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This is to certify that the

thesis entitled

Concentration, Distribution, and Withdrawal of
Ethylene Dibromide (EDB)
in Eggs and Tissues Obtained from Chickens
Fed Diet Containing EDB—Contaminated Flour

presented by

Ellen J. Lehning

has been accepted towards fulfillment
of the requirements for

M.S. degree in Animal SCience

   

Major professr

Date

040 6?, '996

\

0-7639

MS U is an Affirmative Action/Equal Opportunity Institution

 

MSU

 

 

 

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remove this checkout from

 

 

     

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be charged if book is
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flaw
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7v~“”“‘”’v~a

 

 

CONCENTRATION, DISTRIBUTION, AND WITHDRAWAL OF
ETHYLENE DIBROMIDE (EDB)
IN EGGS AND TISSUES OBTAINED FROM CHICKENS
FED DIET CONTAINING EDB‘CONTAMINATED FLOUR
by

Ellen J. Lehning

A THESIS
Submitted to
Michigan State University
in Partial Fulfillment of the Requirements
for the Degree of
Master of Science

Department of Animal Science

1986

ABSTRACT

CONCENTRATION, DISTRIBUTION, AND WITHDRAWAL OF
ETHYLENE DIBROMIDE (EDB)
IN EGGS AND TISSUES OBTAINED FROM CHICKENS
FED DIET CONTAINING EDB-CONTAMINATED FLOUR
by

Ellen J. Lehning

Hens were fed diet containing 6.7 ppm, EDB for 21 days
followed by 21 days Of non-contaminated diet (days 0 to 21 Of
withdrawal). EDB residues in egg, whole body, fat, muscle, liver,
kidney, and skin were quantified with head space GC methodology.
Tissues and eggs contained less than one percent Of EDB intake.
Eggs contained detectable EDB by char 3 Of feeding contaminated
diet and reached. a plateau Of 28 ppb ‘by day 8. By day (5 Of
withdrawal EDB Was not detectable in eggs. Concentration of EDB on
day 0 Of withdrawal in whole body, fat, and muscle was 11, 54, and
0.44 ppb, respectively. Fat contained 95% Of whole body residues.
EDB was not detected in liver, kidney, and skin on day 0 Of
withdrawal and was not detected in tissues on day 21 of
withdrawal. Activities of hepatic mixed function oxidases were not

induced by feeding EDB at 6.7 ppm for 21 days.

,+

ACKNOWLEDGEMENTS

I would like to thank the members Of my guidance committee,
Dr. Donald Polin, Dr. Steven Bursian, and Dr. ththew Zabik, for
their valuable assistance during the preparation Of this
manuscript. Sincere thanks are extended to my major professor, Dr.
Polin, for the encouragement, thoughtful guidance, and unlimited
patience that made possible the attainment Of this degree.

Special thanks are extended to the Michigan State Department
Of Agriculture for sponsoring this research. Special thanks are
also extended to TOHI Whaleni Of the Department. of .Agriculture
Laboratories. His Skill as a teacher enabled. me t1) learn to
operate a GC effectively.

Special acknowledgement belongs to Dr. John Gill for taking
time to discuss with me the principles involved in conducting
"efficient" research.

I would like to thank. my .fellomr graduate Students, Barb
Olson, Paul Bernthal, Patricia Wiggers, Mike Underwood, and Dave
Pullen, for their willingness to participate in the many
discussions we have had during my association with them.

I am grateful to Brad Clark, Julie Mackie, Marina Garza, and
Bridget Gregus for their aid in conducting the research. and
analyses.

1 would especially like to thank my parents, brother, sister

and brother-in-law for their unending support and for their
delight in having a graduate student in the family.

ii

TABLE OF CONTENTS

Page

_—._.

LIST OF TABLES

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

LIST OF FIGURES

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

INTRODUCTION .........................................................
LITERATURE REVIEW ....................................................
Chemical Properties Of EDB .........................................
Production and Uses Of EDB .........................................
Sorption and Residues of EDB in Grain ..............................
Residues Of EDB in Tissues and Blood Of Rats and Chicks ............
Toxicology Of EDB in Poultry .......................................
Toxicology of EDB in Mammals .......................................
Summary ......... ...... .

MATERIALS AND METHODS ................................................
Experimental Methods ..... . ..................................... ....
Mixed Function Oxidase Assay .......................................
Headspace GC Analysis of EDB Residues in Egg, Tissues, and Diet

RESULTS ..............................................................
Feed Consumption, Body Weights, Egg Production, and Egg Weights
Residues Of EDB in Diet and EDB Intake .............................

Residues Of EDB in Egg, Whole Body, and Tissues ....................
Distribution Of EDB Residues .......................................

Activity Of Hepatic Mixed Function Oxidases ........................

DISCUSSION
Residues ...........................................................
Distribution and Metabolism .........................................
Research Design . ...................................................

00000000000000000000000000000000000000000000000000000000000

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

APPENDICES ...........................................................
A. Codes of Hens Used for Whole Body and Tissue Analysis ..........
B. Raw Data for Feed Consumption, Body weights, Egg Production, and

Egg Weights ........ . .............
C. Statistical Methodology for Linear Regression ..................
D. Raw Data for Calibration Curves and Predicted Concentration Of

EDB in Unknowns for Eggs, Tissues, and Diets .......... . .......

iii

vii

16
21

23
28
36

71
71
71
72
81
85

87
87
89
9O

91

32a
92a

93
98

101

 

B. Preparation of Microsomal Isolation, Biuret, and MFO Reagents .. 114
F. MFO Assay Raw Data ............................................. ll7

BIBLIOGRAPHY ......................................................... 125

iv

 

LIST OF TABLES

Table Page
1. Summary of the EPA survey on EDB residue data in grains and grain

products ......................................................... 11
2. Composition of experimental diets ................................ 22
3. Experimental schedule ............................................ 26
4. Volumes Of reagents used to establish a three point Standard curve

for the biuret protein determination assay ....................... 31
5. Volumes Of reagents used to establish a standard curve for the

aminopyrene N-demethylase assay .................................. 33
6. Hewlett Packard 3390A integrator conditions ...................... 38
7. Summary Of pooling method for eggs collected days 1 to 14 and 22

to 42 ............................................................ 43
8. Summary Of pooling method for eggs collected days 15 tO 21 ....... 44
9. Standard addition prediction equations (with confidence intervals)

used to calculate concentration of EDB in fat .................... 59
10. Standard addition prediction equations (with confidence intervals)

used to calculate concentration Of EDB in muscle ................. 62
11. Summary Of prediction equations and detection limits used to cal-

culate concentration of EDB in eggs, whole body, liver, kidney,

skin, diet, and flour ............ . ................................ 69
12. Residues of EDB in eggs Obtained from EDB hens on days 1 tO 14 Of

residue build up and days 1 to 21 Of withdrawal .................. 73
13. Residues of EDB in eggs Obtained from EDB hens days 15 to 21 of

residue build up ................................................. 74
14. Residues Of EDB in scrambled eggs before and after frying egg

samples Obtained from 4 EDB hens during days 15 to 21 of residue

build up ......................................................... 76

15.

l6.

l7.

l8.

19.

Page

Residues of EDB in egg, whole body, and tissues of EDB hens on day
0 of withdrawal ... ...............................................

Total residue Of EDB (ng) deposited into eggs Obtained days 1 to 21
of residue build up from the 4 hens used for whole body analysis on
day 0 Of withdrawal ...............................................

Distribution Of EDB in whole body and egg during residue build
up ................................................................

Activity Of aryl hydrocarbon hydroxylase (AHH) and aminopyrine
N-demethylase (AND) in liver of broilers fed diet at 80 ppm poly—
brominated biphenyls (PBBS) for 7 days ..........................

Activity Of aryl hydrocarbon hydroxylase (AHH) and aminopyrine

N—demethylase (AND) in liver Of hens on day 0 and 21 of with—
drawal ..........................................................

vi

77

79

80

83

10.

ll.

12.

13.

14.

15.

LIST OF FIGURES

Figure Page
1. Metabolism of EDB in rats ......................................... 18
2. Identification of EDB peak on chromatograms using egg as an

example ............................................................ 4O

Detection limit Of EDB in 5 ml 20 N H2804 + 2 g control egg ....... 46

Gas chromatograph dose—response Of EDB in 5 ml 20N H SO + 2 g

control egg ........................................ g..é ........... 47
Chromatograms of EDB in tissues ................................... 49
Gas chromatograph dose-response of EDB in 5 ml 20 N HZSO4 + 2 g
control whole body ................................................ 50
Gas chromatograph dose-response of EDB in 5 ml 20 N H2804 + 2 g
control liver ..................................................... 52
Gas chromatograph dose-response of EDB in 10 ml 20 N H2804 + 1 g
control kidney ....................................... . ............ 54
Gas chromatograph dose—response of EDB in 10 ml 20 N H2804 + 0.5 g
skin .............................................................. 56
Standard addition dose-response lines for fat ..................... 60
Standard addition dose-response lines for muscle .................. 63
Chromatograms Of EDB in diet and flour ............................ 65
Gas chromatograph dose—response Of EDB in 10 ml 20 N H2804 + 0.1 g
control flour ..................................................... 66
Gas chromatograph dose-response of EDB in 10 ml 20 N H2804 + 0.1 g
control diet ...................................................... 67
ResidUes of EDB in eggs obtained from EDB hens during residue

buildup, plateau, and withdrawal .................................. 75

vii

INTRODUCTION

In 1938 ea food law was passed that prohibited sale of insect
infested grains. This prompted the development Of several grain
insecticides, one Of which was the fumigant ethylene dibromide
(1,2—dibromoethane (n: EDB), sum aliphatic halogenated hydrocarbon.
EDB was first registered for use as a grain fumigant in 1948. It
was found to be an effective method for control Of insect
infestations in stored grains (Girish et_ 31: 1972). However,
problems with its use were encountered in 1958 when several
poultrymen in South Carolina reported substantial reductions in egg
size and egg production after feeding their flocks oats that had
been fumigated with an EDB fumigant (Caylor and iLaurent 1960).
Restrictions were run: set against use (A? EDB as 21 grain fumigant
because: 1) at that time there was no indication that EDB was toxic
to humans and 2) it was assumed that proper processing would
eliminate EDB residues from grains (Environmental Protection Agency
1977). Use of EDB as a grain fumigant was continued until February
3, 1984 when William D. Ruckelshaus, Administrator Of the
Environmental Protection. Agency (EPA), issued aur emergency <yrder
suspending registration (fl? EDB pesticides used to fumigate grain
milling equipment and stored grains. Mr. Ruckelshaus issued this
order because Of evidence that consumption Of EDB-contaminated grain

"posed increased risk Of cancer, heritable genetic damage, and

 

adverse reproductive effects" to the general public (EPA 1984). On
April 23, 1984, the EPA established tolerance limits for EDB per_
_se in grain as follows: 1) 900 ppb in non-processed grains, 2) 150
ppb 2hr milled products such as flour, and 3) 30 ppb in finished
ready—to—eat products such as cereals. Any grain products that
contain more EDB than what is specified by the tolerance limits
cannot be marketed (Food and Drug Administration 1984).

On February 2, 1984, the Michigan State Department Of
Agriculture confiscated 50 pounds Of EDB-contaminated flour from
the Amendt Milling Company Of Mbnroe, Michigan. The .flour
contained 31.1 ppm EDB and was made available to ascertain if
Chickens fed this food item would deposit EDB into eggs and tissue
and thus, present a source of EDB contamination to humans. The
objectives Of this study were:

1) To develop an assay with detection limits by which
residues Of EDB in tissues and eggs could be quantified.

2) To quantify residues of EDB in tissues and eggs Obtained
from hens fed diet containing EDB-contaminated flour.

3) TO determine if subsequent withdrawal Of EDB-contaminated
diet from hens would reduce EDB residues in tissues and

eggs.

4) To determine if dietary exposure to EDB 'would induce
hepatic mixed function oxidase activity in chickens.

LITERATURE REVIEW

I. Chemical Properties of EDB

Ethylene dibromide (1,2-dibromoethane, symdibromoethane,
glycol ‘bromide, (n: EDB) :R; an aliphatic halogenated hydrocarbon
produced commercially by reacting gaseous ethylene with liquid
bromine. EDB is known commercially as Bromofume, Orthofume, Dowfume
W-85, Escobrome-D, Bromotox, Celmide, Nephis, or ‘Kop Fume. Its
chemical formula is Br-CHZ-CHZ-Bg and its molecular weight is
187.86. It is a heavy, colorless 'non—flammable liquid. at 'room
temperature ‘which turns brown 'when. exposed to Slight. It ‘has a
chloroform-like Odor detectable in air by humans at a concentration
from 10 to 25 ppm (77 mg/m3 to 192.5 mg/m3). EDB is readily soluble

ha most organic solvents euui is slightly soluble :hi water (0.43

g/100 g at 30°C). EDB also has the following properties:

25
Specific gravity = 2.17225
Melting point = 9.6°C
Boiling point = 131.4°C at 760 mm Hg
Density = 2.18 g/ml at 20°C

11 mm Hg at 25°C
6.5 (air = 1.0)
1.65 centipoise at 20°C

Vapor pressure
Vapor density
Viscosity

 

Heat Of vaporization = +53 cal/g at 25°C with no flash point

The preceding information was taken from Girish _et al.

(1972), EPA (1977), and the Department (Dept.) Of Labor (1983).

II. Production and Uses of EDB
A. Production and uses prior to banning Of EDB as a pesticide
The EPA estimated that prior to bans against use Of EDB as a
pesticide approximately 135,000 to 160,000 metric tons (fl? EDB were
produced annually in the United States by PPG Industries, Ethyl
Corporation, Great Lakes Chemical Corporation, Dow Chemical
Corporation, and Velsicol Chemical Corporation. About 30% Of the EDB
produced was exported, 50 to 60% was used as a pesticide, and a small
amount was used. as an intermediate in. the synthesis. of dyes and
pharmaceuticals and as 21 solvent for resins, gums, and waxes (Brown
1984, EPA 1977, Dept. Of Labor 1983).
Pesticide uses of EDB have included: 1) soil nematocides (85 to
90%), 2) fumigation Of stored grain and grain milling equipment (6 tO
107.), 3) fumigation Of citrus fruit (1 to 27;), and 4) control Of
pinebark beetles, termites, and wax moths in honey combs (17.). In
1983, 160 million bushels of wheat, 14 million bushels Of corn, and 30
million bushels Of other grains (oats, barley, rice, rye, and sorghum)
were fumigated with EDB alone or in admixture with one or more of the
following chemicals: carbon tetrachloride (CT), ethylene dichloride
(EDC), methyl bromide (MB), chloroform, carbon disulfide (CD), sulfur
dioxide (SD), or benzene. Some typical combinations were: CT:EDC:EDB
(Dowfume EB—5, 63:30:7 w/w), EDBzMB (70:30 w/w), and
CT:CD:SD:EDB:pentane (80-9:16.l:1.5:1.2:0.4 w/w). The application rate
Of EDB varies with the facility or grain being fumigated but usually

ranges from 0.7 to 1.4 kg per 30 cubic meters for spot milling or 0.9

 

to 1.8 kg per 1000 bushels Of grain with reapplication at the first
Sign Of reinfestation (Brown 1984, EPA 1977; EPA 1984, Dept. of Labor
1983, Gilby 1983, Girish et_a1. 1972).

Pesticidal use of EDB has been for control and not prevention of
insect infestations. EDB is applied as a liquid which then
volatilizes, and as such is absorbed thorugh the insect's respiratory
system. It acts as an asphyxiant by interfering with enzymatic
functions associated with cell respiration (Christensen 1974).

B. Bans, current uses, tolerance limits, and alternatives to EDB

On December 14, 1977, the EPA issued a rebuttable
presumption against registration (RPAR) and continued registration of
pesticide products containing EDB. Issuance of a RPAR is the first
step towards suspending use of a pesticide. It is sisued when evidence

exists that a. pesticide meets or exceeds risk criteria relating to
acute and chronic toxic effects outlined in the Federal Insecticide,
Rodenticide, and Fungicide Act. In the case of EDB, a RPAR was issued
because of evidence that l) EDB is sorbed by food during fumigation
and 2) prolonged consumption of EDB—contaminated food could cause
cancer or interfere with reproduction. Thus, in 1984, all but the
following uses of EDB as a pesticide were cancelled: 1) fumigation of
exported fruit and 2) control of wax moths, beetles, and termites.
Non—pesticidal uses of EDB have not been cancelled. Tolerance limits
of EDB in raw grain, milled grain, ready—to-eat grain products, whole
fruit, edible pulp Of fruit, and ready-to—eat honey are 900, 150, 30,
250, 30, and 30 ppb, respectively.

Alternatives to pesticidal use of EDB include: 1) the liquid

fumigants CT, (EL. SD, EDC, and chloropicrin, 2) the solid fumigants
aluminum and magnesium phosphide, 3) the gaseous fumigant inethyl
bromide, 4) cold treatment, and 5) irradiation. As Of July 1, 1986 use
(fl? CT, CD, SD, enui EDC will be cancelled. Use Of methyl bromide is
likely tO be cancelled in the near future also. Irradiation is a
promising alternative, but grain and citrus producers have been
unwilling to adapt their storage areas to accomodate its use. Thus, as
of now, insect infestations in inills, grain, and fruit are 'being
controlled with cold treatment and the fumigants chloropicrin..and

aluminum or magnesium phosphide (Tangley 1984 and EPA 1985).

