18 is the disposition
of the pesticide assumed present in the body fat and
blood at parturition. The assumptions made for body

fat composition and leQi volume required for calculations

of these data were dj-;to;ed earlier. Of the amount pre—
sent in the body at pa tt:itior, 116% was excreted in

the milk, Z% eliminated via feces,and 8% remained in
body fat and blood after 24 weeks of lactation.

e. Excretion of HEOD in Feces and Urine.-—To in—

 

vestigate possible excretory pathways of HEOD, total
collections of urine and feces were made during two
5—day periods; one at the midpoint of the contamination
period for the l80-day group and the second at 30 days

postpartum for two animals in each of the 60—day groups.

 

 

 

 

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.m:m>mmo om whommom mo oooan one pom moon cm ogzom momm mo opom-l.om mamas

 

   

104

Shown in Table 19 are data on excretion of HEOD by
non-lactating dairy heifers during contamination. A mean
concentration of 0.52 ppm HEOD found in the feces ac—
counted for an average of 2.5% of the daily intake. Only
trace amounts (<.OOl ppm) of HEOD were found in the
urine. Mean HEOD excretion for two heifers in each of
the 60—day treatment groups at 30 days postpartum are
presented in Table 20. Milk was definitely the major
excretory pathway of HEOD from lactating animals. A
much smaller amount was eliminated in feces while only
trace amounts were found in urine.

5. Placental Transfer
of HEOD

 

a. Tissue Residue Levels in Newborn Male Calves.“—

 

Placental transfer of HEOD occurred in each of 33 dams,
resulting in contamination of all newborn calves. Seven—
teen male calves were prevented from nursing and slaught—
ered immediately after birth. Mean levels in body tissues
from these calves are presented in Table El. Neither
differences in HEOD in blood or body fat of the dams at
parturition significantly affeCted the mean residue level
in whole carcass, carcass fat, brain, or renal fat of the
male calves. HEOD concentrations in the kidneys of the
calves born to heifers Contaminated at 180 days were
significantly greater than those of the bO-day group

(P < .01). However, residues in livers of calves born

 

 
 

105

TABLE 19.v—Daily excretion of HEOD in feces and urine of
non-lactating heifers at midpoint of 60 day contamination

period.

 

HEOD Concentration HEOD Excreted in Feces

 

 

 

 

Heifer
Feces Urine Total % of Amount Fed
mN/kg mg
H—786 .47 <.OOl 1.18 2.3
H—788 .59 <.OOl 1.27 2.6
H—783 .65 <.OOl 1.68 3.2
H—814 .35 <.OOl 1.04 2.0
Mean .52 <.OOl 1.29 2.5
aData are from four heifers contaminated 180 to

120 days prepartum.

 

106

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d

 

 

108

in the 60—day group were higher for those born to 120—
and 180—day animals.

Calculated recoveries of HEOD in the male calves
averaged 0.9% of the total ingested by the dams. Total
placental transfer was probably higher than the recovery
figures indicate because the calves were skinned prior
to grinding of the carcasses for HEOD analysis. Further—
more, HEOD in the placental tissues and fluids was not
determined.

Blood residue levels taken at birth and averaged
for all calves are given in Table 22. It is apparent
that calves born to heifers contaminated during the last
60 days of gestation had higher blood HEOD levels than
those born to heifers contaminated for a similar time,
but starting at 120 and 180 days prepartum. A direct
relationship between blood residue levels in the dams
and their calves was shown.

Seven female calves were sacrificed or died during
the growing period. Concentrations of HEOD in liver,
kidney, brain, and renal fat from these calves averaged
0.75, 0.29, O.lO,and 26.20 ug/g, respectively. These
values agreed closely with the comparable means observed
for newborn males as shown in Table 21.

b. Age Effect on HEOD Concentrations in Blood

 

and Omental Fat of Female Ca1ves.——The effect of age on

 

blood residues in female calves is presented in Table 23.

 

 
 

109

TABLE 22.——Comparison of HEOD concentrations in body fat
and blood of dams at parturition with blood levels in
all newborn calves.a

 

HEOD in Dams

 

 

HEOD Fed at Calving Blood HEOD in
Prepartum a Calves at Birth
Body Fat Blood
days ug/g mug/m1 mug/ml

60—0 9.9C 1 .8b 15.2c _ 1.2 6.2f i .9
(16) (14) (12)

120—60 16.8d i 1.2 6 9d 1 1.7 1.8 i 1.2
(7) (7) (7)

180—120 31.7e i 1.3 7.ud i 1.8 2.8 i 1.8
(6) (6) (3)

 

aNumber of animals
in parentheses.

each value represents is given

bStandard error of

the mean.
c d e . .
’ ’ Means sharing common superscript are not
significantly differen; ’P ~ .01)
f . . . . . . .
The general analysis Ci variance test indicates

a significant dllfer,n a if .05) among treatment means
but Duncan's New Multiple Range Test does not indicate
so at the same level J1 pxibability. 1f, in fact, these
differences are real 1: is apparent which means are
different.

 
 

110

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111

Calves born to dams in the 60—day group contained signifi—
cantly higher levels of HEOD in their blood than those of
dams from the other two groups (P < .05). HEOD in the
blood disappeared more quickly than that found in the
omental fat (Table 24). At 16 weeks of age HEOD in
blood was detectable in only one of seven calves, but
none was found at 24 weeks in the four calves sampled.
HEOD in omental fat removed from female calves at
various ages was not significantly affected by the stage
of gestation during which their dams were contaminated
nor by the concentration of HEOD in the body fat of the
dams (Table 24). The average rate of decline in HEOD
concentration in body fat was similar for calves born to
both the 60—day and l20—day groups. Prediction equations
for rate of decline of HEOD in omental fat for calves

from the 60— and l20—day groups were

Y = 7.13 + (—.221)X

t< \
||

9.69 + (—.210)x ,

respectively. Thus, under the conditions of this study,
approximately 40 weeks were required before residues in
omental fat of calves contaminated in utero declined
below detectable limits. At 52 weeks of age, HEOD was

detectable in only one of five calves. Neither did

   

 

112

 

 

 

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113

stage of gestation during which dams were contaminated
affect residue levels in samples of shoulder fat taken
simultaneously with the omental fat from female calves.
These data are consistent with the similar concentration
of HEOD found in renal and carcass fat of male calves,
regardless of when the dams were contaminated.

6. Effect of Feeding Dams'

Contaminated Milk or Non—

Contaminated Milk to Calves
Contaminated In Utero

 

Residue levels in omental fat of female calves fed
either contaminated milk from their dams or non—contami-
nated milk for 12 weeks are shown in Table 25. Mean
TABLE 25.——Comparison of HEOD levels in omental fat of

calves contaminated in utero when fed dams’ HEOD—contami—
nated milk or non—contaminated milk for 12 weeks.a

 

 

Age of Calves

 

 

 

 

Milk HEOD
Fed Intake Birth 8 wks 16 wks 24 wks 52 wks
ms ug/g
Non—Con—
taminated O 6.38 5.06 3.23 2.66 0.17
(5) (2) (2) (l) (2)
‘Dams' Con—
taminated 79 8.60 4.97 2.58 2.37 <.OOl

(4) (3) (2) (3) (2)

 

a . . .
Number of animals each value represents is given
in parentheses.

 

 

 

1.1: 8'

.11-

calf during the 12-week feeding ff“:

a.
‘*3 concentration in the whole milk av~wxgau

 

ppm during the first week and declined to 0.06 ppm dur—
ing the twelfth week of feeding. No detectable resi-
dues were found in the non-contaminated milk fed to
calves. No significant difference in residue concen—
trations<ifomenal fat resulted from the two milk feed—

ing regimes.

   

  

”r earlierijigestation dairy heifers were contaminated with

dieldrin, the greater (P < .005) the storage of HEOD in
their body fat. For example, storage resulting from the
120- and 180-day prepartum contamination was approxi—
mately two and three times, respectively, that found
for heifers contaminated 60 days prepartum, even though
total pesticide intake was essentially equal (Table 26).
Explanations for the stage of gestation effect upon
HEOD storage are not readily apparent, but various theor-
ies are proposed. First, the body weight data in Table
27 indicate that the 60—day prepartum heifers gained more
weight during the contamination period (56 vs 43 and 28
kg, respectively) than the 120— and l80—day groups. Thus,
the HEOD available for storage might have been diluted by
a larger body fat mass in the 60— than the 120— and 180—
day groups. However, much of this weight gain, especially
for the 60—day heifers, was undoubtedly due to the rapidly
developing fetus, with minimal fat deposition, since more
than half of the fetal—weight increase occurs during the

last two mcnths of gestation (114). Therefore, the

 

  
  

 

 

116

 

 

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118

dilution effect of increased body fat deposition on HEOD
storage would be negated.

Secondly, the possibility exists that the HEOD was
stored in dissimilar body fat depots during the three
different stages of contamination. Hence, the external
body fat biopsies might have not truly represented the
total body burden of the pesticide.

Alternatively, the endocrine changes caused by
advancing pregnancy might have stimulated a greater
metabolism of the pesticide resulting in less unchanged
HEOD. Data in Table 28 indicate a dramatic increase
(6 to 16 fold) in estrogen production during the last
five weeks of pregnancy (47, 70) which corresponds to
the contamination period of the 60—day groups. Nalbandov
(104) pointed out that estrogens cause profound changes
in liver metabolism such as increased lipemia. Estro—
gens also have a marked effect on certain enzyme systems
in the rat as shown by a quantitative increase in seven
different enzyme systems activating amino acids of rat
uteri three hours folloWing a single injection of the
hormone (104). Enzyme activities continued to increase
for the ensuing 24 hours. The estrogen was thought to
act by stimulating enzyme synthesis d3 ngvg, by activat—
ing already existing enzymes, or by uncoupling the
enZymes which, after being "released," could participate

in cellular metabolism (97). It is conceivable that the

   

 

 

119

TABLE 28.——Re1ative amounts of estrogen found in the
urine of cows at various stages of pregnancy compared
with prepartum contamination periods.

 

Ratio Relative

 

 

 

Stage of to Amount Contamination Period
Pregnancy Found at
101—123 Days Days Prepartum
days a b

101—123 1.0 1.0 ———————————————180
165—175 1.9 3.1

}——————-———~————l20
200—212 1.9 O 7
226—237 2.4 —-—
250—254 6.7 6.1 }—————————-———— 60
271—285 11.3 16.1

a

R. E. Erb et al. (47)

F. L. Hisaw, and R. K. Meyer (70).

high levels of estrogen in late pregnancy might stimu—
late enzyme systems capable of metabolizing dieldrin;
thus, less intact dieldrin would remain for storage as
was the case with the 60—day heifers compared with those
contaminated earlier in gestation.

Coinciding with the rapid rise in estrogen at about
250 days of pregnancy is a decrease in progesterone.
Short (118) found that progesterone levels in cows re—
mained remarkably constant from the 32nd to about the

256th day of pregnancy, ranging from 0.74 to 0.98 pg/lOO

   

 

 

120

ml plasma. Subsequently, there was a decrease in the
level and on the day before calving only 0.4 to 0.1
ug/lOO ml was found. The effect of decreased progester—
one and a shift in the estrogenzprogesterone ratio on
dieldrin—metabolizing enzymes needs investigation.