III. Sorption and Residues Of Ethylene Dibromide in Grain

Grains chemically' and. physically sorb EDB. Chemical. sorption
occurs primarily by the protein fraction Of teh grain (endosperm) via
alkylation anui release Of 51 bromide molecule. Chemical sorption is
irreversible and an: 25°C accounts for lO—ZOZ of the total amount of
EDB sorbed. It has been determined that chemically sorbed EDB and the
bromide released are not the cause. Of toxic effects Observed in
poultry and mammals. Physical sorption Of EDB is reversible, does not
chemically alter EDB, involves both absorption and adsorption,
accounts for up to 90% of the total amount Of EDBV sorbed at 25°C, and
is the cause Of toxic effects Observed in poultry and mammals
(Olomucki and Bondi 1955, Bondi_EE El: 1955, Berck and Gunther 1970,
EPA 1977).

The amount of EDB sorbed by grain has been quantified by

several methods. Some researchers have measured the decrease in
concentration Of EDB over time in an airtight atmosphere following
fumigation (Berck 1965, Vincent and Lindgren 1971). EDB lost from the
atmosphere was assumed to have been sorbed by the grain. These
studies could not distinguish between chemically and physically
sorbed EDB, but they did show that the amount of EDB sorbed increases
with decreasing temperature, increasing moisture content of the
grain, and increasing surface area (decreasing particle size). These
studies also showed that up to 557. less EDB is sorbed when it is
applied in an admixture than when it is applied singly. Several
studies have reported residues of EDB in grain as total bromide
content instead of as EDB pg _S_e (Girish and Kumar 1975, Girishet
31. 1972), but the bromide levels reported did not distinguish
between bromide originating from chemically sorbed EDB and bromide
bound to physically sorbed EDB . Once it was determined that it is
chemically unaltered EDB which is responsible for toxic effects
observed in poultry and mammals, researchers began quantifying
residues Of EDB p_e_i; se in whole grain and grain products. These
studies have consisted of 2 types: 1) whole grain was fumigated with
EDB, aerated, processed, and then analyzed for EDB content and 2)
random samples of grain products were removed from commercial
enterprises and analyzed for presence of EDB.

Berck (1974) quantified residues of EDB E E in wheat and
milled wheat products after fumigating whole grain with 28 kg of
Dowfume EB—5 per 1000 bushels. Dowfume EB-5 is 7% EDB so this is

equivalent to a dose of 2.0 kg of EDB per 1000 bushels. The wheat

 

contained 14% moisture, was Stored in a paper laminate bin, and was
fumigated on the third, seventh, fourteenth, twenty-fifth,
forty-second, and forty-ninth day Of storage. After the last
fumigation, concentration of EDB was measured in whole grain and in
bran, middlings, flour, and bread derived from the whole grain. Whole
grain contained 10 to 1360 ppb EDB, bran and middlings contained 20
to 220 ppb EDB, and flour contained 10 to 20 ppb EDB. Bread samples
did not contain detectable IHHB (detection limit = 0.05 nanograms).
Therefore, after prolonged exposure to EDB some Of the samples
analyzed contained more EDB than allowed by the current grain
tolerance limits (see introduction for grain tolerance limits).

Anderson it. _a_1_. (1985) quantified residues of EDB pg}; _s_e_ in
whole corn and milled corn products after fumigating whole grain with
an admixture Of CT, CD, SD, EDB, and pentane (80.9:16.0:l.5:1.2:0.4
w/w). Every 91 kg Of corn received 40.5 m1 of admixture. This is
equivalent to a dose of 1.3 g EDB per 91 kg of corn, or approximately
360 g EDB per 1000 bushels Of corn. The corn was exposed to the
fumigant for 5 days at 25°C in an air tight steel drum. Residues Of
EDB were measured at 0, 30, and 180 days post-fumigation in whole
grain and in hulls, germ, starch, gluten, flour, bran meal, Oil,
hominy, masa, and tortillas derived from whole grain. It was found
that EDB concentrated in the germ and hull, but all residues detected
(detection limit = 1 ppb) were below current grain tolerance limits
of EDB.

Several other researchers have measured EDB per se in grain and

milled pmoducts after fumigation. Chylor and Laurent (1960) found
10-15 ppm EDB in fumigated oats at several weeks post—fumigation. Wit
35. a1. (1969) fumigated wheat for 10 days with 4 kg EDB per 1000
bushels. At 12 weeks post-fumigation, they found on average 5 ppm EDB
in whole grain and 2 ppm EDB in flour, 18 ppm EDB in bran, and .002
ppm in bread derived from the whole grain. McMahon (1971) fumigated
wheat with an admixture of CT, CD, EDB, anui methylene chloride
(70.5:l6.5:6.6:6.4 w/w). The admixture was applied at 3.8 liters per
1000 bushels i.e. 550 g EDB per 1000 bushels. Residues Of 2.5 ppm EDB
in whole grain and 1.3 ppm EDB in milo at 2 and 3 months
post—fumigation, respectively, were reported.

Rains and Holder (1981) quantified residues Of EDB in flour and
biscuit samples that would have been used in a school lunch program.
They found up to 4.2 ppm EDB and 0.3 ppm EDB in flour and biscuits,
respectively. Out Of 22 flour and 22 biscuit samples analyzed, 5
samples of each exceeded current tolerance limits for EDB.

In 1984, the EPA conducted a survey on residues of EDB in grain
products. The data were Obtained from several government and industry
sources. See Table 1 for a summary. Thirty to 75.2% Of raw grains,
17.4 to 69.3% of milled grain products, and 6.0 to 39.5% of
ready-to-eat grain products analyzed contained detectable residues of
EDB. Detection limits were 1 ppb or less. Residues detected ranged
from ND to more than 10,000 ppb in raw grains, ND to 990 ppb in
milled grains, and ND to 51.5 ppb in ready—to—eat grain products. The
percentage Of samples analyzed which exceeded current tolerance

limits for EDB was not reported. In this survey, the EPA. also

10

estimated that prior tO bans against use of EDB as a pesticide: l)
60% of wheat products marketed in the United States contained
detectable EDB and 2) the public Fem;exposed to 8.6 X 10‘5mg EDB per
kg Of diet per day from.wheat products alone. NO estimates were given
as to time needed for EDB-contaminated food products to pass from the
market (Brown 1984, EPA 1984).

All studies reviewed have reported a marked decrease in EDB
residues after aeration, processing, or cooking. These losses could
be due to: 1) evaporation of physically sorbed EDB and/or 2)
conversion Of physically sorbed EDB to chemically sorbed EDB. If the
latter occurs, then the concentration Of free bromide should
increase. Olomucki and Bondi (1955) aerated EDB—fumigated sorghum for
45 chum» During that time, free bromide residues increased 10-15%
while EDB residues_pe£_§e decreased 95-99%. Morris and Fuller (1963)
stored an EDB—fumigated laying inash :hi a. non-hermetically sealed
container for seven days. EDB residues pe£_§e dropped about 40% while
free bromide increased 5-10%. Ambient temperature did not fluctuate
significantly during the study period in either trial. Thus, losses
of EDB during aeration and storage Of grain are primarily due to
evaporation Of physically sorbed EDB. It is not known whether losses
Of EDB in grain due to high temperature are a result Of evaporation
or Chemical sorption. It is likely, however, that more phySiCally
sorbed EDB is converted to chemically sorbed EDB during exposure to
high temperatures than during aeration.

In summary, the concentration Of EDB per _s_e_ in grain after

11

Table 1. Summary of the EPA survey on EDB residue data in grains and grain
products (Brown, 1984).

 

 

l

 

 

 

 

 

EDB residues found Range of residues
%
N0. of detectable Average Median

Commodity samples residues minimum maximum residue residue
Raw Grain Products

wheat 862 75.2 N02 1842 40.3 4.0

Corn 290 60.7 ND >10.000 55.8 1.4

Other 112 29.5 ND >10,000 109.9 ND
Milled Grain Products

Wheat 638 69.3 ND 450 14.4 2.0

Corn 303 55.1 ND 990 44.1 1.5

Other 46 17.4 ND 128 4.0 ND
RTE Grain Products3

Wheat 272 21.7 ND 49.4 2.3 ND

Corn 86 39.5 ND 51.5 4.0 ND

Other 100 6.0 ND 3.8 ND ND

 

1 All residues are in ppb.
2 N0 = not detected, detection limit = 1.0 ppb.
3 RTE = ready-to-eat.

 

12

fumigation is dependent on the grain's chemical composition and varies
with dose, length Of exposure, and amount Of processing, cooking, and
aeration. Directly fumigated grains contain the highest concentration
Of EDB and residues decrease upon further processing. All but one Of
the studies reviewed reported residues Of EDB which exceeded the
current tolerance limits.
IV. Residues of Ethylene Dibromide in Tissues and Blood of Rats and

Chicks

In contrast to grains, little: work. hsa been done. to quantify
residues of EDB in tissues Of animals acutely or chronically exposed to
EDB. Morris and Fuller (1963) fed 2-week Old chicks a diet containing
280 ppm EDB for 2 weeks. They found 118 ppm EDB in liver and 123 ppm
EDB in kidney. They did not determine if residues decreased upon
withdrawal from contaminated diet. Nachtomi and Alumot (1972) gave a
single oral dose of EDBV to chicks (14 mg EDB per 100 g of body weight)
and rats (22 mg EDB per 100 g of body weight). At 5 minutes post-dose,
chick blood and liver contained 4 ug EDB per m1 and 24 ug of EDB per
100 g of body weight, respectively, while rat blood and liver contained
7.1 ug EDB per ml and 70 ug EDB per 100 g Of body weight, respectively.
EDB was non-detectable (detection limit was less than 2 ug) in rat
blood, rat liver, chick blood, or chick liver by EL 113, 24, and 24
hours post-dose, respectively. This indicates an efficient metabolizing

process.

V. Toxicology Of Ethylene Dibromide in Poultry

The effects of EDB on growth, production, and reproduction in

13

poultry have been studied. Mbrris and Fuller (1963) fed 2-week old
male chicks diet at 40 ppm EDB for 2 weeks and Observed a decrease in
growth rate, feed consumption, and feed efficiency. 1hr a paired
feeding trial, the decrease in growth rate was found not only to be a
result of reduced feed intake, but was also due to a growth
depressant effect Of EDB. Alumot et_al, (1968) pair fed three-day Old
male chicks diet at 0, 80, or 180 ppm EDB for 12 weeks and found EDB
reduced feed consumption but not growth.

Bondi E£.§i3 (1955) fed diet at 10, 25, or 60 ppm bromide to
hens. Bromide was incorporporated into the diet by fumigating sorghum
with EDB. Then, free (physically sorbed) EDB was extracted from the
sorghum, and the grain was blended into a laying mash. Thus, the
diets fed tX) hens contained. only chemically sorbed. EDB anui free
bromide and did not contain free EDB. Diets were fed to hens for 16
weeks, and it was reported that there was no effect on egg production
or egg weight. Bondi_e£_al. (1955) also fed hens diet at 10 ppm EDB
pe£_§e for 12 weeks and reported a decrease in egg weight. Thus, they
showed that it is physically sorbed, chemically unaltered EDB which
is responsible for toxic effects in poultry.

Fuller and Morris (1962) dosed hens orally with 0.5, 1, 2, 4,
or 8 mg EDB per hen per day (mg EDB/h/d) over several weeks. Hens
averaged 100 grams Of intake per day so the doses were equivalent to
5, 10, 20, 40, or 80 ppm EDB in the diet. EDB was dissolved in a
water—ethanol solution and injected into the crop daily as follows:
12 weeks (fl? EDB injections followed by 8 weeks of non-contaminated

injections followed by 12 weeks Of EDB injections. At the end of the

14

treatment period, egg production, and egg weights of EDB-treated birds
were compared to controls. Doses from 0.5 to 4 mg EDB/h/d (5 to 40 ppm
EDB) had no effect on egg production but hens which received 8 mg
EDB/h/d (80 ppm EDB) produced 25% fewer eggs than controls. All doses
of EDB reduced egg weight. The loss in egg weight followed a
dose-response pattern. Eggs from hens that received 0.5 mg EDB/h/d (5
ppm EDB) weighed 5% less than controls, whereas eggs from hens that
received 8 mg EDB/h/d (80-ppm EDB) weighed 40% less than controls. Hens
were fed non-contaminated diet for several months after EDB treatment
was complete. Egg production returned to normal after 12 weeks but egg
weight did not equal that of controls until 6 to 10 months
post-treatment. In 1963, Fuller and Morris repeated the 1962 study with
one change. Hens were fed IHHS hi the diet instead Of via daily oral
doses. As in their 1962 study, it was found that 5 ppm EDB reduced egg
weights, but egg production was not affected at doses less than 80 ppm
EDB. They also found that at all doses of EDB there was no effect on
feed consumption, body weight, or mortality.

Olomucki (1957) showed that decreases in egg weight were due to
impaired follicle growth. Fuller and Morris (1962) found that follicles
in ovaries cflf EDB treated hens were only partially developed. Alumot
and Mandel (1969) showed that the impaired growth of follicles was not
due to impaired synthesis or release Of gonadotropic hormones. Alumot
and Harduf (1971) found that the decrease in egg size may be related to
impaired follicular uptake (fl? serum proteins (albumin and globulin).
Hens were fed 100 Inmi EDB until egg weight had dropped to 33% below

controls. then, follicular uptake Of 1251 labeled serum proteins was

 

15

measured. Uptake Of serum proteins per whole yolk or per unit Of
membrane area was only half that Of controls. The authors hypothesized
that membrane permeability was impaired.

Alumot et_ 31, (1968) conducted several feeding trials which
assessed the effects Of EDB on reproduction in chickens. In 2 Of the
trials they fed male chicks diet at 0, 80, or 180 ppm EDB and female
chicks diet at 40 ppm EDB from hatch until sexual maturity and found nO
delay in age Of onset of egg or sperm production. In another trial they
fed adult males 150 or 300 ppm EDB for 12 months and found no effects
(n1 Spermiogenic activity, spermatozoa count, or testes weight but a
decrease in comb weight was reported. Semen from those males was used
to artificially inseminate control females and no effect was observed
on fertility or hatchability Of eggs. In a final trial laying hens were
fed diet at 100 ppm EDB for 4 weeks after which they were artificially
inseminated with semen from control males. Only 12% (fl? eggs laid by

EDB-treated females were fertile. None Of the fertile eggs hatched.

Westlake (1981) orally dosed Japanese quail (Coturnix coturnix)

 

In order to determine the LD50 and LCSO' The Single oral LD50 for

EDB in Japanese quail was 130 mg EDB per kg of body weight. A 95%

confidence interval on the LD50 ranges from 107.4 to 157.3 mg EDB per

kg Of body weight. For a five day exposure, the chornic LC50 for EDB in

Japanese quail was lldl rm; EDB per bird per day; Quail consumed 6.73

grams of diet per day. Therefore the LC is equivalent to diet at 1650

50

ppm EDB. A 95% confidence interval on the LC ranges from 8.9 to 13.9

50

mg EDB per bird per day (1320 to 2020 ppm EDB in diet).

16

In summary, the only' no—effect levels determined for EDB in
poultry have been for egg production and male reproduction. These were
found to be, respectively, 40 ppm and 150 ppm EDB or less in the diet.
The dietary levels Of EDB which have no effect on egg weight, growth,
and female reproduction in poultry have not been determined but are
less than 5 ppm, 40 ppm, and 100 ppm, respectively.

Current tolerance limits for EDB only allow grain containing less
than these levels to be marketed. Therefore, it. is ‘not ‘known if
prolonged consumption Of grains containing less EDB than currently
allowed by tolerance limits would have any significant effect on egg

weight, growth, fertility, or hatchability in poultry.

VI. Toxicology of Ethylene Dibromide in Mammals
A. LD50 and LC50

The LC50 of EDB in mammalian species has not been determined.

The acute oral Single LD for guinea pigs, male rats, female rats, and

50
female mice is 110, 146, 117, and 420 mg EDB per kg Of body weight
(Rowe et_al. 1952).
B. Metabolism of EDB in rats

The metabolic half-life of EDB in intravenously injected rats
is 2 hours. EDB is metabolized in rats by: l) conjugation with
glutathione (GSH) and/or 2) oxidative dehalogenation (Figure l). GSH
conjugation occurs more frequently than oxidative dehalogenation, is

catalyzed by GSH S-transferases, and occurs primarily in the liver. One

or 2 GSH'S can be transferred t1) EDB. If 12 are transferred, the

l7

resultant compound, S-S'ethylene-bis(glutathione) is split
hydrolytically ix) fornr S-(B-hydroxyethyl) glutathione (HEG) or
the sulphoxide Of HEG. If only one GSH is transferred to EDB,
then HEG and its sulphoxide are formed directly. Both routes to

HEG result in the release Of 2 bromides which are excreted in

urine. HEG and its sulphoxide can either bind to polynucleotides

or' be further 'metabolized :hi the ‘kidney' by 21 2-step process.

First, glutamic acid and glycine are removed from the GSH portion

of HEG to form S-(B-hydroxyethyl)cysteine (HEC) or the sulphoxide

Of HEC. Then, HEC and its sulphoxide are metabolized to

S-(B—hydroxyethyl) mercapturic acid (HEM) and its sulphoxide. HEM
is the primary metabolite of EDB. HEC, HEM and their sulphoxides

are excreted :hi urine and bile (EPA 1977, Nachtomi et 31, 1966,

Nachtomi 1970, Shih and Hill 1981).