Conney et_al. (27) studied the effects of pesti—
cides on drug and steriod metabolism. They emphasized
that the drug—metabolizing enzymes in liver microsomes
may also play a role in the metabolism of other body
substrates and thereby regulate important biochemical
reactions in the body. If such is the case, drugs and
insecticides may alter normal body metabolism through
their action on liver microsomal enzymes.

In general, the biotransformations of foreign
chemicals which are catalyzed by the liver microsomal
enzymes result in the conversion of foreign substrates
to less toxic, more water—soluble (or less lipid—soluble)
materials (17, 55). These changes usually involve an
increase in urinary excretion of the foreign compound by
decreasing tubular reabsorption, a process that depends
on solubility in the lipoidal membranes of the kidney
tubules. Also, the lipid-insoluble metabolites are less
able to penetrate other membranes to reach sites of
storage. Thus, the more rapid excretion rate and the
lower capacity for penetration of membranes would de—

crease the amount of both the original compound and its

 

121

metabolites found in body storage sites. It is con-
ceivable that in our studies biotransformation of HEOD
was stimulated by hormonal changes characteristic of
impending parturition. Hence, less unchanged HEOD would
have been available for storage in body fat during the
latter stages of gestation with the resulting meta—
bolite(s) excreted via the urine and escaping detection
by our analytical methods.

Non—specific oxidizing enzymes in the microsomal
fraction of liver cells are responsible for oxidative
conversion of many lipid—soluble compounds. Brooks (18)
recently found a species difference in the action of
these enzymes. He showed that pig liver, in addition to
producing the normal oxidation products, cleaved the
epoxide ring of cyclodiene metabolites to yield the diol
products; whereas, rat liver was incapable of the epoxide
change. This reaction is not catalyzed by microsomal
mixed—function oxidases; thus, the pig has some additional
mechanists) whi‘h elicitks) this cleavage. If such a
mechanism exists in the bovine, cleavage of the epoxide
ring of HEOD would yield a more hydrophilic compound that
could be excreted in the urine leaving less unchanged
HEOD available for body storage. The possibility that
Similar mechanisms exist in other species, including

the bovine, merits investigation.

 
 

 

122

A most interesting phenomenon in these studies was
the lack of change in the level of stored HEOD during
the two—or four-month period between end of contami—
nation and parturition in the l20— and l80—day groups
(Table 9, Figure 7). These results are contrary to
data obtained in other studies which showed a rapid de—
cline in body storage of chlorinated hydrocarbon pesti—
cides upon cessation of intake (l0, 52, 54, 87). If
estrogen does stimulate metabolism of HEOD, it would
appear to do so before the pesticide molecule is stored
in the fat depot, but the hormone probably is much less
effective once storage has occurred.

In contrast to the highly significant differences
in body fat residues found at the end of contamination,
blood HEOD levels were not significantly different.

Mean blood levels were approximately 15, 18, and 16 ppb
(parts per billion) at the end of contamination for the
three stages oi gestation (Figure 8, Table ll). Thus,

it would appear that similar levels of circulating pesti—
cide were presented to body storage sites of all groups.

The finding of about 7 ppb HEOD in the blood two
and four months after cessation of intake in the 120— and
l80—day groups indicates either continuous turnover and
release of stored pesticide, or a very long circulation
time following absorption. Why blood HEOD levels did

not increase during lactation in the 120— and lBO—day

 
 

m ..-.

 

123

animals, thus reflecting the greater amount of HEOD re—
moved from their body fat compared to the 60—day groups
in not clear at this time. It is peculiar that HEOD
levels were highest in the blood and lowest in the milk
fat at parturition for the 60—day groups. The situation
was reversed for the 120— and l80—day heifers.

A change might have occurred in the partition
coefficient or rate of transfer by which the HEOD was
removed from the blood by the mammary gland. Also it
is possible that the mammary gland performed selective
withdrawal of certain lipid fractions which might trans—
port a disproportionate amount of HEOD in the blood. All
blood—lipid fractions contributing to milk fat might
have been saturated with HEOD because of the higher body
storage in the 120— and l80—day heifers compared with a
lesser saturation of these particular fractions in the
60—day heifers due to lower body stores.

Perhaps the HEOD carrier in the blood involves not
only the lipid portion, but also the soluble proteins or
lipoproteins as suggested by Moss and Hathway (102) for
rat and rabbit blood. Factors such as chain length and
saturation of specific fatty acids might also be in—
volved in the transfer mechanism of HEOD between body

fat, blood, and milk. These details need elucidation.

   

 

12;:

Another factor complicating the comparison of body
storage of HEOD between the three stages of pregnancy is
that the 60—day heifers were fed dieldrin up to and in—
cluding the day of parturition (if parturition had not
occurred by 8:00 A.M., the daily dose was administered).
Therefore, part of the ingested dieldrin was still in
the digestive tract or circulating in the blood at the
time lactation was initiated. Thus, milk secretion
would have provided an immediate, major route of elimi—
nation and minimized body storage of dieldrin ingested
during the concluding days of contamination.

However, even though less pesticide might have
reached body storage sites in the 60—day heifers, it still
should have appeared in milk following parturition. After
2A weeks of lactation, only 18% of the dieldrin consumed
by the 60-day groups could be accounted for in the
milk, compared with mere than A0% for the 120— and 180—
day groups (Table 15); suggesting that conversion to a
metabolite(s) was much greater in the heifers contami—
nated immediately tefore calving.

The 16 cows in Experiment 1 which were contaminated
postpartum had body fat residues comparable to those of
the 60—day prepartum heifers (Table 26) even though
about 19% of the pesticide ingested by the cows was
excreted during contamination (Table 3). For animals

in both experiments the ratio between body fat and milk

 
 

 

125

fat was about 2:1 when body storage was 10 ppm. With
body fat HEOD residues of about 18 ppm or above, milk
fat levels were approximately equal to body fat concen—
trations. The narrowing of the ratio at higher levels
of HEOD in body fat could have been due to saturation
of all blood lipid fractions contributing to milk fat.