Oxidative dehalogenation Of EDB occurs 'primarily in the
liver. The reaction is catalyzed by the microsomal oxidase that is
induced by phenobarbital. The end-product is 2-bromoacetaldehyde
that either: 1) binds to proteins or 2) converts to 2—bromoacetic
acid. 2—bromoacetic acid is excreted in ‘urine (Shih. and 'Hill
1981).

C. Mutagenicity and oncogenicity of EDB in rats and mice

Several researchers (Rannug 1980, Anonymous 1977, Dept.
Of Labor 1983) have reviewed in detail the mutagenic and oncogenic
actions of EDB. EDB acts as a mutagen by covalently binding to DNA
via an alkylation reaction which releases a bromide molecule. The

result is formation Of a "half-mustard" reagent that can undergo a

18

 
  
  
 

Happy, 02 uanp+ + ”20
u-az-az-I: . . lr—Cla-bw— usual—coon
EDB MLICd functton oxidase 2-brououceta1dehydc 2-brouoacecic acid
(binds Iicrosoual proteins) (excreted in urine)
,0
E ““2
mm H mkaka§4mf<m4mwi
f 2 GSH 2 Br-(excreted in urine) T-CHZ-CH-CO-NB-Cflz-COOH
t G541,
r GSH S-transfbraae |
3 S-CHZ-CH-CO-NH-CHZ-COOH
r.
3 NB-CO—Cfl -CH -CH-COOH
2 2

f |
9 NH

2 Br— r 2
a S-S'-ethylene-bis(glutethione)
s
e 658

. m5
NH-CO-CBZ-CBZ-Cfl-COOH
S-CHZ-CH-CO-NH-CBZ-COOH
CIz-Clzfoa

S-(fi-hydroxyethyl)glucothione (binds polynucleotides)

glutamic acid, cysteine

m5

S-CHZ-CB-COOH

S-(B-hydroxyethy1)cysteine (excreted in urine and bile)

NH-CO-CH3

-CH2-CH-COOH

c"2"3‘2‘0“

S-(B-hydroxyethyl)mercapturic acid (excreted in urine and bile)

Figure 1. Metabolism Of EDB in rats.

19

second alkylation. Binding Of EDB to DNA in this manner causes: 1)
separatflni of strands, 2) base pair transitions, and 3) single
strand breaks. EDB can be activated to a mutagen (fl? greater
potency by conjugation to GSH. It is thought that EDB might be
responsible for contact tumors while its GSH conjugate might be
responsible for tumors in remote organs.

EDB has also been found to be a potent carcinogen. Tumors
have lxxnr Observed in the forestomach, adrenals, mammary glands,
lungs, and nasal cavities of rats and mice exposed to EDB by
inhalation, intraperitoneal injections, gavage,or dermal routes.
Nitsche_e£_al. (1981) exposed male and female rats to 0, 3,10, and
40 ppm airborne EDB as follows: 6 hours per day and 5 days per
week for 13 weeks. They found that the no—effect level for tumor
incidence after inhalatflmn Of EDB as outlined is 3 ppm airborne
EDB. The no—effect levels for tumor incidence after dermal or oral
exposure have not been determined.

D. Effects Of EDB on reproduction

The EPA published a detailed review on the reproductive
effects of EDB in rats, bulls, cows, Sheep, and mice (EPA 1977).
EDB has been found to interfere with both male and female
reproductive processes. For example, Amir and Lavon (1976) dosed
bulls with 4 mg EDB per kg Of body weight on alternate days over a
20—day period and found that sperm production and motility were
reduced while the number Of sperm that had Udsshapen beads was
increased. Short e£_§13 (1976) exposed pregnant rats and mice to

32 ppm airborne EDB for 23 hours per day during days 6 to 15 Of

20

gestatbmi. It was reported that EDB decreased fetal weight, the
number Of implants per dam, and the number Of fetuses per dam. The
no-effect levels for male and female reproduction in mammals via
exposure by oral, inhalation, or dermal routes have 'not been
determined.
E. Toxicology Of EDB in humans

Humans are exposed to EDB by ingestion, inhalation, or
dermal contact. Ingestion Of EDB is 21 result Of consumption Of
EDB—contaminated grain or fruit while inhalation exposure to EDB
comes from leaded gasoline fumes which can contain trace
quantities Of EDB. Both inhalation and dermal exposure occur
occupationally' during production and/or application (fl? EDB. The
Occupational Safety and Health Administration in“; published 21
detailed review (Hi occupational exposure Ix) EDB (Dept. Of Labor
1983). Several other studies have reviewed the toxicology Of EDB
in humans. Olmstead (1960) reported that a woman who had ingested
a single dose (4.5 m1) of EDB experienced vomiting, abdominal pain,
diarrhea, and nausea. She died 54 hours after ingestion. An
autopsy showed massive centrilobular liver necrosis and damage to
tubular epithelium (fl? kidneys. Dermal contact with EDB has been
found to cause severe burns (Peoples 33 313 1978). Studies that
have surveyed populations that are exposed to EDB have only given
limited evidence that EDB decreases fertility or increases the
risk. Of cancer (Ott 51E 3&3. 1980, Takahashi. 1981, ‘Wong 11979).
However, :hi 1983, using 21 one-hit carcinogen model derived from

animal carcinogen studies with EDB, the EPA predicted that the

21

levels of EDB in the nation's food supply at that time would lead
to an additional 3 cancer deaths per 1000 people. They' also
predicted that lifetime occupational exposure to 0.4 ppm airborne
EDB would lead to an almost 100% chance Of developing cancer. It
is these estimates which caused the EPA to ban uses of EDB as a
pesticide. Much controversy surrounds the EPA'S estimates (Ramsey
et_313 1979), and it is unclear at this time what amount or length
Of exposure tO EDB in the diet or through inhalation will increase
cancer risk to the general public.
VII. Summary

It has been found that EDB residues per §e_are sorbed by
grain during fumigation. This results in EDB exposure to the
general public through consumption Of EDB-contaminated grain.
Therefore, much work was done to categorize the mutagenic,
oncogenic, and reproductive effects of EDB in animals. The results
Of this research were used to estimate possible toxic effects Of
EDB in humans. However: 1) most Of the research was conducted with
levels Of EDB that are much higher than are usually found in food
products after fumigation with EDB, 2) few no-effect levels have
been determined, and 3) little work has been done to quantify
residues of EDB in tissues of food-producing animals after
consumption (fl? EDB-contaminated grain. If time latter occurs, it
would. represent. another route for EDB. exposure to tine general
public. Thus, the purpose of this research was to feed chickens

EDB—contaminated grain Obtained from the Michigan food supply to

determine if and to what extent EDB per §e_is deposited into tis-

sues and eggs.

 

 

MATERIALS AND METHODS

1. Experimental Methods
A. Composition and blending of experimental diets

An EDB-contaminated diet and 21 non-contaminated. diet
were prepared. Both diets contained equal amounts Of all
ingredients except for flour. The EDB source was a. flour
confiscated by the Foods Division Of the Michigan State Department
of Agriculture. It anui a non-contaminated cookie flour Obtained
from Michigan State University FOOd Stores were incorporated into
their respective diets t1) account for .55.7% cu? the diet. The
contaminated flour was confiScated on February 2, 1984 from the
Amendt Milling Company of anroe, MI and was an unbleached cake
flour marketed as Honey Queen. The confiscated cake flour
contained 31.1 ppm EDB. The resulting EDB diet assayed at 6.7 ppm
EDB although it theoretically should have contained 17.3 ppm EDB.
The non-contaminated cookie flour was e1 non-brominated. pastry
flour produced by Michigan. Bakery Supply and. was marketed :as
Cookie Maker. Both poultry diets were blended in Mix-MillT°M° 250
CT Nutri Blenders. TR) prevent contamination of the control diet,
it was blended in one Mix—Mill blender, and. the EDB. diet ‘was
blended in another.

B. Husbandry

Single Comb White Leghorn female chickens in their first

22

23

Table 2. Composition of experimental diets.

 

 

Non-contaminated
flour diet1

EDB-contaminated
flour diet2

 

Ingredient parts/1000 parts/1000
Alfalfa, dehy, 17% 40 4O
Soybean meal, 44% 240 240
Cookie flour3 557 0
Cake flour4 0 557
Limestone 72 72
Dicalcium phosphate 22 22
Corn oil 55 55
Ethoxyquin 0.125 0.125
dl-methionine l 1
Magnesium oxide 5 5
Choline chloride, 50% l 1
Vitamin mix5 3 3
Mineral mix6 0.5 0.5
Selenium mix7 0.5 0.5
Iodized salt 3 3

 

l Metabolizable energy = 2.65 kcal/g; Crude protein = 17%
2 Metabolizable energy = 2.65 kcal/g; Crude protein = 18%
3 Non-contaminated flour purchased from Michigan State University Food

Stores.

4 contained 31.7 ppm EDB; confiscated from the Amendt Milling Co. of Monroe,

MI.

5 Supplied per kg of diet: Vitamin A, 11,000 I.U.; Vitamin 03, 1,100
I.C.U.; Vitamin E, 11 I.U.; Vitamin K, 22 mg; Thiamin, 2.2 mg; Riboflavin,
4 mg; Pantothenic acid, 14.1 mg; Nicotinic acid, 31.5 mg; Pynidoxine,
4 mg; Biotin, 0.1 mg; Folic acid, 1.3 mg; Choline, 13.2 mg; Vitamin

B12, 0.0] mg.

5 From Calcium Carbonate Company; Supplied per kg of dietszManganese,
60 mg; Zinc, 40 mg; Iron, 30 mg; Copper, 5 mg; Iodine, 0.5 mg.

7 From Calcium Carbonate Co.; Supplied 0.1 mg Selenium/kg diet.

 

24

year Of production were used in this study. They were Obtained
from.51 laying flock maintained an: the Michigan State University
Poultry Research and Teaching Center (PRTC). Control hens were
housed in Anthony Hall, Michigan State University and were
confined singly in 41.9x20.3x40.6 cm (waxH) cages with 5.1x2.5 cm
wire mesh. EDB-treated hens were housed at the PRTC. They were
isolated to prevent the possible spread Of EDB. EDB—treated hens
were confined singly in 45x45x45 cm (LxWXH) cages with 5.1x2.5 cm
wire mesh. Artificial lighting was supplied to both rooms on a
schedule of 16 hours light:8 hours dark, daily. The control room
was maintained at 23ilO°C while the EDB room. was maintained at
l8.3:2.8°C.

Control and EDB-treated hens received feed and water ad
libitum throughout the study. Feed intake and body weights were
Obtained weekly. Eggs were collected daily, marked in pencil with
date, cage number, and treatment, and stored in plastic egg flats
at room temperature until processed for EDB residue analysis.

C. Schedule

The study consisted Of a pre—experimental and an
experimental time period. The experimental time period was
subdivided into residue build—up and residue withdrawal time
periods based on dietary treatment and expected concentration Of
EDB residues in eggs and tissues. See Table 3 for a summary of the
schedule.

The pre-experimental period, from March 7 to March 20 (days

-14 to -1 Of the studY), was to allow the hens to adapt to their

25

respective environments and to convert the hens from the usual
commercial—type mash to the semi-purified type diet containing the
flour. The transition of one diet to the other occurred over a 7
day period the first 2 days of which hens were fed a blend of
commercial laying mash (CLM) and non-contaminated flour diet
(NCFD) in a ratio Of 3:1 (CLM:NCFD). This was followed by 2 days
of feeding the blend at 1:1 and one day Of feeding at 1:3. The
next day hens were fed 100% NCFD completing the transition to the
semi-purified type diet.

The feeding of the EDB-contaminated diet occurred from
March 21, 1984 to April 11, 1984 (Days 0 to 21 of residue
build-up). The duration (M? 21 days was considered sufficient to
allow maximum build up of EDB in eggs and hens. On March 21, the 8
best laying hens in Anthony and the 16 best laying hens at PRTC
were chosen 1x1 receive experimental diets. Hens not chosen were
returned to the PRTC laying flock. The 8 hens in Anthony received
the diet with non—contaminated flour, and the 16 hens at PRTC
received EDB-contaminated diet from March 21 until the evening of
April 10 iduni feed was removed. Hens were fasted for 18 hours
prior 11) necropsy to zfljrnv feed. residues 11) pass through. the
gastrointestinal tract. This eliminated the possibility that
during assay procedures feed residues of EDB would contribute to
the EDB residues in the chickens.

On April 11, 4 control and 8 EDB hens were randomly selected

and euthanized bloodlessly with excess 002. Of the hens

 

Table 3. Experimental schedule.

26

 

 

 

 

Experimental Number
Dates days of hens Treatment
Pre-experimental
Control hens 3/ 7/84-3/20/84 -14 to -1 8 None]
EDB hens 3/ 7/84-3/20/84 -14 to -1 16 None1

Control hens 3/21/84-4/11/84
EDB hens 3/21/84-4/11/84

Control hens 4/11/84-5/ 2/84
EDB hens 4/11/84-5/ 2/84

Residue build-up

 

0 to 21 82
0 to 21 163

Residue withdrawal

 

21 to 424 42
21 to 424 83

Non-contaminated
flour diet

EDB-contaminated
flour diet

Non-contaminated
flour diet

Non-contaminated
flour diet

 

l Hens were acclimated to the semi-purified flour diet during this period.

2 4 control hens were sacrificed on the final day of each specified time
period, 2 of which were used for analysis of EDB residues in whole body
and 2 of which were used for tissue analysis.

3 8 EDB hens were sacrificed on the final day of each Specified time period,
4 of which were used for analysis of EDB residues in whole body and
4 of which were used for tissue analysis.

4 Days 0 to 21 of withdrawal.

27

euthanized, 12 control and £1 EDB hens were randomly selected for
determination of whole body EDB residues. They were vacuumed to
remove dust from their feathers and then sealed in plastic bags
and frozen at -20°C. From the other 2 control and 4 EDB hens
euthanized, liver, kidney, abdominal fat, breast skin, and right
breast muscle (pectoralis superficial) were removed for analysis
of EDB residues in those tissues. The livers were weighed and had
11) grams excised for measurement Of deed function oxidase (MFO)
activity. The tissues were sealed in plastic bags and frozen at
-20°C. After necropsy, the EDB room. was cleaned enui vacuumed,
thereby completing the time period for residue build-up.

Residue withdrawal extended from April 11, 1984 to May 2,
1984 (Days 0 to 21 of withdrawal). It was predicted that
concentration Of EDB in eggs and tissues would be reduced during
this time. The remaining 4 hens in Anthony and 8 hens at PRTC were
fed non-contaminated flour diet from April 11 until the evening of
May 1 when feed was removed. Hens were fasted for 18 hours, and on
May 2 the study was terminated when the remaining hens (4 control
and 8 EDB) were euthanized bloodlessly with excess C02. The
necropsy procedure used (n1 April 11 was also followed on May 2,
i.e., samples were obtained for whole body, tissue, and MFO
analysis. The number of hens euthanized and tissue samples taken
at each time period is presented in Table 3.

D. Safety methods and contaminated waste disposal

Because EDB is a xenobiotic, its use is regulated. Thus,

EDB—treated luau; were isolated.:u1 room 4E PRTC, and any part of

 

28

the research which involved possible contamination from EDB was
conducted within. room. 4E. All personnel who entered [d3 wore
protective clothing (disposable coveralls, hair nets, masks,
gloves, and plastic boots). ResearCh equipment used in the room
was rinsed with hexane to remove EDB residues. Droppings were
collected on. disposable plastic Sheets, and .all inorganic. and
organic waste was sealed’in barrels and disposed of in accordance
with state and federal laws by Michigan State University's

Laboratory Animal Care Service.

II. Mixed Function Oxidase Assay
A. Introduction

There are two classes Of xenobiotics (XB) which induce
activity in mixed function oxidase (MFO) enzymes:
phenobarbital—type inducers and 3-methy1cholanthrene—type
inducers. Activity of aminopyrine N-demethylase (AND) is measured
to determine if a XB is a phenobarbital—type inducer, and activity
of aryl hydrocarbon hydroxylase (AHH) is measured to determine if
a xenobiotic is a 3-methylcholanthrene-type inducer. Both enzymes
are found. in the cytosol of liver cells associated with rough
endoplasmic reticulum (Bresnick 1978). AND and AHH activity were
accounted for in microsomes (rough endoplasmic reticulum) and were
related 11) protein content of lime microsomes. The methods for
determining AND and AHH activity were adapted from Anders and

Mannering (1966) and Van Cantfort £5.2i3 (1977), respectively

NW)

28

the research which involved possible contamination from EDB was
conducted within room 4E. All personnel who entered 4E ‘wore
protective clothing (disposable coveralls, hair nets, masks,
gloves, and plastic boots). Research equipment used in the room
was rinsed with hexane to remove EDB residues. Droppings were
collected on disposable plastic sheets, and. all inorganic and
organic waste was sealed in barrels and disposed of in accordance
with state and federal laws by Michigan State University's

Laboratory Animal Care Service.

II. Mixed Function Oxidase Assay
A. Introduction

There are two classes of xenobiotics (XB) which induce
activity in mixed function oxidase (MFO) enzymes:
phenobarbital—type inducers and 3—methylcholanthrene-type
inducers. Activity of aminopyrine N—demethylase (AND) is measured
to determine if a XB is a phenobarbital—type inducer, and activity
of aryl hydrocarbon hydroxylase (AHH) is measured to determine if
a xenobiotic is a 3-methylcholanthrene—type inducer. Both enzymes
are fOund :hi the cytosol 11f liver cells associated with rough
endoplasmic reticulum (Bresnick 1978). AND and AHH activity were
accounted for in microsomes (rough endoplasmic reticulum) and were
related to protein content of the microsomes. The methods for
determining AND and AHH activity were adapted from Anders and

Mannering (1966) and Van Cantfort et_al, (1977), respectively

29

B. Miscellaneous

See Appendix E for preparation of reagents. Reagents were
prepared the day before the livers were procured and the
microsomes were isolated. During the assay, Gilson Pipetman
adjustable-volume ‘pipets (Models Kr79-16l43, Kr79—11742,
K-79-11158, and K-79-l6907) and Rainin disposable pipet tips
(types RC20, RC200 and RCZOOO) were used to pipet volumes between
20 ul and 5 ml. Volumes of 5 ml or greater ‘were dispensed
with. graduated cylinders or :1 Lab Industries Repipet®. Samples
were on ice during all steps of the assay except incubation. See
Appendix F for raw' data Obtained during crude protein
determination and assay termination.

C. Isoation Of microsomes

Livers were excised from hens, weighed, and profused with
cold 150 mM KCl. Approximately 10 g of each profused liver was
placed into a polycarbonate centrifuge tube and minced into small
pieces with a pair Of scissors. Approximately 20 ml (2 times the
wet weight of the liver sample) of homogenizing buffer was then
added to the centrifuge tube, and the liver was homogenized for 5
seconds, twice at speed 5 with a polytron homogenizer (Type PT 10
OD). Inbetween samples, connective tissue was cleaned out of the
polytron blade, and the blade was rinsed with double-distilled
water (DD H20). After homogenization, the samples were spun for
20 minutes at 12,000 rpm in a Sorvall® Superspeed RC—2 Centrifuge

with SA-600 rotor. The resultant supernatant was poured through a

30

triple layer of cheesecloth into 21 thick-walled. polycarbonate
centrifuge tube and spun for 75 minutes at 30,000 rpm in a Beckman
L2—65B ultracentrifuge with type 30 rotor. The supernatant was
discarded and the microsome pellet was .1eft :hi the centrifuge
tube. Ten milliliters of 200 me Tris-HCl was added to the
centrifuge tubes, the tubes were covered, and the samples were
stored overnight at 4°C. The following day, the pellet was
scraped Off the side of the centrifuge tube with a glass rod and
suspended into 200 mM Tris—H01 by homogenization with the
polytron at speed 5 for 2 seconds.
D. Determination of crude protein in microsomes

After the microsome pellets were suspended into 200 mM
Tris-HCl, the concentration of crude protein in the microsomes was
determined by the Biuret method (Gornall E£_al, 1949). Solutions
of bovine serum albumin (BSA) at 0, 3, and 5 mg were used to
establiSh a dose—response curve of mg of protein versus
spectrophotometric absorbance from which the weight (mg) of
protein in Udcrosome samples was calculated. The steps involved
were:

1. Duplicates of each standard curve solution and each microsome
sample were analyzed.

2. Volumes of DD H O, 6% NaOH, and 180 mM KCl were added to
standard curve and microsome sample test tubes as outlined
in Table 4.

3. BSA was added to standard curve test tubes as outlined in
Table 4.

4. 100 ul Of microsomes was added to each microsome sample
test tube.

31

Table 4. Volumes of reagents used to establish a three point standard
curve for the biuret protein determination assay.

 

 

‘ -Protein - mg,
Reagents1 UnknownsZ 037 35 53

 

 

Microsomes - ml 0.1 0.0 0.0 0.0
Bovine serum albumin — ml 0.0 0.0 0.6 1.0
Biuret - ml 0.2 0.2 0.2 0.2
Double-distilled H20 - ml 1.9 1.9 1.4 1.0
6% NaOH - ml 2.0 2.0 2.0 2.0
150 mM kcl - ml 0.0 0.1 0.0 0.0
Total volume - ml 4.2 4.2 4.2 4.2

 

1 See Appendix E for preparation of reagents.

2 Refers to microsome samples for which protein concentration was being
determined.

3 Standard dilutions used to establish a dose-response curve of mg of
protein versus spectrOphotometric absorbance from which concentration
of protein in microsome samples was calculated.

10.

11.

ul,

 

32

200 ul of Biuret was added to each test tube.

All test tubes were vortexed for 15 seconds at full speed
with a K—550-G Vortex—Genie.

Color was allowed to develop for 10 minutes.
Absorbance of each sample was read in a Gilford Stasar II
1367x5 spectrophotometer at A = 540 nm. DD H O was used

2
as zero.

From the standard curve solutions a dose-response line was
calculated for x = mg protein and y = absorbance.

The amount of protein (mg) in 100 ul of microsomes was
inversely predicted from the dose—response line. After
duplicates were averaged, this number was divided by 0.1

to obtain mg protein/ml microsomes.

Each microsome sample was then diluted with 200 mM Tris-HCl
so that there was 1 mg protein/200 ul microsomes.

E. Incubation of microsomes with substrate

After microsome samples were diluted to 1 mg protein/200

microsomes were incubated with aminopyrine and benzo(d)—

pyrene substrates so that activity of AND and AHH, respectively,

could be measured.

1.

The water bath in a Dubnoff Metabolic Shaking Incubator was
preheated to 37°C.

For the AND assay, 2 samples and 2 blank 12x75 mm test tubes
were labeled for each microsome sample. Two 12x75 mm test
tubes were labeled for each point of the AND standard curve
(Table 5).

For the AHH assay, 2 samples and 2 blank 8 ml scintillation
vials were labeled for each microsome sample.

16 ml of 200 mM Tris-HC1, 1.28 ml of glucose—6—phosphate (G6P),
640 m1 of 200 mM MgCl and 64 ul of glucose—6—phosphate dehydro—
genase (G6PD) were blended to make a premix. In the premixes
the reagents must be in the ratio 250:20:10:1 (Tris-HC12G6P:
MgClZ: G6PD).

33

Table 5. Volumes of reagents used to establish a standard curve for the
aminopyrine N-demethylase assay.

 

Standard curve solutionsg- mM formaldehyde

 

 

Reagents1 04 34 6‘ 124
30 mM formaldehyde - ul 0 100 200 400
Double-distilled water - ul 1000 900 800 600

 

] See Appendix E for preparation of reagents.

2 30 mM formaldehyde (CHZO) and double-distilled water were blended as
outlined in the table. Then 20 ul of each solution was used to develop
a standard curve of X = nmoles formaldehyde and Y = spectrophotometric
absorbance. 20 ul of 3 mM CH20 = 60 nmoles CH20; 20 ul of 6 mM CH20
= 120 nmoles CHZO; 20 ul of 12 mM CH20 = 240 nmoles CHZO.

10.

ll.

12.

 

34

280 ul of premix was pipetted into AND and AHH blank tubes and
Vials and AND standard curve tubes.

16 mg of B-nicotinamide adenine dinucleotide phosphate (NADP,
Sigma, N-0505) was blended into the remaining premix, i.e.
0.5 mg Of NADP was added for each 280 ul of premix remaining.

280 ul of NADP premix was added to AND and AHH sample tubes
and vials.

A four point AND standard curve was established as outlined
in Table 5 by pipetting 20 ul Of each standard solution into
its corresponding test tube. Then, 200 ul of 150 mM KCl was
added to each standard curve test tube.

20 ul of tritium—labeled benzo(o)pyrene was pipetted into
three 8 ml scintillation vials labeled as total count vials.
5 ml of benzO(d)pyrene cocktail was added, and the vials
were capped and saved until assay termination.

200 ul of microsomes was pipetted into AND blank and sample
tubes and AHH sample vials. Tubes and vials were placed in
the water bath.

Sample, blank, and standard curve tubes and vials were oscil—
lated in the water bath for 5 minutes at 37°C and 60 rpm.

Oscillation was stopped, a timer was set for 30 minutes,
and at 10 second intervals, 20 ul of aminopyrine substrate
was pipetted into AND blank and sample tubes. Substrate
was never added to AND standard curve tubes. Also at 10
second intervals, 20 ul of benzo(o)pyrene substrate (BP)
was added to AHH blank and sample tubes. After substrates
were added, tubes and vials were incubated at 37°C and

60 rpm for the time that remained out Of the 30 minutes.

F. Assay terminations and calculation of enzyme activity

After the nflcrosomes were incubated with substrate, AND

and AHH enzymatic reactions were terminated and amount of product

formed was measured as follows:

I.

AND termination and activity calculations

1.

After 30 minutes of incubation, at 10 second intervals, 1
ml of 20% ZnSO was added to AND blank, sample, and
standard curve tébes.

II.

10.

AHH

35

Tubes were removed from the water bath, and 1 m1 Of saturated
BaOH was pipetted into each tube.

Tubes were vortexed at full speed for 15 seconds.

Tubes were centrifuged at full speed for 30 minutes in
a Sorvall® GLC—4 centrifuge.

The water bath was heated to 60°C.

After tubes had finished spinning, 7 m1 of supernatant
and 1 m1 of nash reagent were pipetted into a 12x75
mm test tube, and tubes were vortexed at full speed
for 15 seconds.

Samples were heated in the water bath for ten minutes
and then cooled to room temperature.

Absorbance was read in the Gilford spectrophotometer at
A = 412 nm. DD H20 was used as zero.

From the absorbance of the standard curve solutions, a
dose-response line was calculated for x = nmoles formal—
dehyde produced/30 minutes and y = absorbance.

The amount Of formaldehyde produced by AND in 30 minutes
in nmoles was inversely predicted from the dose-response
line. Duplicates were averaged, and blank values were
subtracted from sample values. The resultant number was
divided by 30 to Obtain net nmoles formaldehyde produced
by AND/mg microsomal protein/minute of incubation.

termination andvactivity calculations

. After 30 minutes of incubation, ath second intervals, 1

DMSO-KOH was added to AHH blank and sample vials.

. Vials were removed from the water bath and 200 ul of

microsomes were added to AHH blank vials.

. 5 ml Of glass—distilled hexane was added to blank and

sample vials.

Vials were rotoracked for 20 minutes at full speed in a
Fisher 343 Roto-Rack.

. The hexane layer was aspirated. Any emulsion formed during

rotoracking was not aspirated.

III.

36

6. Steps 3 and 4 were repeated.
7. The hexane and emulsion layers were aspirated.

8. 500 ul of the DMSO-KOH phase was transferred to an 8 ml
scintillation vial, 5 ml of benzo(o)pyrene cocktail was
added, and vials were capped.

9. AHH blank, sample, and total count vials were counted for
'10 minutes on channel 11 in a Searle 68700 Isocap/300
Temperature Controlled Liquid Scintillation Counter.

10. After the vials were counted, 100 ul of tritiumrlabeled
toluene (New England Nuclear, NBS—006, 2.07x105 dpm/100
ul in April 1984) was added to each vial, and vials were
recounted for 10 minutes on channel 11. B channel counts
per minute (cpm) before and after toluene spike were
calculated by dividing by 10. Efficiency was calculated
for sample, blank, and total count vials as follows:

Efficiency = cpm after toluene spike - cpm before toluene
spike
2.07 x 10D dpm

 

ll. AHH produces hydroxylated benzo(d)pyrene (BP-OH) as an
end product. The pmoles of BP—OH produced by AHH/mg
microsomal protein/minute of incubation in sample and
blank solutions was calculated as follows:

pmoles BP—OH produced by AHH/mg protein/minute =

 

 

 

1
sample or blank cpm x efficiency x 64,000 pmoles BP/20 ul
1
average cpm in 20 ul BP x efficiency 1
x x l x l

 

 

3
l 1 mg protein in 200 ul microsomes 30 minutes

12. Duplicates were averaged, and net pmoles BP-OH produced

by AHH/mg microsomal protein/minute of incubation was
calculated by subtracting blanks from samples.

Headspace GC Analysis of EDB Residues in Egg, Tissues, and
Diet
A. Introduction

Headspace GC was developed as a means of accurately

37

quantifying trace volatiles in solid matrices. One application of
headspace analysis involves dissolving the solid in a liquid in a
gas-tight vial. The resultant solution is heated driving volatile
compounds into the ‘headspace of the vial. After thermodynamic
equilibrium between the gaseous and liquid phase is reached, an
aliquot of the gaseous phase is swept onto a column for analysis
(Vitenberg ‘et .31. 1974). Because EDB is volatile under the
conditions described, headspace analysis was chosen as the method
for quantification of EDB residues in egg, tissues, and diet.
B. Headspace gas chromatograph and integrator conditions

Residues of EDB in egg, tissues, and diet were
determined with a Perkin Elmer F45 headspace GC (HSGC) equipped
with: l) a 63Ni electron capture detector emitting a 3.0 mV
signal, 2) a 6'x1/8" i.d. glass column containing; 1% SP—lOOO
liquid phase on 60/80 flesh Chrbopack.l3 solid support, and 3) a
Hewlett Packard 3390A integrator. Analysis temperatures were: 1)
needle = 150°C, 2) injector = 150°C, 3) colunui = 160°C, 4)
detector == 200°C, zuul 5) automatic turntable oil bath == 90°C.
Argonzmethane (9:1) was the purge gas for egg, tissue, and diet
samples and. was the carrier gas for egg samples while ‘helium
(99.99% pure) was the carrier gas for tissue and diet samples. Gas
flow rate was 30 cc/minute for both carrier and purge. See Table 6
for integrator parameters.

C. General procedure
Egg, tissue, or diet samples were weighed with a Mettler

top-loading balance into a 24 ml Perkin Elmer crimptop vial. A

38

.umseFxm pcmmcmF m
.mcFmema op mcFmeam F

 

 

mm mm mF mF mF mF mF NmF me waxy xmwa
ooF ooF ooF ooF ooF ooF ooF ooF ooF uumnmc amt<
F F o F- o F F F F- uFo;mmch
mF.o oF.o oF.o mF.o oF.o oF.o mF.o oF.o oF.o sputsxama
m.o m.o m.o m.o m.o m.o m.o m.o m.o :FE\EO . woman peace
o o o o F m N F F- :onmacmea

m m m m m m m m m as . ocmN
FmFo caoFm mFumzz cm>Fu xmchx Fan :Fxm xvon mam tomeMLma

mFoez

 

 

.aeaFaFaeao taaaeaaaeF <ommm aeaeoae aaanaz .a aFaaF

39

Tipet automatic transfer pipet was used to dispense 20 N H SO into

2 4
the vial. EDB standard (for spiked samples only) was then pipetted

into the vial with a Hamilton 701N microliter syringe. Vials were
sealed with teflon—liquid washer septa and metal seals. Samples
were digested at 90°C in the automatic turntable of the HSGC.
After digestion, the vials were shaken manually, replaced in the
turntable, and heated at 90°C until EDB was in equilibrium between
the gas and liquid phases. Then, an aliquot of gas was injected
into the column by the HSGC automatic injector. This “method
required that for egg, tissues, and diet several analysis
parameters had to be determined: 1) identification of the EDB peak
on. chromatograms, 2) 'weight Of sample and. voluuma of acid, 3)
digestion time, 4) equilibration time, and 5) analysis time.

The EDB peak on egg chromatograms was identified by obtaining

chromatograms Of the following samples: 1) air, 2) 5 m1 20N H2804,

3) 5 Ifl. 20 N H2804 + 5 ifl. methanol, 4) 55 ml 20 N H2S04-+ 5 ul
methanol + 5 ng EDB, 5) 5 ml 20 N H2804 + 5 111 methanol + 2 g
control egg, and 6) 5 ml 20 N H S0 -+ 5 ul methanol + 2 g

2 4

control egg + 70 ng EDB (Figure 2). The peak at retention time
5.20 or 5.21 in the chromatograms of Figure 2 is found only on
chromatograms Of samples which contained EDB. Therefore, it was
identified as the EDB peak. This procedure was also followed when
identifying the EDB peak on chromatograms of tissues and diets.
Determining the weight of sample and volume of acid to use for

egg, tissues, and diet involved determining what volume of

.AI

 

 

 

Air

‘2’
“‘3‘1—
1- E
9!

5 ml ZON H2804

{B

 

,1. 1y

 

SURF

5 ml 20N H2804

5 ul methanol

1.11

 

 

 

40

2 .36
1.12.

 

 

 

ffifiL——

--.-_u;.-_~—. _-. .‘I,,. vF _ - .F _ '

5 ml 20N H2804

5 ul methanol
5 ng EDB

2J1

ll ’3.
n
fifi ‘fiDB peak
.-

1g:

:1

ll

 

 

 

 

 

SUE?

:igil

 

 

 

 

t

5 ml 20N H2804

5 ul methanol
2 g control egg

2J2

4J3

217

5 ml 20N HZSO4

5 ul methanol
2 g control egg
70 ng EDB

EDB peak

/

 

 

 

Lo

Figure 2. Identification of EDB peak on chromatograms using

egg as an example.

41

headspace in the sample vial would give optimum resolution of the
EDB peak. Sample weight had to be great enough to detect EDB while
acid volume had to be great enough to digest the sample and to
keep the sample in solution, but enough headspace was needed for
optimum partition Of EDB into the gaseous phase. Several
combinations of weights and volumes were tried until the weight of
sample and volume Of acid which gave optimum. resolution. and
detectability had been determined.

Sample digestion was considered complete when it appeared that
all particles had been broken up and were in solution.

Equilibration time, the time needed for maximum partition Of
EDB into the headspace, was determined for egg, tissues, and diet
as follows. Twelve control samples were spiked with equivalent
amounts Of EDB, digested, heated at 90°C (equilibrated), and
injected onto the column at 15 minute heating intervals up to 180
minutes. Integrated peak area for EDB was recorded for each time.
The point in time at which peak area plateaued was chosen as the
minimum time needed for equilibration.