Shown in Table 29 and Figures A and 9 are the HEOD
concentrations in milk fat for the various dietary treat—
ments used in both experiments. The initial pesticide
concentrations were similar for all seven treatment
groups. Within—cow variation of HEOD residues in milk
fat from samples taken at weekly intervals fluctuated
considerably as was noted also with DDT (83).

The pattern of residue decline was similar for the
seven treatment groups. For all treatments, concen—
trations had decreased to about one—half their initial
levels after three to four weeks and to one—third by
five to six weeks. Thereafter, the rate of decline was
slower with plateaus occurring between weeks 5 to 10
and 12 to 18. The plateaus were less definite in Experi—
ment 2 than Experiment 1.

Laben e§_al. (83) stated that DDT residues in milk
fat declined exponentially after cessation of intake at
rates of 9 to 11% per week when maximum concentrations
were 19, 231, and 812 ppm for the three treatment groups.

Rate of decline was 6.6% per week for their control

   

 

126

TABLE 29.——HEOD decline in milk fat during decontamination
for all dietary treatments.

 

 

 

 

Experiment 1a Experiment 2b
% of TDN Requirement Diet
week Y 150 + .
150 75 100 gigiggn Normal giggn Eglfigg—
ug/g

1 24.9 18.2 21.0 24.0 18.8 17.4 23.9

2 19.9 19.1 16.2 19.9 13.4 9.9 11.4.

3 13.4 11.8 11.8 10.7 7.3 8.2 12.5

4 9.7 11.7 8.8 12.2 8.4 10.3 11.9

5 8 0 6.9 9 1 8.6 7.0 9.6 10.9

6 6 4 7.5 8.8 12.2 5.2 6.3 8.8

7 4.6 6.4 7.4 8.0 ——— —-- —-—

8 6 5 7 7 5.5 8.6 4.5 3.6 8.1
9 7 1 10 0 8.9 9.5 —-— —__ ___
10 7.2 7 7 8.6 8.8 4.9 3.8 7.3
12 1 6 4 » 1.6 1 0 4.0 3.4 7.8
14 2 2 2 5 1.8 0 4 ——— ——— --—
16 1 2 2 c 0.9 1 0 2.2 3.3 6.3
18 1 3 2 6 0.8 o 2 --- ___ ___
20 -—— —-— ——- ——— 1.3 1.6 2.8
24 ——— ——— ——— ——— 1.0 1.2 1.7
30 ——~ ——— ——— ——— 0.8 0.2 0.3

 

 

 

a
Four animals per treatment.

bSix animals per treatment.

 

 
 

 

127

group which had a maximum concentration in milk fat of
2 ppm. However, when their data on milk fat residues
were plotted on a semi—log scale a plateau similar to
that observed in our study occurred between weeks 5 to
18 for the high, medium, and control groups. Further—
more, these workers (83) did not indicate statistical
treatment of regression lines to test if the rate of
residue decline observed in their studies could be
described but by a linear function.

Other workers (30, 51, 100, 138) have found that
after discontinuation of DDT intake, levels in the milk
fat fell rapidly for the first three to five weeks, then
leveled off, and thereafter declined at a much slower
rate. In our study linear regression coefficients were
calculated for milk fat residue decreases by transforming
to the log Y + 1 basis. Tests for goodness—of—fit showed
that, because of the plateauing, a straight line did not
accurately d€b”T1bL 5;: hEUD disappearance pattern.

Witt e£_al. ll 6) reported that the depletion rate
of DDT was uniform for low—level accumulations (< 2 ppm
in milk fat), but found two disappearance rates for
higher levels of storage (6—8 ppm) as well as for storage
resulting from six—day compared to one—day exposures,
regardless of the contamination level reached. The
presence of two distinctly different dissipation rates

Of DDT suggests a two—compartment storage with different

 

 

128

rates of exchange. Therefore, it is possible that immedi—
ate storage could occur in a site with rapid depletion
potential; and then upon longer exposure the pesticide
would accumulate in less labile sites.

Wertheimer (131) has pointed out that the fat cell
consists of two compartments: a large one serving as a
relatively inert storage site in which the exchange or
turnover is slow; and a second, smaller compartment,
containing lipids in a very rapid state of turnover and
in close metabolic association with the serum and liver
lipids. The existence of these two compartments in the
fat cell might also explain the different rates of
elimination of dieldrin suggested in this study, and of
DDT shown by Witt et_al. (138).

It is known also that adipose tissue occurs in
sites where mechanical support is needed and the metabolic
activity of this adipose tissue is much lower than that
of mesenteric fat. The metabolic activity of sub—
cutaneous fat is intermediate between these two extremes
(3). Thus, a differential in fat mobilization from
storage sites could contribute to two different elimi—
nation rates for residues and explain the plateauing of
HEOD levels found in each of the nine treatment groups
used in Experiments 1 and 2.

There is a dearth of information concerning methods

which might effect a more rapid decontamination of

129

pesticide residues from the animal. The possibility of
increasing the rate of decontamination by an increased
depletion of body fat has been suggested. Conversely,
it was theorized that energy intake in excess of re—
quirements might minimize body fat turnover, thereby
reducing release and decreasing concentrations of
residues in milk fat. Increasing the exchange in body
stores of residue—free lipids for contaminated fat by
increasing the catabolic rate via the use of thyroprotein
in conjunction with a high energy intake has also been
suggested. Diets designed to depress milk fat, thereby
reducing the rate of pesticide elimination in milk, is
another possible way of reducing concentrations below
detectable levels.