Analysis time, the time needed for a sample to completely pass
through the column, was determined in egg, tissues, and diet as
follows. A control sample was spiked with EDB, digested,
equilibrated, and injected. The point in time at which peaks no
longer appeared on the chromatogram was chosen as minimum analysis

time.

—‘_

Jr.

42

D. Preparation and storage of EDB standard solutions

Calibration curves in egg, tissues, and diet were
developed by spiking samples with EDB (Aldrich Gold Label; 99%
pure; 24,065-6) dissolved in reagent grade methanol (CHBOH). A
stock solution prepared at 15 mg EDB per 50 m1 CH3OH was diluted
with CH3OH to develop standard solutions used to spike samples.
Stock and standard solutions were prepared fresh weekly, stored in
50 ml volumetrics at -25°C, and warmed to room temperature before
use.

E. Analysis of EDB residues in egg

Eggs collected from hens fed experimental diets were
weighed individually with a Mettler top-loading electronic
balance. Then egg contents (yolk + albumen) were pooled by day
(over hens) within treatment for experimental days 1 to 14 and 22
to 42 (1 to 21 Of withdrawal) and were pooled by hen within
treatment over experimental days 15 to 21. This pooling regimen
was followed because it was expected that concentration of EDB in
eggs would increase from days 1 to 14 of feeding EDB—contaminated
diet, be at maximum. concentration from days 15 to 21 Of feeding
EDB-contaminated diet, and would decrease during days 1 to 21 of
withdrawal from EDB-contaminated diet. See Tables 7 and 8 for a
summary Of how eggs were pooled. Samples were pooled (after egg
contents had been broken out of the shell) by homogenization in a
Waring blender at low speed for 15 seconds. Homogenized samples

were stored at —20°C in glass bottles with screw caps. Prior to

43

Table 7. Summary of pooling method for eggs collected days 1 to 14 and

 

 

 

 

22 to 421. .
# of hens that laid3
Day Diet Control treatment EDB treatment

1 EDB-contaminated2 7 15
2 " 6 4
3 " 7 l4
4 " 7 10
5 " . 5 9
6 " 4 7
7 " 8 l3
8 " 7 9
9 ” 5 5
10 " 7 8
11 " 7 9
12 " 3 5
13 " 6 8
14 “ 6 10
22 Non-contaminated l 3
23 " 4 5
24 " 3 5
25 " 4 4
26 " 2 1
27 ” 3 5
28 " 3 4
29 " 4 4
30 " 2 5
31 " 4 5
32 " l 3
33 " 4 6
34 ” 3 3
35 " 2 4
36 " 2 6
37 " 3 3
38 " 1 6
39 " 3 6
40 " l 4
41 " 1 6
42 " 2 4

 

1 Days 0 to 21 of withdrawal.
2 Control hens received non-contaminated diet days 1 to 14.
3 Represents number of eggs pooled per day for each treatment.

44

Table 8. Summary of pooling method for eggs collected days 15 to 21.

 

 

# of eggs laid by each

 

Treatment Hen number hen from days 15 to 211
Non-contaminated diet 1 5
2 5
3 5
4 6
5 4
6 5
7 6
8 4
EDB-contaminated diet 1 0
2 0
3 0
4 4
5 4
6 2
7 O
8 4
9 2
10 2
11 5
12 5
13 0
14 5
15 5
16 4

 

l Represents number of eggs pooled per hen over days 15 to 21.

._.——.

 

45

HSGC analysis, samples were thawed and gravimetrically transferred
with a disposable pasteur pipet to a sample vial.

HSGC analytical parameters for egg samples were: 1) sample
weight = 2.0 g, 2) 20 NHZSO4 = 5 ml, 3) digestion time = 15

minutes, 4) equilibration time = 75 minutes, 5) analysis time = 15

minutes, and 6) injection time - 6 seconds.

Concentration of EDB in eggs was inversely predicted from a
calibration curve developed. in EDB-spiked control eggp Samples
were spiked with 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0,
5.5, 6.0, 6.5, 7.0, 7.5, 15, 22.5, 30, 45, 60, 75, 90, 105, or 120
ppb EDB, digested, equilibrated, and injected. Integrated area of
the EDB peak was recorded for each dose. An analysis of variance
was conducted on areas obtained for doses 0.5 to 7.5 ppb EDB.
Areas from doses 0.5 to 4.0 ppb EDB were not significantly
different from each other (P<0.05) while area at 4.5 ppb EDB was
significantly greater than areas from 0.5 to 4.0 ppb EDB (P<0.05)
(Figure 3). Therefore, the calibration curve with x=ppb EDB and
y=integrated area of the EDB peak was calculated from 4.5 to 120
ppb EDB. The prediction equation is 9=707x -509 with r=.99 (Figure
4). The 95% confidence interval (C.I.) on r extends from .983 to
.994, lflma 95% CLI. (hi the y-intercept (b0) is -509i989, and the
95% C.I. on the slope (bl) is 707:27. Since the C.I. on bO
includes zero, and the C.I. on bl does not, one can conclude that
the origin is zero and the regression line is not horizontal. 95%

C.I.'s on hy/x and y/x are pictured. in Figure 4. There is

(My w-v- ,~ .. _

 

46

.mwo Fonoaoo mm + acmmm zow Fe m ea new no oaaaF nonnoonon .m acumen

    

 

one 1 mam
m.© m.m m.¢ m.m m.N m.F m.o
III-Ill
Rea . eaoz _----
N
oEon m + xcman coo:
.mm F
»..
.0
O. .0 I. O 0
MW.
oaomuoouoa w vacuuoouon uoz

m.q m.m

 

oFx

weaa gag go 931V p9391833u1

Integrated area Of EDB Peak

x10

100

80

60

40

20

47

H ,

x ‘ .0
_ Y = 707X - 509; r = .99 ’0'.
;
' o
O
___ 95% confidence limits on 01 IX 0! /
Y " 4/ Q
//
O
...-...... 95% confidence limits on ylx .0. . / 0

Figure 4.

   
 

Mean blank + 3 si;-a

2 .5 3 .5 5 .5 67.5 82.5 97.5 112.5
EDB - ppb

Gas chromatograph dose—response of EDB in 5 ml 20N

H2804 + 2g control egg.

1"'_' a—q-Ce-Lv—q

48

substantial heterogeneity of variances among areas at each dose.
Therefore, C.I.‘s and predictions based (x1 the preceding
prediction equation are biased and are only approximately correct.

The detection limit of EDB in egg was calculated as follows.
Mean area +1 3 standard, deviations was calculated. froni random
fluctuation Of 20 non-spiked control egg samples analyzed in the
HSGC. That area (2117) is equivalent to a dose of 3.7 ppb EDB.
However, since the regression line is not linear until 4.5 ppb
EDB, 4.5 ppb EDB is the true detection limit Of EDB in egg and
values of EDB below 3.7 ppb are not detectable while values of EDB
in between 3.7 and 4.5 ppb fall on a portion of the curve in Which
detectability is uncertain (Figure 3).

See Appendix C for statistical formulas used to calculate the
prediction equation and confidence intervals. See Appendix D for
egg calibration curve raw data.

F. Analysis of EDB residues in whole body

Chickens chosen for whole body analysis were removed from
the freezer, thawed overnight at 4°C, sawed into several small
pieces with a Hobart 5212F electric saw, and ground to hamburger
consistency (feathers included) in a Hobart 4732 SS telectric
grinder. Samples were put through the grinder 5 times to obtain a
homogeneous sample. Grab samples were removed and frozen in whirl
pack bags at -20°C. Prior to HSGC analysis, samples were thawed
and gravimetrically transferred with forceps to a sample vial.

HSGC analytical parameters for whole body samples were: 1)

49

1:1:

WHOLE BODY

30

n'
EDB peak

9J7

 

 

 

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MUSCLE

 

 

 

 

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l R
in
LIVER
a
n$
. EDB peakfi
" 3
.1 :t 8’ .3 g
'l- 36" _;
3
KIDNEY
S‘sEDB peak
'
/ '°‘
1‘)
15

 

 

10
db
)0)

 

 

 

 

 

 

Figure 5. Chromatograms of EDB in tissues.

Integrated Area of EDB Peak

50

x10

18

— {z = 1336X - 5039; r = .95 .

    

15 -- 95% confidence limits on '

A

u X 0
Y

nun-m 95% confidence limits on

 

9 X /
12
i
.0
.0
O
.0
.0
.0
9
6
. .0.
Mean blank + 3 sigma / .o'
/ °.
0
J "
0..
3 I
.0
.0
.0
.0
.0

   

EDB - ppb

Figure 6. Gas chromatograph dose-response of EDB in Ssml

20N H2804 + 2g control whole body.

51

sample weight = 2.0 g, 2) 20 N H2S04 = 5 m1, 3) digestion time

ll

30 minutes, 4) equilibration time = 75 minutes, 5) analysis time
75 minutes, and 6) injection time — 6 seconds. See Figure 5 for a
chromatogram.

Concentration of EDB in whole body was inversely predicted
from a calibration curve developed in EDB-spiked control samples.
Samples were spiked with 7.5, 10, 12.5, or 15 ppb EDB, digested,
equilibrated, and injected. Integrated area of the EDB peak was
recorded for each dose. The prediction equation with x=ppb EDB and
y=integrated area Of the EDB peak is y=1336x - 5039 with r=.95
(Figure 6). TUNE 95% C.I. (H: r extends from .79 to .98, the 95%
C.I. on bO is -5039i3639, and the 95% C.I. on bl is 1336:32. Since
the C.I.‘s on b0 and b1 do not include zero, one can conclude that
the origin is not zero and that the regression. line :u; not
horizontal. 95% C.I.‘s on hy/x and y/x are pictured in Figure 6.

The detection limit Of EDB in whole body was calculated as
follows. A mean area + 3 standard deviations was calculated from
random fluctuation (H? 20 non—spiked control samples analyzed in
the HSGC. That area (4570) is equivalent to a dose of 7.2 ppb EDB
and is the detection limit. See Appendix D for whole body
calibration curve raw data.

G. Analysis of EDB residues in liver

Liver samples were removed from the freezer, thawed and
homogenized individually with a Tekmar SDT Tissumizer. Homogenized

samples were stored.:h1 whirl pack bags EM: —20°C. Prior to HSGC

Integrated Area of EDB Peak

x10

48

36

24

12

52

— x} = 33o7x + 12279; I: = .99

'-- 95% confidence limits on fiylx

 

.' Mean blank + 3 sigma

 

Figure 7.

2.5 5.0 7.5 10.0
EDB - ppb

Gas chromatograph dose-response of EDB in 5 ml

ZON H2504 + 2g control liver.

53

analysis, samples were thawed and gravimetrically transferred with
a spatula to a sample vial.

HSGC analytical parameters for liver samples were: 1) sample
weight = 2.0 g, 2) 20 NHZSO4 = 5 m1, 3) digestion time = 30
minutes, 4) equilibration time = 75 minutes, 5) analysis time = 40
minutes, anui 6) injection time == 6 seconds. See Figure 5 for a
chromatogram.

Concentration of EDB in liver was inversely predicted from a
calibration curve developed in EDB-spiked control samples. Samples
were spiked with 2.5, 5.0, 7.5, or 10 ppb EDB, digested,
equilibrated, and injected. Integrated area (M? the EDB peak was
recorded for each dose. The prediction equation with x=ppb EDB and
y=integrated area (M? the EDB peak is y=3307x + 12279 with r=.99
(Figure 7). The 95% C.I. on r extends from .908 to .995, the 95%
C.I. (n1 bO is 12279i3347, and the 95% C.I. (x1 bl is 33071506.
Since the C.I.'s on b0 and b1 do not include zero, one can
conclude that the origin is not zero and that the regression line
is not horizontal. 95% C.I.‘s on fly/x and y/x are pictured in
Figure 7.

The detection limit of EDB in liver was calculated as
follows. Mean area + 3 standard deviations was calculated from
random fluctuation (H? 20 non-spiked control samples analyzed in
the HSGC. That area (14925) is equivalent to a dose of 0.8 ppb EDB
and is the detection limit. See Appendix D for liver calibration

curve raw data.

x10

60

48

U
0)

Integrated Area of EDB Peak
N
A

12

54

— 1? = 3388X - 2468; r = .96

...... 95% confidence limits on fiylx

"""' 95% confidence limits on 9]): .0 /

 

 

Figure 8.

2.5 5.0 7.5 10.0 12.5
EDB - ppb

Gas chromatograph dose-response of EDB in 10 m1 20N H
+ lg control kidney.

2

SO

4

15.0

55

H. Analysis of EDB residues in kidney
Kidney samples were removed from the freezer, thawed, and
gravimetrically transferred with forceps to a sample vial. HSGC
analytical parameters for kidney samples were: 1) sample weight =

1.0 g, 2) 20 N H2804 = 10 ml, 3) digestion time = 30 minutes, 4)

equilibration time == 75 minutes, 5) analysis time = 75 minutes,
and- 6) injection time = 8 seconds. See Figure 5 for a
chromatogram.

Concentration of EDB in kidney was inversely predicted from a
calibration curve developed in EDB-spiked control samples. Samples
were spiked with 5.0, 7.5, 10.0, 12.5, or 15.0 ppb EDB, digested,
equilibrated, and injected. Integrated area (n: the EDB peak was
recorded for each dose. The prediction equation with x=ppb EDB and
y=integrated area of the EDB peak is y=3388x - 2468 with r=.96
(Figure 7). fflua 95% C.I. (x1 r extends from .72 to .99, the 95%
C.I. on bO is —2468ill,047, and the 95% C.I. on bl is 3388:1071.
Since the C.I. on bO includes zero while the C.I. on bl does not,
one can conclude that the origin is zero and that the regression
line is not horizontal. 95% C.I.‘s on hy/x and y/x are pictured in
Figure 8.

The detection limit Of EDB in kidney was calculated. as
follows. In. kidney, the integrator did not recognize separate
peaks at retention times 3.80 (unidentified peak) and 4.12 (EDB
peak) (Figure 5) at doses below 5.0 ppb EDB. Therefore, 5.0 ppb is
the detection limit in kidney. See Appendix D for kidney

calibration curve raw data.

Integrated Area of EDB Peak

x10

40

30

20

10

56

 

— 1? = l338x + 1458; r = .98

--" 95% confidence limits on fiylx

""“" 95% confidence limits on FIX

      

15 20 25
EDB - ppb

5 10

Figure 9. Gas chromatograph dose-response of EDB in

10 ml ZON H2804 + 0.5g control skin.

57

1. Analysis of EDB residues in skin
Skin samples were removed from the freezer, thawedpand
gravimetrically transferred with forceps to 21 sample vial. HSGC

analytical parameters were: 1) sample weight = 0.5 g, 2) 20 N

H2804 - 10 an” .3) digestion time = 60 minutes, 4) equilibration

time 120 minutes, 5) analysis time = 15 minutes, and 6)
injection time = 8 seconds. See Figure 5 for a chromatogram.
Concentration Of EDB in skin was inversely predicted from a
calibration curve developed in EDB-spiked control samples. Samples
were spiked with 15, 20, or 25 ppb EDB, digested, equilibrated,
and injected. Integrated area Of the EDB peak was recorded for
each dose. The prediction equation with x=ppb EDB and y=integrated
area of the EDB peak is y=1338x + 1458 with r=.98 (Figure 9). The
95% C.I. on r extends from .63 to .99, the 95% C.I. on bO is

l458i11,928, and the 95% C.I. on b is 1338:584. Since the C.I. on

1

b0 includes zero while the C.I. on bl does not, one can conclude
that the origin is zero and that the regression line is 'not
horizontal. 95% C.I.'s on hy/x and y/x are pictured in Figure 9.
The detection limit of EDB in skin was calculated as follows.
In skin, the integrator did not recognize separate peaks at
retention times 3.62 (unidentified peak) and retention time 4.05
(EDB peak) (Figure 5) at doses below 15 ppb EDB. Therefore, 15 ppb

is the detection limit in skin. See Appendix D for skin

calibration curve raw data.

58

J. Analysis of EDB residues in fat
Fat samples were removed from the freezer, thawed, and
gravimetrically transferred with a spatula to a sample vial. HSGC
analytical parameters were: 1) sample weight = 2.0 g, 2) 20 N H2804
= 5 ml, 3) digestion time = 15 minutes, 4) equilibration time = 75
minutes, 5) analysis time = 15 minutes, and 6) injection time = 6
seconds. See Figure 5 for a chromatogram.

Concentration of EDB :hi fat was calculated. frOU1 standard
addition to each fat sample obtained from hens fed
EDB-contaminated diet instead of from a calibration curve
developed :hi EDB-spiked control samples because control fat was
depleted during determination of fat HSGC analysis parameters.
Quantification by standard addition involved spiking known amounts
of EDB into samples which contained unknown quantities of EDB. A
fat sample for which concentration of EDB was to be determined was
partitioned into several 2 g samples. Then 2 of the samples were
analyzed in the HSGC as is, i.e. without an EDB spike. The rest of
the 2 g Samples were analyzed in the HSGC with one of 3 other EDB
spikes of varying concentrations. A linear regression equation was

calculated from x = 0, x

1 = amount of first EDB spike in ppb, X

2 3

= amount of second EDB spike in ppb, x4 = amount of third EDB
spike in ppb, and y1 = area from unknown EDB concentration, y2 =
area from unknown EDB concentration plus area due to first spike,

y3 = area from unknown EDB concentration plus area due to second

spike and y4 = area from unknown EDB concentration plus area due

59

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A

*......*Y

x10 995x + 102,419; r = .99

A

e---e‘Y

697K + 42,369; r = .98

 

 

500 ..
...-.... 1? = 46514 + 13,610; je-
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r = .99 .3

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Mean blank + 3 sigma

 

-200 -100 0 100 200 300 400 500

EDB — ppb

Figure 10. Standard addition dose-response lines for fat.

61

to third spike. The linear regression parameters (b0, b r) were

1’
calculated as usual, and the concentration of EDB in ppb in
unknowns was calculated from lx-intercept' (Harvey 1950).

Four standard addition lines were developed for the 4 fat
samples which contained detectable EDB (Figure 10). Two of the fat
samples were spiked with 0, 25, 50, or 100 ppb EDB, and 2 of the
fat samples were spiked with 0, 100, 200, or 400 ppb EDB. Table 9
contains the standard addition linear regression equations with
95% C.I.‘s for r, b0, and bl' By comparing the C.I.‘s on bl’ it
can. be seen that only one pair (fl? C.I.‘s (n1 bl overlap. This
indicates that the regression lines are not homogeneous.

The detection limit of EDB cannot be calculated on a ppb
basis from standard addition lines because the line representing
the area of .mean blank + 3 standard deviations crosses the
standard addition lines at 21 point corresponding to 21 negative
x—value (Figure 10). However, the mean area + 3 standard
deviations obtained from analyzing 20 non-spiked fat samples in
the HSGC was 1705. Any areas 'below 11th; obtained. during :fat
analysis were considered as random fluctuations and
non—detectable.

K. Analysis of EDB residues in muscle

Muscle samples were removed from the freezer, thawed, and
gravimetrically transferred with forceps to 21 sample vial. HSGC

analytical parameters were: 1) sample weight = 3.0 g, 2) 20 N H SO

2
= 5 m1, 3) digestion time = 30 minutes, 4) equilibration time = 75

minutes, 5) analysis time = 75 minutes, and 6) injection time = 6

4

62

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Integrated Area of EDB Peak

Figure 11.

 

4459K + 1439;

63

4296X + 2685; r = .99

4042X + 2351; r = .99

4174X + 868; r = .99

O
t
g
0'13
00/
O
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Mean blank + 3 sigma
5 10
EDB - ppb
muscle.

Standard addition dose-response lines for

64

seconds. See Figure 5 for a chromatogram.

Concentration (fl? EDB in muscle was calculated from standard
addition to muscle samples obtained from hens fed EDB-contaminated
diet instead Of from. a calibration curve in control samples
because control muscle was depleted during determination of muscle
HSGC analysisv parameters. Four standard addition lines were
developed (following the general standard addition procedure
outlined in the fat analysis section) for the 4 nmscle samples
which contained detectable EDB (Figure 11). Table 10 contains the
standard addition linear regression equations with 95% C.I.‘s for
r, b0 and b1' By comparing the C.I.'s on bl’ it can be seen that
all of the C.I.‘s on bl overlap indicating homogeneity of
regression among the muscle samples obtained from different hens.

The detection limit of EDB in muscle could not be calculated
on a ppb basis for the same reasons as outlined under fat
analysis. However, the mean area + 3 standard deviations obtained
from analyzing 20 non-spiked muscle samples in the HSGC was 696.
Any areas below this obtained during muscle analysis were
considered as random fluctuations and non-detectable.

L. 'Analysis of EDB residues in flour

Subsamples of flour were taken just prior to blending of
experimental diets, placed in whirl pack bags, and stored at
-20°C. Prior to HSGC analysis, samples were warmed to room
temperature euui gravimetrically transferred ndth.za spatula to a

sample vial. HSGC analytical parameters were: 1) sample weight =

65

 

 

 

 

 

 

 

 

8 «=1
l ” *
FLOUR . ‘ DIET
8
‘:
5?
fl
EDB peak
8’
3.
N
(:10 8
9’ l ~56 ' 551:;

E"

Figure 12. Chromatograms of EDB in diet and flour.

Integrated area of EDB Peak

x10

1?

66

.= 3271835 + 794,167; I.“ = .99 .0

 

Figure 13.

EDB - ppb

Gas chromatograph dose-response of EDB in 10 ml

2ON H2804 + 0.19 Control flour.

Integrated Area of EDB Peak

67

    

x106
.... i? = 359,894 + 1,279,471; r = .99
8 .g
.._... 95% confidence limits on .0.
fi Ix
Y
""”' 95% confidence limits
on 91x '
6
-'/
4
.0
Mean blank + 3 $10 a
2

 

5 10 15
EDB - ppb

Figure 14. Gas chromatograph dose-response of EDB
in 10 ml 20N H 80 + 0.1g control diet.

2 4

68

0.1 g, 2) 20N H 807 = 10 ml, 3) digestion time = 60 minutes, 4)

2 4
equilibration time == 75 minutes, 5) analysis time = 20 minutes,
and 6) injection time = 8 seconds. See Figure 12 for a
chromatogram.

Concentration of EDB in flour was inversely predicted from a
calibration curve developed in EDB—spiked control samples. Samples
were spiked with 25, 30, or 35 ppm EDB, digested, equilibrated,
and injected. Integrated area of {jug EDB peak was recorded for
each dose. The prediction equation with x=ppm EDB and y=integrated
area (H? the EDB peak is y=327,835x + 794,167 with r=.99 (Figure
13). The 95% C.I. on r extends from .841 to .998, the 95% C.I. on

bO is 794,167i2,510,249, and the 95% C.I. on b is 327,835:82,9ll.

1
Since the C.I. on bO includes zero while the C.I. on bl does not,
one can conclude that the origin is zero and that the regression
line is not horizontal. 95% C.I.‘s on uy/x and y/x are pictured in
Figure 13.

The detection limit of EDB in flour was calculated as
follows. Pban anxm1-+ 3 standard deviations was calculated from
random fluctuation (fl? 20 non-spiked control samples analyzed in
the HSGC. That area (858,235) is equivalent to a dose of .19 ppm
EDB and is the detection limit. See Appendix D for flour
calibration curve raw data.

M. Analysis of EDB residues in diet

Subsamples of diet were taken just after blending of

experimental diets was completed. Samples were stored in whirl

pack bags at —20°C. Prior to HSGC analysis, samples were warmed to

69

Table ll. Summary of prediction equations and detection limits used to
calculate concentration of EDB in eggs, whole body, liver,
kidney, skin, diet, and flour.

 

 

 

 

Prediction Detection
Sample equation1 r2 limit3-ppb
Egg i = 707x - 509 .99 4.5
Whole body i = l336X - 5039 .95 7.2
Liver i = 3307x - 12279 .99 0.8
Kidney i = 3388X - 2468 .96 5.0
Skin 9 = l338X + 1458 .98 15.0
Diet i = 359,894X + 1,279,471 .99 230.0
Flour 9 = 327,835X + 794,l67 .99 190.0
1 X = ppb EDB, Y = integrated area of the EDB peak.
i r = product-moment correlation.

Calculated from random fluctuation of non-spiked control samples for
egg, whole body, liver, diet, and flour. Calculated from smallest dose
which could be integrated in skin and kidney.

70

room temperature and gravimetrically transferred with a spatula to
a sample vial. HSGC analytical parameters were: 1) sample weight =

0.1 g, 2) 20 N H SO = 10 ml, 3) digestion time = 60 minutes, 4)

2 4
equilibration time == 75 minutes, 5) analysis time = 20 minutes,
and 6) injection time = 8 seconds. See Figure 12 for a

chromatogram.

Concentration of EDB in diet was inversely predicted from a
calibration curve developed in EDB-spiked control samples. Samples
were spiked with 5, 10 or 15 ppm EDB, digested, equilibrated, and
injected. Integrated area of the EDB peak was recorded for each
dose. The prediction equation with x=ppm EDB and y=integrated area
of the EDB peak is y=359,894x + 1,279,471 with r=.99 (Figure 14).
The 95% C.I. on r extends from .985 to .995, the 95% C.I. on bO is
1,279,471 1: 300,820, and time 95% C.I. (n1 bl is 359,894:30,863.
Since the C.I.‘s on b0 and b1 do not include zero, one can
conclude that the origin is not zero and that the regression line
is not horizontal. 957o C.I.‘s on {By/x and y/x are pictured in
Figure 14.

The detection limit of EDB in diet was calculated as follows.
Mean area +- 3 standard deviations 'was calculated. froui random
fluctuation of 20 non-spiked control samples analyzed in the HSGC.
That area (1,362,978) is equivalent to a dose of .23 ppm EDB and
is the detection limit. See Appendix D for diet calibration curve
raw data.

See Table 11 for a summary of prediction equations .and

detection limits in egg, tissues, diet, and flour.

RESULTS

1. Feed Consumption, Body Weights, Egg Production, and Egg Weights
Because it was necessary to house EDB and control hens in
different environments, this study could not be designed to
determine if feeding EDB—contaminated diet for 21 days would have
an effect on feed consumption, body weights, egg production, or
egg weights. Therefore, although data on those parameters were
collected (see Appendix B), statistical analysis of it is not

valid.

II. Residues of EDB in Diet and EDB Intake

EDB loss occurs during mixing, storage, and aeration of diet
in feeding troughs (Fuller and Morris 1963 and Morris and Fuller
1963). These losses must be quantified if an accurate estimate of
the concentration of EDB in diet at the time of ingestion is
desired. During this study, flour samples were obtained just prior
to blending of experimental diets while dietary samples ‘were
obtained after blending was completed. EDB-contaminated flour (EF)
contained 31.1 ppm EDB. This level of EDB is much higher than has
been typically found in flour samples obtained from
EDB-contaminated grain implying that the flour was directly
fumigated. EDB-contaminated diet (ED) contained 6.7 ppm EDB. Since

EF constituted 55.7% of ED, theoretically, ED should have

71

72

contained 17.3 ppm EDB. Thus, when EF was blended into diet, it
retained 38.7% anui lost 61.3% of its EDB residues. Most of this
loss was probably due to evaporation of EDB. Losses during storage
and from aeration in feeding troughs throughout the course of this
study cannot be quantified because dietary samples were not
obtained during those periods. It can be assumed that some losses
did occur so that actual concentration of EDB in diet at the time
of ingestion by hens was less than 6.7 ppm.

EDB hens consumed a total of 2.19 kg of ED per hen over the
21 day period during which ED was fed. Assuming that EDB content
in diet (6.7 ppm) was not reduced, during that time, this is
equivalent to a total intake of 14.7 mg of EDB per hen. Since some
evaporation of EDB from ED probably did occur, actual EDB intake

per hen would have been somewhat less than 14.7 mg.

111. Residues of EDB in Egg, Whole Body, and Tissues

Residues of EDB were not detected in any control egg, control
whole body, or control tissue samples. See Table 15 for detection
limits. Egg samples obtained from EDB hens were homogenized with a
Waring blender prior to analysis. EDB residues in egg could have
been reduced during this process via evaporation. Thus, residues
reported below are biased to the degree that residues may have
declined during homogenization.

The average concentration (ppb) and burden (ng) of EDB in egg

for each day from days 1 to 21 of residue build-up (RB), i.e. days

72

contained 17.3 ppm EDB. Thus, when EF was blended into diet, it
retained 38.7% and lost 61.3% of its EDB residues. Most of this
loss was probably due to evaporation of EDB. Losses during storage
and from aeration in feeding troughs throughout the course of this
study cannot be quantified because dietary samples were not
obtained during those periods. It can be assumed that some losses
did occur so that actual concentration of EDB in diet at the time
of ingestion by hens was less than 6.7 ppm.

EDB hens consumed a total of 2.19 kg of ED per hen over the
21 day period during which ED was fed. Assuming that EDB content
in diet (6.7 ppm) was not reduced during that time, this is
equivalent to a total intake of 14.7 mg of EDB per hen. Since some
evaporation of EDB from ED probably did occur, actual EDB intake

per hen would have been somewhat less than 14.7 mg.

111. Residues of EDB in Egg, Whole Body, and Tissues

Residues of EDB were not detected in any control egg, control
whole body, or control tissue samples. See Table 15 for detection
limits. Egg samples obtained from EDB hens were homogenized with a
Waring blender prior to analysis. EDB residues in egg could have
been reduced during this process via evaporation. Thus, residues
reported below are biased to the degree that residues may have
declined during homogenization.

The average concentration (ppb) and burden (ng) of EDB in egg

for each day from days 1 to 21 of residue build-up (RB), i.e. days

 

74

 

 

 

 

Table 13. Residues of EDB in eggs obtained from EDB hens days 15 to 21 of
residue buildup.
Average egg Nanograms of
Treatment Hen # EDB—ppb1 weight-grams EDB per egg3
Residue
buildup l NE4 -- ---
2 NE -- ---
'3 NE -- ---
4 25.3 i 10.8 52.1 1318 i 563
5 23.8 i 10.8 54. 6 1299 i 590
6 23.0 i 10.8 52.1 1198 t 563
7 NE -- --—
8 32.7 t 10.8 51.8 1694 r 559
9 27.6 i 10.8 54.7 1510 i 591
10 22.9 i 10.8 57.5 1317 i 621
11 23.9 t 10.8 55. 6 1329 i 600
12 35.1 i 10.8 54. 7 1920 i 591
13 NE -- ---
14 36.5 t 10.8 55.0 2008 t 594
15 25.5 t 10.8 51. 5 1313 i 556
16 29.5 i 10.8 56. 4 1664 i 609
Mean : 95% 0.1.5 27.8 e 3.3 -- 1506 i 185

 

1 Values represent concentration of EDB (with a 95% confidence interval)
in egg sample pooled from all eggs laid by each hen over days 15 to 21.

2 Represents average weight of egg contents (yolk + albumen) of eggs used

for residue analysis for each hen.

by subtracting shell weight from total weight.

3 Nanograms of EDB per egg (with a 95% confidence interval) was calculated
by multiplying concentration of EDB in egg times average egg weight.

4 NE= no. eggs laid by that hen during days 15 to 21.

5 C.I. =

confidence interval.

Weight of egg contents was calculated

75

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76

Table 14. Residues of EDB in scrambled eggs before and after frying egg
samples obtained from 4 EDB hens during days 15 to 21 of residue

 

 

 

 

buildup.
Concentration Concentration % of % of
before after residues residues

Hen # frying1 frying1 retained lost
9 27.6 17.3 62.7 37.3
10 22.9 13.9 60.7 39.3
11 23.9 13.7 57.3 42.7
12 35.1 19.7 56.1 43.9

Mean : SE2 27.4 1 2.8 16.2 i 1.4 59.2 t 1.5 40.8 e 1.5

 

1 ppb EDB.
2 SE = standard error of the mean.

77

Table 15. Residues of EDB in egg, whole body, and tissues of EDB hens on

day 0 of withdrawal.

 

 

 

 

 

Whole

Egg1 bodyZ Fat3 Musc1e4 Liver5 Kidney6 Skin7
-8 10.3(NL)9 29 0.58 N010 ND ND
- 9.9(NL) 103(NL) O.63(NL) ND ND ND
- 10.8 61 0.32 ND ND ND
- 12.1 24(NL) 0.21(NL) ND ND ND
27.8 10.8 54 0.44 -- —- --
t 3.311 t 1.511 t 5811 t 0.3211

1 Detection limit is 4.5 ppb EDB.

2 Detection limit is 7.2 ppb EDB.

3 Detection limit is equivalent to an area of 1405.

4 Detection limit is equivalent to an area of 696.

5 Detection limit is 0.8 ppb EDB.

6 Detection limit is 5.0 ppb EDB.

7 Detection limit is 15.0 ppb EDB.

8 See Table 13 for individual values for egg.

9

11

NL indicates hen was not laying for at least 7 days at the time the
sample was obtained.

Not detected.
Represents mean i 95% confidence interval.

78

1 to 21 of feeding EDB-contaminated diet (ED), and days 1 to 21 of
withdrawal are given in Table 12. The average concentration (ppb)
and burden (ng) of EDB in eggs obtained from EDB hens days 15 to
21 of residue build-up are given in Table 13. The average
concentration of EDB found in egg days 15 to 21 was 27.8 ppb (1506
ng EDB per egg) with the 95% confidence interval (C.I.) ranging
from 24.5 to 31.1 ppb (1321 to 1691 ng EDB per egg). Since this
range is the maximum concentration of EDB expected in egg, it is
apparent that maximum concentration of EDB in egg was reached by
day 8 of feeding ED. EDB was first detected in eggs at 9.8 ppb
(524 ng) (n1 day 3 of feeding ED. Residues increased linearly in
egg from day 3 to day 8 of feeding ED at which time plateau
concentration was reached. Concentration of EDB in egg dropped 37%
the first day after withdrawal of ED and then decreased linearly
until EDB was no longer detectable in egg by day 6 of withdrawal.

The concentratbmn of EDB :ni four egg samples obtained from
EDB hens during days 15 to 21 of residue build-up was determined
in scrambled eggs before and after frying (Table 14). On average,
frying reduced residues 40.8%.

Concentration of IHH3 in whole body and tissues is presented
in Table 15. (hi day 0 of withdrawal, whole body, abdominal fat,
and breast muscle obtained. from: EDB 'hens contained (n1 average
(with the 95% C.I.) 10.8:1.5, 54:58, and 0.44:0.32 ppb EDB,
respectively. The C.I.'s for fat and muscle are large because of

high variability of response among hens. This variability is not

79

Table 16. Total residue of EDB (ng) deposited into eggs obtained days 1
to 21 of residue buildup from the 4 EDB hens used for whole body
analysis on day 0 of withdrawal.