Thyroprotein feeding in Experiment 1 caused a
rapid and marked loss of body weight (57 kg/cow within
four weeks) (Figure 2), and decreased the time required
to reach a mean residie level in milk fat of 1.0 ppm to
12 weeks compazed with 16 weeks for the control group.
A slower decline was noted for the groups receiving 150%
and 75% energy requirement which averaged 1.3 and 2.6
ppm, respectively, alter 18 weeks of decontamination
(Table 29). The effect noted for thyroprotein is in
contrast to the data of Miller (100) who reported that
the hormone did not decrease the time required for the

milk fat levels of either pre— or postpartum cows

   

 

 

 

130

contaminated with DDT to reach 1.25 ppm. A difference
in mobilization of dieldrin and DDT may partially explain
this apparent conflict.

Residue levels in milk fat for the 75% energy
group in Experiment 1 declined at a slower rate.than all
other groups and did not change significantly during
the last six weeks of decontamination. This observation
is also in contrast with Miller's data for DDT (100),
which indicated that cows on a low—energy ration de—
clined at a more rapid rate than those receiving normal
energy. This difference, however, might be explained
by the different effect of the low-energy rations on
milk yields in the two studies. Miller did not present
production figures or exact energy levels; however, it
was stated that cows on the low—energy treatment produced
about the same amounts of milk and milk fat during the
experimental period as in previous lactations. These
cows received only 9 kg of alfalfa hay as the sole energy
source for the 7th through the 42nd day of lactation and
lost an average of 68 kg body weight. Apparently the
inherent stimulatory effects of early lactation main—
tained nearly normal milk yields at the expense of body
fat. It is well known that cows tend to mobilize large

quantities of body stores to maintain high—level milk

 

production during the first four to six weeks of lact—

ation° However, in the latter stages of lactation,

 

 
 

 

 

 

131

milk yields decline as the result of low energy intake
even though body fat is being deposited. -

2 Our 75% group averaged six months postpartum at
initiation of the low—energy diet. These cows responded

with a marked decline in production which preceded small
losses in body weight (Figures 2 and 3). Because of a
decreased excretion of HEOD during the early weeks of

decontamination in the low—energy group, a greater body

 

burden of pesticide resulted in the more persistent
levels noted at 18 weeks. It would appear that the
efficacy of a low-energy diet on enhancing the rate of
elimination of chlorinated hydrocarbon pesticides from
dairy cows depends on the stage of lactation at which
the low—energy ration is imposed.

After 18 weeks of decontamination all dietary
treatment groups in Experiment 1 had excreted a similar
percentage of the ingested dieldrin but concentrations
in milk fat were still highest for the low-energy group.
This should emphas1ze the importance of not imposing a
treatment to cause an appreciable decrease in milk pro-
duction while attempting to eliminate residue from cows
contaminated with chlorinated hydrocarbon pesticides.

In Experiment 2, heifers contaminated during the
last 60 days of pregnancy were assigned to either the
normal (A), high grain (B), or pelleted hay (C) diets.

ReSidue levels in milk fat of heifers in groups A and

 

   

 

132

B were not significantly different; whereas, group C
animals, which averaged 2.2% fat for the first 24 weeks
(compared with 3.2% fat for the control group A) ex—
hibited higher HEOD concentrations on a milk fat basis
(P < .01). Despite this marked depression of milk fat
for group C, it is noteworthy that the mean residue
level in the whole milk was not different from groups A
and B. Heifers fed the pelleted hay produced the most
milk (Table 8) which contributed to a greater removal

of pesticide. Although the total amount of HEOD excreted
during 24 weeks of lactation was about 43% greater for
the pellet—fed than the other 60—day groups, differences
were not statistically significant (P < .10).

Since it is generally accepted that HEOD is associ—
ated with the lipid portion of milk, the observation that
a depression in percent milk fat did not alter the resi—
due level in whole milk is of special significance. It
indicates that HEOD excretion via the bovine mammary
gland is related to iactors other than the total amount
of fat synthesized in the gland.

Laben gt_al. (83) suggested that various pools or
fractions of body fat may vary widely in their concen—
trations of residues and that a particular lipid fraction
may be an important precursor of milk fat and also con—
tain a disproportionately higher concentration of
pesticide than would be revealed by analysis of external

body fat samples.

 

 

 

133

Stanley and Morita (120) reported that blood lipids
contributed to a larger percentage of the total milk fat
in thyroprotein—fed cows than in those on a control
ration. Hence, it is possible that HEOD concentration
in milk can be altered by relative amounts of the
different fat precursors taken up by the mammary gland.
This is a possible explanation for the observation on
the pelleted ration that, even though fat content of
milk was drastically reduced, HEOD levels in whole milk
did not change.

As the pesticide was eliminated from the body via
secretion in the milk, residue levels in samples of ex—
ternal shoulder fat declined. Only two animals in each
group of four were biopsied for fat residue analysis in
Experiment 1, thus, estimates based on these data are
not complete, but definite trends were noted. Residue
concentrations in samples taken at 0, 8, and 16 weeks
showed an exponential decline. Equations for the re—

spective energy treatments of 150%, 75%, 100%, and 150%

plus thyroprotein were: Y = 11.6eg0'0198t,
_ 08% _\ >
Y = 10.8e O'Ol"', Y = 10.0e 0'0178L, and
_ —0.02241 . . .
Y — 10.0e . Thus, the coefficients of these

equations indicate that residue levels declined at rates
of 1.78% to 2.24% per day. Although body fat levels of
HEOD in thyroprotein—fed cows declined at the fastest

rate, numbers were insufficient to detect a significant

difference.