 

 

 

 

 

 

 

 

 

 

Days an Nanograms
Hen 4 egg was laid1 EDB per egg2
1 3 524
5 854
7 1147
Total EDB deposited in egg 2525 (or 2.5 ug)
2 3 524
4 719
7 1147
Total EDB deposited in egg 2390 (or 2.4 ug)
4 3 524
4 719
6 1100
7 1147
8 1397
10 1290
11 1335
13 1364
14 1381
15 1318
17 1318
19 1318
20 1318
Total EDB deposited in egg 15,529 (or 15.5 ug)
5 3 524
4 719
6 1100
7 1147
9 1349
10 1290
11 1335
13 1364
14 1381
15 1299
17 1299
19 1299
21 1299
Total EDB deposited in egg 15,405 (or 15.4 ug)

 

1 Represents days from 1 to 21 of residue buildup.
2 Values were obtained from Table 12.

80

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81

related to the hen's state of production because for both fat and
muscle, the highest and lowest responses occurred in non—laying
hens. EDB was not detected in liver, kidney, or skin on day 0 of
withdrawal. EDB was not detected :UI whole body or tissues on day

21 of withdrawal.

IV. Distribution of EDB Residues

The total amount (fl? EDB that was deposited into egg during
days 1 to 21 of RB by each of the hens used for whole body residue
analysis on day 0 of withdrawal is presented in Table 16. Two of
the hens deposited only small amounts of EDB into egg because they
went out of production. The 2 hens which remained in production
during residue build-up deposited per hen a total of 15.5 ug of
EDB into egg and 22.3 ug of EDB into whole body (Table 17). Thus,
using data iknr laying hens only, 51 total of 37.8 ug of EDB was
deposited into tissues and egg by each hen with tissues receiving
59.0% and egg 41.0% of the total burden. The amount of EDB
deposited into tissues and eggs by laying hens accounts for only
0.26% of EDB intake. Since actual EDB intake was probably less
than. calculated intake (for reasons «outlined :hi Section. II of
Results), percent of EDB intake deposited into tissues and egg is
probably higher than 0.26%. However, even if EDB intake was 75%
less than reported, deposition into tissues and eggs would still
account for only 1% of intake. EDB not deposited was either: 1)
not absorbed or 2) efficiently metabolized.

EDB which was deposited into whole body was distributed in

82

breast muscle and abdominal fat. The percent of total body burden
found in each of these tissues is calculated as follows. The
average body weight of EDB hens and the average concentration of
EDB in whole body on day 0 of withdrawal were 1980 g and 10.8 ppb,
respectively. The product of those 2 numbers (21.4 ug) is the
average total body burden of EDB in each EDB hen at day 0 of
withdrawal. Knowing that abdominal fat and breast muscle contained
54 enul 0.44 Ink) EDB, respectively, and assuming that hens: 1)
deposited comparable amounts of EDB into all muscle and fat and 2)
contained 19% fat and 50% muscle (Maynard_etfial. 1979), it follows
that fat and muscle contained approximately 95% (20.3 ug) and 2%
(0.4 ug), respectively, of the whole body residues. The 3% of
residues which are unaccounted for were probably deposited in the
yolks of developing follicles.

A11 EDB residues were withdrawn from muscle and fat by day 21
of withdrawal, i.e. 21.4 ug of EDB was mobilized from tissues
during withdrawal. Using the calculation method outlined in Table
16, it can be shown that on average, a total of 2.5 ug of EDB (12%
of the total body burden) was deposited into egg per hen during
withdrawal. Therefore, mobilization from tissue into egg was not
the primary withdrawal route. This indicates that the main route
for withdrawal. of EDB 'residues fron1 fat and 'muscle. must have

involved mobilization from tissue followed by metabolism.

83

Table 18. Activity of aryl hydrocarbon hydroxylase (AHH) and aminopyrine
N-demethylase (AND) in liver of broilers fed diet at 80 ppm
polybrominated biphenyls (PBBS) for 7 days.

 

 

 

 

 

 

AHH AND
Treatment activity1 activity2
Control broilers None 103.8 0.51
95.9 1.33
Mean 1 553 99.9 t 2.95a5 0.92 : 0.41a
PBB broilers 80 ppm PBBs 745.8 2.57
458.6 1.69
Mean t SE3 602.2 t 143.6b 2.13 t 0.44
(6.0)4 (2.31

 

1 pmoles hydroxylated benzopyrene produced/mg protein/minute.
2 nmoles CHZO produced/mg protein/minute.
3 SE = standard error of the mean.

4 The number in parentheses represents the increase in activity over
controls.

5 Numbers in the same column with a different subscript are significantly
different (P < .10).

84

Table 19. Activity of aryl hydrocarbon hydroxylase (AHH) and aminopyrine
N-demethylase (AND) in liver of hens on day 0 and 21 of with-

 

 

 

 

 

 

 

 

drawal.
AHH activity1 AND activity2
Day 0 Day 21 Day 0 Day 21
Control hens 1.02 0.63 35 142
0.94 1.38 48 120
Mean : SE3 0.98 1.01 41.5 131.0
: 0.04,4 t 0.38a : 6.5a : 11.0b
EDB hens 1.41 0.83 65 62
1.47 0.67 80 107
0.61 0.22 57 23
0.79 2.12 23 231
Mean 1 SE 1.07 0.96 56.3 105.8
i 0°22a i 0.41a i 12.1a : 45.1a

 

1 pmoles hydroxylated benzopyrene produced/mg protein/minute.
2 nmoles CHZO produced/mg protein/minute.
3 SE = standard error of the mean.

4 Means in the same row for the same enzyme with different subscripts are
significantly different (P < .01).

85

V. Activity of Hepatic Mixed Function Oxidases

To confirm the validity of the assay by which activity of AHH
and AND was measured, broilers at 6 weeks of age were fed diet
containing a mixture of polybrominated biphenyl isomers (PBBS)
that are known to induce activity of AHH and AND in poultry
(Bursian ‘35 .31. 1983- and Bursian and Polin 1986, personal
communication). The mixture consisted of 62.8% hexabromobiphenyl,
13.8% heptabromobiphenyl, 10.8% pentabromobiphenyl, 2.0%
tetrabromobiphenyl, and 1144% (H? other bromobiphenyls. Two
broilers were fed non-contaminated broiler starter and two were
fed broiler starter at 80 ppm PBBs for 7 days. Then, broilers were
killed, livers 'were excised, euui mixed function. oxidase (MFO)
activity was determined. The activity of AHH and AND in livers
obtained from broilers fed PBBs was, respectively, 6.0 and 2.3
times greater than that of (controls (Table 18). Using the
student's t-test, these increases were found to be significant (P
.10). Thus, because an increase in activity was detected as was
expected, it was decided that results obtained. with tine assay
would be valid.

Activity of hepatic AHH and AND was determined in 2 control
and 4 EDB hens on both day 0 and 21 of withdrawal (Table 19). It
is not valid to statistically compare control and EDB MFO activity
because hens ‘were housed :hi different environments during the
experimental period. However, residues of EDB were shown to have

been withdrawn from all tissues by day 21 of withdrawal. That

86

implied that activity of hepatic AHH and AND in EDB hens on day 21
of withdrawal couLd be used as the control for activity in EDB
hens on day 0 of withdrawal. This same comparison (day 21 versus
day 0 of withdrawal) is also valid for control hens. The student's
t-test was used to make the comparisons. AHH activity in control
liver significantly increased from day 0 to 21 of withdrawal (P
<.01). The reason for an increase in AHH activity in controls is
not known. EDB hens also exhibited an increase in AHH activity
from day 0 to 21 of withdrawal, but it was not found to be
significant (P >.15)because of the high variability in response
AND activity did not significantly change (P>.25) from day 0 to 21
of withdrawal :hi either control or EDB hens. Since there was no
significant change in hepatic MFO activity in EDB hens from day 0
to 21 of withdrawal, it appears that activity of hepatic AHH and
AND was not induced in hens which had consumed EDB-contaminated

diet for 21 days.

DISCUSSION

1. Residues Versus Tolerance Limits

In review, the EPA has set the following tolerance limits for
EDB: 1) 900 pbb in non—processed grains, 2) 150 ppb in processed
grains and in grain-containing food products that will be cooked
such as flour and cake 'mixes, and 3) 3%) ppb in. ready-to-eat
grain-containing food products such as cereals and breads. At this
time no tolerance limits have been established for EDB in products
obtained from food—producing, animals ‘which. have consumed
EDB-contaminated. grain. However, the possibility exists that the
tolerance limits (M? the preceding could be expanded to apply to
all food products. Therefore, it is useful to compare residues
detected in eggs, muscle, and fat to the grain tolerance limits.

When eggs are considered as a ready-to—eat food, eggs
obtained from hens fed diet containing 6.7 ppm EDB for 21 days
would contain levels (fl? EDB equivalent to line 30 ppb tolerance
level. If eggs are combined with other products as in egg—nog, the
EDB would be diluted, and residues would fall below the
ready—to—eat tolerance limit. 1U? eggs are cooked, the residues
would be much less than the comparable tolerance limit.

Breast muscle does run: contain per unit weight as much EDB

residue as eggs or fat when hens are fed diet originating at 6.7

87

88

ppm EDB for 21 days. Muscle residues were well below all tolerance
levels.

Concentration of EDB in chicken fat was greater than the
tolerance limit for ready-to—eat foods and was the highest of all
tissues analyzed. Fat can represent a source of EDB—contamination
to humans by its use in: l) soaps and paints, 2) poultry diets,
and 3) human foods. Most uses require rendering and dilution with
other goods, processes which should decrease residues below the
lowest tolerance limit.

In summary, if tolerance limits would be expanded to include
all food commodities one can expect that food products obtained
from hens fed diet containing 6.7 ppm EDB for 21 days to contain
residues below or near the lowest tolerance limit of 30 ppb, i.e.
the foods would be marketable. If a linear relationship between
concentration of EDB in diet immediately after mixing and tissue
residues can be assumed, then the concentration of EDB in diet
that would be required to increase tissue residues above the
ready-to—eat tolerance limit are 6.7, 3.7, and 457 ppm for eggs,
fat, and muscle, respectively. The dietary concentrations required
to increaseresidues above the foods that will be cooked tolerance
limit are 36, 19, and 2284 ppm EDB for eggs, fat, and muscle,
respectively. Those levels of EDB are not typically found in grain
products or diets. However, if chickens did consume
EDB—contaminated grain to an extent such that residues increased

above tolerances, 51 withdrawal period from the EDB source would

89

aid in reducing residues below tolerance limits.
11. Distribution and Metabolism

Less than one percent of EDB residues consumed were deposited
into tissues auui eggs. The other 99% (fl? residues consumed were
either absorbed anui metabolized (n: were not absorbed. However,
since EDB is fat soluble, it is likely that most of the residues
consumed were absorbed in conjunction with fat. If that is so,
since such small quantities of EDB were deposited into tissues and
eggs, an efficient system for metabolizing EDB must exist in hens.
If EDB is metabolized in poultry as it is in rats, glutathione and

mixed function oxidases are involved in the process. In this

study, it was found that the mixed function. oxidases 'were 'not
induced. This could be because: 1) the level of EDB exposure was
low enough to be handled by normal enzyme activity or 2) mixed
function oxidation is run: a primary route for EDB metabolism in
poultry. If glutathione (GSH) conjugation is a major route for EDB
metabolism in poultry, their cancer risk is increased because the
GSH-metabolite of EDB has been found to be a more potent
carcinogen than EDB pg£_§e. It would be interesting to determine
in poultry and mammals what percent of GSH-metabolites derived
from EDB are excreted and what percent become involved in
alkylation reactions with DNA strands.

In summary, the majority of the EDB consumed by hens was
probably metabolized. This decreases human exposure to EDB because

hens are not depositing large quantities of EDB into tissues and

90

eggs that would be consumed by humans. However, it could increase
the cancer risk to the animals themselves because of formation of

GSH-metabolites.

III. Research Design

If research similar to this study was to be conducted in the
future, the design should involve the following. All hens should
be housed in a similar environment or a pilot study should be done
to determine what effect different environment has on hens so that
the effect of EDB on feed consumption, body weights, egg
production, egg weights, and activity of mixed function oxidases
can be accurately determined. Dietary samples should be obtained
during storage and feeding so that an accurate estimate of EDB
intake can be determined. It would be interesting to ascertain
what percent of EDB intake is absorbed, metabolized, or deposited
into tissues versus VflEH: is not absorbed. Likewise, residues of
EDB metabolites in tissues and eggs should be quantified, and the
metabolic pathway in avian species should be outlined. Several
levels of EDB could be fed for varying time periods in a factorial
study so the no—effect levels for growth, production, and

reproduction could be measured.

SUMMARY

The liquid fumigant EDB is: 1) strongly sorbed 107 grains
during fumigation, 2) a carcinogen, and 3) interferes with
reproductive processes. The purpose of this study was: 1) to
determine if and to what extent chickens which had. consumed
EDB-contaminated grain. would, deposit EDB :hnx) tissues enui eggs
that could be consumed by humans and 2) to determine if a
withdrawal period from the EDB source after contamination would
reduce residues in tissues and eggs. Therefore, a
practical-oriented study was conducted in which EDB-contaminated
flour obtained from the Michigan food supply was fed to chickens
in diet at 6.7 ppm EDB for' 21 days followed far 21 days of
non-contaminated diet (days 0 11) 21 of vdthdrawal). Methodology
was developed with a headspace CC for quantifying residues of EDB
£53; s3; in. eggs collected daily and tissues (whole ‘body, fat,
muscle, skin, liver, and kidney) obtained on day () and 111 of
withdrawal. Detection sensitivities in all tissues and egg were at
the ppb level. Less than one percent of EDB intake was deposited
into tissues and eggs. Eggs contained detectable EDB by day 3 of
feeding contaminated diet, reached a plateau of 28 ppb by day 8,
and IK) longer contained detectable EDB by char 6 of vfithdrawal.

Frying scrambled eggs reduced residues by 40.8%. Concentration of

91

92

EDB on day 0 of withdrawal in whole body, fat, and muscle was 11,
54, and 0.44 ppb, respectively. Fat contained 95% of whole body
residues. EDB was not detected in liver, kidney, and skin on day 0
of withdrawal. EDB was not detected in any tissues on day 21 of
withdrawal. No tolerance limits have been set for EDB in food
products obtained from animals that were exposed to EDB, but if
the tolerance limits for EDB in grain are applied to the tissues
and eggs analyzed, residues would be below tolerances and the
products could be marketed. Activity of hepatic mixed function
oxidases was not induced. There was evidence that the hens
efficiently metabolized EDB. This decreased. the amount. of EDB
deposited into tissues and eggs but could have increased cancer
risk tx> the animals through formation of glutathione metabolites

that can alkylate strands of DNA.

APPENDICES

92a

Appendix A. Codes of hens used for whole body and tissue analysis.

 

 

 

Day Residue
Band # Cage # Code #1 killed2 analysis
Control hens 24567 65 1 0 T3,MF04
24577 66 2 21 T,MFO
24578 67 3 0 MB5
24579 68 4 21 NB
24580 69 5 0 T.MFO
24581 70 6 21 T,MFO
24584 71 7 21(NL)6 MB
24588 72 8 0 MB
EDB hens 24600 1 l 0(NL) NB
24568 2 2 0(NL) NB
24569 3 3 0(NL) T,MFO
24557 4 4 0 ND
24559 5 5 0 NB
24561 6 6 0 T,MFO
24563 7 7 0(NL) T,MFO
24565 8 8 0 T,MFO
24552 13 9 21(NL) T,MFO
24554 14 10 1 MB
24556 15 11 21 T,MFO
24558 16 12 21 NB
24560 17 13 21(NL) NB
24562 18 14 21 T,MFO
24567 19 15 21 NB
24566 20 16 21 T,MFO

 

1 These codes will be used to refer to hens in tables in the text or in

other appendices.
2 Represents day of withdrawal.

3 T = tissues; tissues obtained include liver, kidney, skin, muscle, and

fat.

4 Liver used for mixed function oxidase assay.

5 M8 = whole body.

6 Not laying for at least seven days prior to being killed.

93

Appendix B. Raw data for feed consumption, body weights, egg production,
and egg weights.

I. Feed Consumption - grams/hen/day.

 

 

 

Experimental Food
days Treatment consumption
Control hens 0- 6 Non contaminated diet 123.4
7-13 " 118.8
14-20 " 124.4
21-271 " 96.5
28-342 " 112.4
35-413 " 115.5
EDB hens 0- 6 EDB—contaminated diet 112.4
7-13 " 106.5
14-20 " 93.9
21—271 Non-contaminated diet 95.6
28-342 " 93.5
35-413 " 94.7

 

 

1 Days 0-6 of withdrawal.
2 Days 7-13 of withdrawal.
3 Days 14-20 of withdrawal.

94

Appendix B (con't.)
II. Body weights1-grams.