   

 

134

Experience gained from Experiment 1 dictated that
every animal in Experiment 2 should be biopsied for body
fat analysis. Body residue concentrations decreased ex—
ponentially for all treatments after lactation was initi—

ated. Equations for the 60—day, A, B, and C, and 120- and

—0.0l47t —0.0ll6t
3 3

l80—day groups were: Y = 10.0e Y = 10.0e

—0.0l33t —0.0l39t —0.0182t
J 3 3

Y = 13.2e Y = 16.0e and Y = 34.0e
respectively. The five groups, therefore, declined at

rates of 1.16 to 1.82% per day (8.1 to 12.7% per week).

 

The rate for the l80—day heifers was significantly faster
(P < .01) than any other group. These elimination pat—
terns compare with 8.1 to 9.8% per week as reported by
Laben et_al, (83) when initial body fat levels of DDT
were 16, 253, and 590 ppm for three treatment groups.

The difference in the disappearance pattern of HEOD
from body fat compared with milk fat (exponential decline
versus plateau periods) might be explained in two ways.
First, the eightnweek interval between fat biopsies might
have masked any plateau in tissue residue concentrations
which could haxe occurred during the intervening weeks.
However, Laben et_ai§ {83) took biopsy samples at two
week intervals and did not find a plateauing of DDT resi—
dues in body iat. Secondly, a decrease in residue con—
centrations of shoulder fat might not accurately describe
the decline of total pesticides in the body because of
differential mobilization and re—location of HEOD within

other fat storage sites.

   

135

From the heifer data (Experiment 2), one might
associate the plateaus observed for milk residues
(Figure 9) with the change in body weight during
lactation (Figure 6). The rapid decline in milk HEOD
during the first four to six weeks corresponds closely
with the precipitous loss of body weight during the same
period. All groups reached minimum weights at approxi—
mately six weeks postpartum and generally stabilized at
this level until about 14 weeks after which some weight
gains were noted.

Conceivably, the large initial losses of body fat
containing low levels of HEOD might account for the steep
decline in milk residues early in lactation. After body
weight and fat loss had somewhat stabilized, mobilization
of storage sites containing higher HEOD concentrations
would result in a leveling-off of the fast rate of de—
crease in milk fat residues.

This suggestion fits the pattern for the thyroprotein—
fed cows in Experiment l, but does not explain the similar
pattern for milk residues observed for the 100 and 150%
groups. These latter groups increased in body weight
from the beginning of dietary treatment. Since decontami—
nation for many cows in Experiment l occurred after six
months postpartum, body metabolism may have been con—
siderably different than in the heifers, which were de—

Contaminated in early lactation. The differences in

   

 

136

metabolic states of the animal in the two experiments,

as influenced by stages of lactation, growth require—
ments, and hormonal changes related to pregnancy and
estrous cycles, advise caution in developing explanations
for decontamination patterns applicable to both experi—
ments.

Limited evidence for differential storage and
mobilization of HEOD was obtained with tissues removed
from six cows at slaughter (Table 6). Residues in shoulder
fat were higher than those in kidney or omental fat.
Thyroprotein—feeding appeared to affect mobilization of
fat without stimulating release of stored HEOD, because
residues in shoulder, kidney, and omental fat were three
to five times higher for the thyroprotein—fed cows. The
markedly higher concentration of HEOD in fat at slaughter
of the thyroprotein—fed cows might be explained on the
basis that thyroprotein differentially stimulated mobili—
zation in the two—compartment fat cell and/or at various
fat depots. Thus, the residual HEOD was more concen—
trated in the lipid remaining in the fat cells of the
thyroprotein—fed cows.

Averaging the values for all heifers in Experiment
2, approximately 34% of the total HEOD ingested was ac—
counted for at parturition as the unchanged compound.
This proportion ranged from about 20% for the 60-day

group to 63% for the l80—day heifers (Table 13).

 

 

 

137

Pesticide stored in the body at parturition was
apparently underestimated, since an average of 131% of
the calculated storage had been accounted for after 2M
weeks postpartum. Only in the 180—day group was less
than 100% of the estimated amount of stored pesticide
accounted for at 2“ weeks of lactation. It is possible
that assumed body fat content of 10% was too low which
would have resulted in more stored pesticide at parturi—
tion than indicated. Differential storage in various
fat depots could have also been a contributing factor to
underestimation of total body residues. However, even
a large error in estimating blood volume would have made
little difference in the total body pool of HEOD due to
the very low blood concentrations (7 to 15 ppb).

Investigation of possible excretory pathways of
HEOD from dairy animals revealed that about 2.5% of the
daily intake was excreted as unchanged HEOD in the feces
during contamination. Heath and Vandekar (67) reported

36

that in rats fed HEOD only 3% of the Cl was excreted
as HEOD in feces unless the bile was cannulated. Only
minute amounts were found in the urine. Once lactation
in the cows was initiated, milk was the primary excretory
pathway (Table 17).

The appearance of HEOD in feces 30 days after

contamination ceased indicates a metabolic origin and

recycling of the pesticide. Biliary excretion has been

   

 

 

138

shown for a number of chlorinated hydrocarbon pesticides
(9, 67, 73, 101, 112, 129). Moreover, it has been sug—
gested that the amount excreted in bile may be quite
high. Heath and Vandekar (67) found that the proportion
of HEOD excreted as the unchanged compound could be in—
creased from 3% to 10% by cannulation of the bile duct.
Cook gt_al. (28) have demonstrated that dieldrin is re—
cycled and appears in the bile, pancreatic juice, and
saliva of goats. These were probable pathways through
which dieldrin appeared in the feces of heifers during
decontamination.

Placental transfer of dieldrin occurred in each of
33 dams, resulting in comtamination of all newborn calves
(l2, 1H, 16). Although total HEOD intake was very similar,
the stage of gestation during which the pesticide was
ingested exerted a highly significant effect (P < .01)
upon the concentration of HEOD in the body fat of the
dams at parturition. However, the differences in HEOD
in body fat of dams did not significantly affect the mean
residue levels in whole carcass, carcass fat, brain, or
renal fat of the male calves.

These data are in contrast to those of Laben et_al.
(83), who found that DDT in the body fat of three still—
born calves was proportional to the estimated body fat
level of their dams. HEOD levels in blood of the dams

at parturition did not parallel those of body fat.

 

139

Blood of heifers contaminated during the last 60 days of
gestation contained 12.8 ppb as opposed to 6.7 and 7.3
ppb for the 120— and 180—day groups, respectively. The
difference between these means approached significance
(P‘< .05).

Since blood is the only avenue by which the dieldrin
could be transferred from the dam to the fetus, the tissue '
residue picture in the newborn male calves is interesting
in view of the differences in dieldrin concentrations
found in blood and fat of dams at parturition. The
similar residue levels found in all calves, regardless
of stage and degree of contamination of the dams, suggests
that fetal residues were a function of both concentration
and length of time that blood containing the pesticide was
in contact with the developing fetus.

Heifers contaminated during the last 60 days of
gestation had higher blood levels and lower body fat
concentrations than those contaminated for a similar
time, but starting at 120— or l80—days prepartum. The
greater HEOD levels found in the blood of calves born to
the 60-day group reflect the higher blood levels present
in the dams at parturition.

Calculated recoveries of HEOD in the male calves
averaged 0.9% of that ingested by the dams. Total
placental transfer was probably higher than recovery

figures indicate because the calves were skinned prior

   

140

to grinding the carcasses for HEOD analyses. King et_al.
(75) reported that heptachlor epoxide concentration in one
newborn calf was three to four times higher in the hide
and hair than in several other tissues analyzed. Further—
more, HEOD in the placental tissues and fluids was not
determined.

Effect of age on blood residues in female calves
was similar to that for the males. Females born to dams
in the 60—day group contained significantly higher levels
of dieldrin in their blood than the other two groups
(P < .05). As with the males, this is a reflection of
the higher blood levels circulating in the dams contami—
nated during the last 60 days of gestation. HEOD in the
blood of females disappeared more quickly than that found
in the omental fat. At 16 weeks of age, HEOD in blood
was detectable in only one of seven females, and none was
found at 24 weeks in the four calves sampled.

No evidence of dieldrin poisoning was found in any
of the calves or dams. Kadis and Jonasson (74) fed
single doses of dieldrin (17 mg/kg body weight) and
aldrin (15 mg/kg body weight) to two calves and found
that blood levels of dieldrin at six weeks in both calves
had declined to about one—half of initial values (from
95 and 200 ppb to 46 and 92 ppb, respectively). They
did not indicate whether any symptoms of dieldrin poison—

ing occurred in the calves, although Brown et a1. (19)

c and dogs. Dale

symptoms and blood die:

 

which corresponded to the threshold suggested by Brown
$15.21- (19).

It is encouraging that HEOD levels in omental fat
and blood of calves contaminated in 22239 declined with
age, and that at 52 weeks of age small residues were
detectable in only one of five calves. This would indi-
cate that at the dieldrin levels used in these studies,
female calves contaminated in 22232 will not contain
sufficient HEOD at parturition for its detection in their
milk.

Based on limited data it appears that whole milk
having up to 0.50 ppm can be fed to growing calves con—
taminated in gtgrg without increasing body fat residues.
These data agree with the findings of Finnegan et_a1.
(48) who showed that dog pups contaminated with DDT
in EESEE and suckling contaminated dams had lower DDT
levels after two weeks of nursing than litter mates at

two days of age.

 

 

 

VI. SUMMARY AND CONCLUSIONS

A total of 51 Holstein cows and heifers were con-
taminated for similar periods of time (56 to 60 days)
with equal levels of dieldrin (about .096 mg HEOD/kg
body weight) in two experiments. Sixteen lactating ani—
mals were fed dieldrin daily for 56 days. Dietary treat—
ments during an l8—week decontamination period as percent
of the NRC requirement for TDN were: 75, 100, 150, and
150 plus 22 mg of thyroprotein daily per kg of body weight.
Thirty—one pregnant heifers were contaminated at the
same level for 60 days beginning either 60, 120, or 180
days prepartum. During contamination and decontamination
samples of milk, external body fat, feces, and urine were
analyzed for HEOD to determine storage and excretory path-
ways.

A major, previously unreported, finding was that
significantly greater KP a .005) HEOD storage occurred in
body fat when pregnant dairy heifers were contaminated
early compared to late in gestation. Storage resulting
from the 120— and 180—day prepartum contamination was
approximately two and three times, respectively, (18 and

34 vs 10 ppm) that found for heifers contaminated 60

142

 

143

days prepartum. It is suggested that endocrine changes
caused by advancing pregnancy might have stimulated a
greater metabolism of the pesticide resulting in less
unchanged HEOD.

Once non—lactating pregnant dairy heifers were
contaminated with HEOD, very little was eliminated from
the body until lactation began. The pattern of residue
decline in milk fat was similar for all groups in both
experiments. Concentrations dropped to about one—half
their initial levels after three to four weeks, and to
one—third by five to six weeks. Because of the plateau—
ing, a linear decline did not accurately describe the
HEOD disappearance pattern in milk fat. The presence of
two distinctly different dissipation rates for HEOD sug—
gests a two—compartment storage with different rates of
exchange.

Varying the dietary energy level did not signifi—
cantly enhance the rate of decontamination, although
thyroprotein—feeding did decrease the time required to
reach a mean residue level of 1.0 ppm in the milk fat
(12 weeks compared to 16 weeks for the control group).

A low—energy intake at six months postpartum caused a
marked decline in production which preceded small losses
in body weight. Because excretion of HEOD decreased with
the lowered production, pesticide residues in milk fat

were more persistent in the low—energy group.

 

 

144

Residues in whole milk did not change when the fat
content of milk was drastically reduced by feeding
pelleted hay. This indicates that HEOD excretion via
the mammary gland is related to factors other than the
total amount of fat synthesized in the gland. Since
HEOD levels in blood did not parallel those in milk fat
it is suggested that a change occurred in the partition
coefficient or rate of transfer by which HEOD was
removed from the blood by the mammary gland. Also it
is possible that the mammary gland performed selective I
withdrawal of certain lipid or lipoprotein fractions
which transport a disproportionate amount of HEOD in the
blood.

As the pesticide was eliminated from the body via
secretion in the milk, residue levels in shoulder fat of
the heifers contaminated prepartum declined exponentially
at rates of 1.16 to l 82% per day (8.1 to 12.7% per week).
Limited evidence for differential storage and mobili—
zation of HEOD was obtained. Thyroprotein—feeding ap—
peared to affect mobilization of fat without stimulating
release of stored HEOD since the residual HEOD was more
concentrated in the adipose tissue of cows fed thyro—
protein.

About 2.5% of the daily intake was excreted as un—

changed HEOD in the feces during contamination. Only

 

trace amounts were found in the urine. The appearance

of HEOD in feces 30 days after contamination ceased

 

 

 

 

145

indicates a metabolic origin and recycling of the pesti—
cide. Bile, pancreatic juice, and saliva were probable
pathways through which HEOD appeared in the feces of
heifers during decontamination.

Placental transfer of dieldrin occurred in each
of 33 dams, resulting in contamination of all newborn
calves. Calculated recoveries of HEOD in the male calves
averaged 0.9% of that ingested by the dams. The similar
residue levels found in all calves, regardless of stage
and degree of contamination of the dams, suggests that
fetal residues were a function of both concentration and
length of time that the dam's blood containing the pesti—
cide was in contact with the developing fetus. HEOD
levels in omental fat and blood of calves contaminated
in 33239 declined with age and at 52 weeks negligible
residues were detectable in only one of five female
calves. This would indicate that at the dieldrin levels
used in these studies, female calves contaminated in
utero will not contain sufficient HEOD at parturition
for detection in milk.

Based on limited data it appears that whole milk
having up to 0.5 ppm can be fed to growing calves con—
taminated in utero without increasing body fat residues.
However, feedstuffs contaminated with chlorinated hydro—
carbon pesticides should not be fed to pregnant dairy
heifers as has been previously recommended in several

states.

 

 

 

LITERATURE CITED

146

 

 

 

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Aronovsky, E., Levari, R., Kornblueth, W., and
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_ APPENDIX

161

 

 

162

TABLE I.——Data on individual cows contaminated with

dieldrin during lactation.

 

 

 

Days Since Avg. Daily Body

Cow A e Calving Calving to Milk Prod. Weight

5 Date June 1, May 12—17 May 10

1965 Incl. & 11

yr—mo kg

286 1—10 1/8/65 144 23.5 431
727 3—1 12/23/64 160 22.2 590
248 3—2 3/1/65 92 31.9 552
188 5-10 4/28/65 _3_4_ 455* 618
Avg. 3—6 107.5 30.8 548
759 1-9 12/31/64 152 18.7 414
717 3-5 1/9/65 143 25.4 499
723 3—4 1/28/65 124 32.2 559
701 4—4 3/28/65 _§5 38.9 5g5
Avg. 3—3 121.0 28.8 499
758 1—11 1/22/65 130 19.1 474
264 2—7 1/15/65 137 21.4 542
257 2—11 2/9/65 112 33.7 582
224 4—1 2/7/65 ;;5 34.8* 545
Avg. 2—11 123.3 27.3 536
247 3—1 2/2/65 119 21.3 524
707 4—0 1/22/65 130 32.8 491
721 3-5 2/5/65 116 32.4 591
250 3—2 4/6/65 _5§ 33.2 55g
AVs- 3—5 105.3 29.9 540

 

*5 day average.

 

 

  

 
   
 
  

121./3775’s

212/2/65

  
 

1/14/66

H—766 602 11/14/65 53 3,008 0.095

M ' 40.8

H—767 533 .12/5/65 55 2,796 0.095 M 37.2
H—768 - 558 12/23/65 55 2,796 0.091 F 39.5
H-307 586 12/24/65 62 3,271 0.090 M 42.2
H—315 512 12/30/65 60 2,917 0.095 M 42.6
H—775 520 2/2/66 61 3,101 0.097 F 38.1
Mean 552 57.7 2,982 0 094 40.1

Group C

H—291 ' 607 ’ 12/1/65 54 3,222 0.098 .Fb 37.2
H-771 520 12/5/65 55 2,796 0.098 M 41.7
H-312 542 12/23/65 52 2,643 0.094 F 35.4
H-311 547 12/28/65 61 3,101 0.093 M 42.2
H-290 566 1/9/66 65 3,592 0.098 F 41.7
, H—761 713 4/15/66 57 3,779 0.093 F 45.4
Mean 582 57 3 3,189 0 095 40.6

 

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~-#4fiel%ifig point:
Odor:

Vapor.Pressure

(mm Hg at 20°C):

Flammability:

Bulk density,
lb./cu. ft.:

Solubility:

Solvent:

Methanol
Acetone
Benzene
Hexane
Base oil

(std. oil 10)
Water

  

1 7 5217 5° C_

Odorless

7.78 x 10‘7

Nonflammable

47 to 51

Moderately soluble in aromatics,
halogenated solvents, esters &
ketones, sparingly soluble in
aliphatic hydrocarbons &
alcohols; insoluble in water

Solubility at 26°C (g/100 ml)

4.9
54.0
75.0

7.7

4.3
insoluble (0.05 ppm)

 

 

 

 

 

 

 

 

   
 

 

 

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