 

 

 

 

 

 

Hen # Day 0 Day 212 Day 423
Control hens 1 1756 1736 NA4
2 2234 2222 2174
3 1802 1712 NA
4 1908 1942 1818
5 1708 1788 NA
6 2016 2106 2032
7 2026 2142 2122
8 2196 2222 NA
Mean 4 SE5 1956 t 70 1984 t 77 2037 t 79
EDB hens6 1 1816 1712 NA
2 2015 2030 NA
3 2098 2006 NA
4 1965 2040 NA
5 1890 1856 NA
6 1646 1758 NA
7 2225 2334 NA
8 2015 2040 NA
9 2168 2130 1894
10 2080 2042 2064
11 1680 1720 1662
12 2130 2220 2488
13 1800 1780 1622
14 1685 1892 1798
15 2025 2060 1896
16 2010 2056 1886
Mean 4 SE 1853 t 45 1980 t 45 1914 t 96

 

1 Values represent weights of individual hens on the specified day.
2 Day 0 of withdrawal.

3 Day 21 of withdrawal.

4 NA = not alive on specified day.

5 SE = standard error of the mean.

5 EDB hens received EDB-contaminated diet days 0 to 21 and non-contaminated
diet days 21 to 42 (0 to 21 of withdrawal).

95

Appendix B (con't.)

III. Weekly egg production - percent.

 

 

 

Experimental %
days Treatment production
Control hens Acclimation Non-contaminated diet 77.1
0- 6 " 78.6
7-13 " 73.2
14-20 " 66.0
21-272 " 71.4
28-343 " 71.4
35-414 " 46.4
EDB hens Acclimation EDB-contaminated diet 75.0
0- 6 " 64.3
7-13 " 48.2
14-20 Non-contaminated diet 38.4
21-272 " 48.2
28-343 " 53.6
35-414 " 62.5

 

1 % production = [(# eggs laid per week per treatment)/(# hens per treat-
ment X 7)] x 100.

2 Day 0-6 of withdrawal.
3 Days 7-13 of withdrawal.
4 Days 14-20 of withdrawal.

96
Appendix B (con't.)

IV. Egg prduction by hen - percent1.

 

 

 

 

 

 

Days Days
Hen # Acclimation 14 to 21 35 to 412
Control hens 1 83.3 71.4 NA3
2 66.7 57.1 57.1
3 83.3 71.4 NA
4 66.7 57.1 85.7
5 66.7 57.1 NA
6 100.0 85.7 42.9
7 66.7 57.1 0.0
8 83.3 71.4 NA
Mean 4 SE4 77.1 t 4.4 66.0 t 3.8 46.4 4 197.9
EDB hens5 1 66.7 0(M6) NA
2 66.7 0(M) NA
3 66.7 0(M) NA
4 83.3 57.1 NA
5 83.3 57.1 NA
6 83.3 28.6 NA
7 50.0 0(M) NA
8 83.3 57.1 NA
9 83.3 28.6 0(M)
10 66.7 28.6 100 0
11 83.3 71.4 85.7
12 83.3 71.4 71.4
13 66.7 0(M) 0(M)
14 83.3 71.4 85.7
15 66.7 71.4 71.4
16 83.3 71.4 85.7
Mean 4 SE 75.0 t 2.6 38.4 t 7.7 62.5 t 14.0

 

1 Values represent production of individual hens during the specified time.
Percent production = [(# eggs laid per 7 days per hen)/7] X 100.

2 Days 14 to 20 of withdrawal.
3 NA = not alive during specified time.
4 SE = standard error of the mean.

5 EDB hens received EDB-contaminated diet days 0 to 21 and non-contaminated
diet days 21 to 42 (O to 21 of withdrawal).

6 M = molting during specified time period.

97

Appendix B (con't.)

V. Egg weights1 - grams

 

 

 

 

 

 

Days Days
Hen # Acclimation 14 to 20 35 to 412
Control hens 1 53.1 52.1 NA3
2 66.4 65.9 62.5
3 59.9 58.3 NA
4 63.6 64.0 64.0
5 62.3 60.3 NA
6 61.2 61.7 56.5
7 57.7 58.1 NE4
8 62.6 61.7 NA
Mean : SE5 60.9 t 1.4 60.3 t 1.5 61.0 t 2.3
EDB hens6 1 60.0 NE NA
2 60.0 NE NA
3 57.6 NE NA
4 55.4 57.0 NA
5 61.4 59.7 NA
6 63.7 56.9 NA
7 63.8 NE NA
8 58.0 56.6 NA
9 61.7 59.8 NE
10 65.3 62.9 67.5
11 58.8 60.8 60.5
12 59.0 59.8 61.8
13 53.0 NE NE
14 57.9 60.1 60.4
15 57.4 56.3 58.3
16 64.6 61.7 64.2
Mean 4 SE 59.9 t 0.9 59.2 t 0.7 62.1 t 1.3

 

1 Values are mean weight of eggs produced by one hen during specified time
period.

2 Days 14 to 20 of withdrawal.

3 NA = not alive during specified time.

4 NE = no eggs laid by a hen during specified time.
2 SE = standard error of the mean.

EDB hens received EDB—contaminated diet days 0 to 21 and non-contaminated
diet days 21 to 42 (0 to 21 of withdrawal).

98
Appendix C. Statistical methology for linear regression1.

A. Linear Model

Y = Bo + B] + 5 where Bo = Y-intercept, B] = slope, E = random error.

Predicted Y = = bo + bix where b0 and b] are estimates of Bo and B],
respectively.

Predicted x = R = (Y-bo)/b]

B. Formulas for SS, r, bo, and b]

X = dose = i to t where t = # of doses used to develOp the dose-response
line

Y = response

r1 = number of responses at each dose
t=otal number of/ numbers = iri

SPx xy = ixy - (Exy

ssy = Zy2- EZY% 2/n”

SSX =

SSR = b]SPx

SSE = ssy-s R

SSp = SS pure error = ESSyi

SSNL= SS error due to non- linearity = 835- SSp
b] = Sny/SSX \

b0 = Y - B1X

rxxy*=P1‘°dUCt'mQH'ent correlation = SPx NSSXSSy
rxy* = rxy[l+(l- rxy)/2(n- 4)] when 4<n<15

$2 = 55 E/n- 2
Y/x
Sy/x ‘ 152y/X1/2

 

1 All formulas are taken from Volume I of the Design and Analysis of
Experiments (Gill 1978).

99

Appendix C (con't.)

C.

Confidence Intervals

1) b0

bo : tq/2,n-2(Sy/X)[l/n + (x-1)2/ssX]t

2) b]

3)

6)

i tq/Z,n'2(Sy/x)/’SSX

A
Uy/x (predicted mean response at one dose)

+ b] (x-x) t tay2,n-2(sy/x)[1 + 1/n + (x-x)2/ssx]%
from a single unknown sample

7 + [b1 (Yo-Vl/glg ta/z "52(Sy/x)(T579)
where g = b]2 - _t4/2 n- 2(Sy/x)/SSX
h [(Yo- Y)2/ssx] + (n+1)g/n
YO observed response used to predict X

A
X from >1 unknown sample

I)? + [b] (70_V)/g] i" tx/2,:+m-3 (SJ/x)(11F/g)

where g = b2 - n+m-3 )/SSX
h = [1% -'412 Z/SSx]( + $21n+m)g/nm
10 = one 0response
Y0 = mean of all responses used to predict X
,m = # of responses used to calcualte Y0
§;/x = [(n 2)(5 2y/x) + (YOj'Y01213/n+m 3

Appendix C (con't.)

7) rXy

Zu

= Zupper = ny+

= ( 2)loge[(l+rx )/(1- -rxy
Use xy Zx xy - [(3 xy + rxy1/4n] 11

(Z144/2))11/((n- 3) (Z critical values are from

100

uthe standard normal table)

ZL =
1‘“ =
Y‘L =

D. Hypothesis testing with a linear regression table.

Zlower
rupper

1‘lower

= 2xy - (ziH/zfll/(n-n
= (eZZu_1)/(e2Zu + 1)
= (eZZL_])/(eZZL + 1)

)] Use#

x here if 4<n<15
4<

n<15

 

 

 

Critical
Source df SS MS f ratio value
Total n—l SSy --- --- ---
Regression 1 SSR MSR MSR/MSE F«,],n-2
Error n-2 SSE MSE --- ---
-NL (n 2)- (r1-1) SSNL MSNL MSNL/MSp FdstL,Vp
-P (rj-l) SSp MSp --- ---

 

101

 

 

 

 

 

 

Appendix D. Raw Data for Calibration Curves and Predicted Concentration
of EDB in Unknowns for Egg, Tissues, and Diet.
1. Egg
A. Raw Data
ppb EDB (x) Integrated area of the EDB peak (y)
0.5 0. 0. 0
1.0 0. 0. 0
1.5 2679. 0. 0
2.0 5116. 1273. O
2.5 2571. 2991. O
3.0 1808. 1613. 0
3.5 2368. 2057. 2727
4.0 3481. 3685. 2809
4.5 4382. 3292. 3462
5.0 3108. 3289. 4222
5.5 3182. 4634. 4067
6.0 3711. 5933. 3795
6.5 5670. 5158. 3923
7.0 4458, 4995, 5145
7.5 6453, 6573, 6488, 6123, 6939
15.0 9375. 14045. 10050. 7868. 8610
22.5 14903. 12981. 15571
30.0 20335. 22002. 22114. 13812. 15142
45.0 31445. 33981. 32972. 38512. 21867
60.0 41075, 43018. 43715. 33848. 30311
75.0 52661. 55015. 55006. 42506. 42714
90.0 62770. 69755. 68082
105.0 73322, 77724. 76094
120.0 83101. 88260. 87559
8. Prediction equation for doses from 4. 5- 120 ppb EDB
y= 707X- 509 r = .990 ru = .994 rL= .983
C. Linear Regression Table for doses from 4.5-120 ppb EDB
Source df 55 MS f Critical Value
Total 59 4. 0018444x1010 -- --
Regression 1 3. 9192518x1010 3. 9192518x1010 27521 f.001,1,58=12.06
Error 58 825,926,000 14,240,103 --
-NL 14 218,865,160 15,633,226 1.1332 f. 25,14, 44= 1. 298
-p 44 607,060,840 13.796.837
ISignificant at P < .001
2Not significant (P > .25). i.e., Non—linearity is not significant

Appendix D (con't.) 102

0. Predicted Concentration of EDB in Eggs Obtained from EDB hens
days 1 to 14 of residue buildup and days 1 to 21 of withdrawal].

 

 

Treatment Day Area2 EDB-ppb3
Residue Buildup 1 ND4 --—

2 ND ---

3 6389 9 75

4 9046 13 51

5 11263 16 64

6 14313 20 95

7 15029 21 97

8 18174 26 41

9 17464 25 41
10 16268 23 72
11 16884 24 59
12 19922 28 88
13 17385 25 30
14 17380 25 29

Withdrawal 1 11832 17 45

2 11299 16 69

3 7934 11.94

4 4865 7.60

5 5142 7.99

6 ND ---

7 ND ---

8 ND ---

9 ND —--
10 ND ---
11 ND ---
12 ND ---
13 ND ---
14 ND ---
15 ND ---
16 ND ---
17 ND ---
18 ND ---
19 ND ---
20 ND ---
21 ND ---

 

lNo EDB was detected in eggs obtained from control hens

2Represents integrated area of the E08 peak. A single analysis was
done on egg samples obtained for each day.

3Inversely predicted from 9 = 707x-509

4Not detected; detection 1imit = 4.5 ppb EDB

 

103

Appendix D (con't.)

E. Predicted Concentration of EDB in Eggs obtained from EDB hens

days 15 to 21 of residue buildup.1

 

 

Treatment Hen # Area2 EDB-ppb3

Residue Buildup 1 NE4 ---
2 NE ---
3 NE ---
4 17400 25.32
5 16294 23.75
6 15790 23.04
7 NE ---
8 22645 32.73
9 19020 27.61
10 15714 22.93
11 16423 23.94
12 24287 35.05
13 NE ---
14 25329 36.53
15 17527 25.50
16 20334 29.46

 

1No EDB was detected in eggs obtained from control hens.

2Represents integrated area of the E08 peak. A single analysis was
done on the homogenized egg sample obtained from eggs laid by each

hen over days 15 to 21.
3Inversely predicted from 9 = 707x-509.

4No eggs laid by that hen during days 15 to 21.

F. Predicted concentration of EDB after scrambling in Eggs obtained

from EDB hens days 15 to 21 of residue buildup.

 

Hen # Area before scramb1ing1 EDB-ppb2 Area after scrambling1 EDB-ppb3

 

 

9 19020 27.61 11696 17.3

10 15714 22.93 9348 13.9

11 16423 23.94 9206 13.7

12 24287 35.05 13414 19.7
1Represents integrated area of the E08 peak.
2Concentration before scrambling. Inversely predicted from 9 = 707x-509.
3Concentration after scrambling. Inversely predicted from 9 = 707x-509.

Appendix D (con't.) l04
11. Whole body

 

 

 

 

 

 

 

A. Raw data
ppb EDB (x) Integrated area of the E08 peak(Y)
7.5 6962,4488. 3623
l0 84l7.9995.8826
l2.5 l1274,llOl3.9l76.10305
15 16738.15555.15234
8. Prediction equation
1 = 1336x - 5039 r = .95 ru = .98 rL = .79
C. Linear regression table.
Source df SS MS 1 f Critical value
Tota1 12 191038175 -- -- ----
Regression 1 169998304 169998304 88.91 f.001.1.11=19.7
Error 11 21039871 1912716 -- ----
-NL 2 9797983 4898002 3.922 f.05.2.9=4.26
-P 9 11241888 1249098 -- ----

 

1 Significant at P < 0.01.
2 Not significant (P > .05)

0. Predicted concentration of EDB in whole body of EDB hens on days
0 and 21 of withdrawal. EDB was not detected in whole body of
control hens.

 

 

 

Day of Average EDB-
withdrawal Hen # Areas1 area ppb3
0 1 6070.7494,12571 8712 10.3
2 9368,7498,7962,7974 8201 9.9
5 9911,12721,9374,7927,7095 9406 10.8

4 14150.10738.10437.8978 11076 12.1

21 10 N04 -- --

12 ND -- --

13 ND -- --

15 ND -- --

 

1‘Represents integrated area of EDB peak.

on each liver.

Multiple analyses were done

2 Represents average of all areas obtained for each liver.
3 Inversely predicted from ?‘= 1336X-5039.
4 N0 = not detected; Detection limit = 7.2 ppb EDB.

:00

‘0

u.

\

\n

.1 no“:osiu

Appendix D (con't.) 105

 

 

 

III. Liver
A. Raw data
ppb EDB (x) Integrated area of the EDB peak (Y)
2.5 21753,17273.21892
5.0 28040.29996
7.5 35734.38319.38539
10.0 42846.46817

 

8. Prediction equation

A

v = 3307x + 12279 r = .99 ru = .995 rL = .908

C. Linear regression table

 

 

 

Source df SS MS f Critical value
Total 9 877.4l8.893 --- --- ----
Regression l 847,549,377 847,549,377 227.01 f.001,l.8=25.4
Error 8 29,869,516 3,733,690 --- ----

-NL 2 1.390.616 695.308 0.152 f.5.2.6=.78

“P 6 2894789900 4,746,483 "’ ""

 

1 Significant at P < 0.001.
2 Not significant (P > .5).

0. Predicted concentration of EDB in livers obtained from

EDB hens on days 0 and 2l of withdrawal. EDB was not detected

in livers of control hens.

 

 

 

Day of
withdrawal Hen # Area EDB-ppb
0 3 N01 --
6 ND --
7 ND --
8 ND --
21 9 ND --
ll ND --
14 ND --
16 ND --

 

1 Not detected; Detection limit = 0.8 ppb EDB.

Appendix D (con't.)

 

 

 

106
IV. Kidney
A. Raw data
ppb EDB (X) Integrated area of the E08 peak (Y)

5 14374

7.5 20033.20854
10 26204.36160
12.5 38182
15 45120.52837

 

8. Prediction equation

A

Y = 3388X - 2468 r = .96 ru

.99 rL = .72

C. Linear regression table

 

 

 

Source df SS MS f Critical value
Total 7 1,174,026,628 ---- -- ---—
Regression l 1,067,133,182 1,067,133,182 59.91 f.001.1,6=35.5
Error 6 106,893,446 17,815,574 -- ----

-NL 3 4,293,413 1,431,138 .0422 f.5.3.3=l.00
-P 3 102,600,033 34.200.011 -- ----

 

1 Significant (P < .001).
2 Not significant (P > .5).

D. Predicted concentration of EDB in kidneys obtained from
EDB hens on days 0 and 21 of withdrawal. EDB was not detected
in kidneys of control hens.

 

 

 

Day of
withdrawal Hen # Area EDB-ppb
0 3 N01 --
6 ND --
7 ND --
8 ND --
21 9 ND --
11 ND --
14 ND --
16 ND --

 

1 Not detected; Detection limit = 5.0 ppb EDB.

Appendix D (con't.) 107

 

 

 

V. Skin
A. Raw data
ppb EDB (X) Integrated area of the E08 peak (Y)
15 22614.20726
20 26129.29722
25 32900.37197

 

8. Prediction equation

 

 

 

 

? = 1338X + 1458 r = .98 ru = .99 rL = .63
C. Linear regression table

Source df SS MS f Critical value
Total 5 196.704.315 ---- -- ----
Regression 1 178,984,262 178,984,262 40.4 f.005,1,4=313
Error 4 17,720,053 4,430,013 -- ----

-NL 1 250,851 250,851 .043 f.5.1.3=.58

“P 3 1734699202 598239067 "" ""

 

1 Significant (P < .001).
2 Not significant (P > .5).

D. Predicted concentration of EDB in skin obtained from EDB
hens on day 0 and 21 of withdrawal. EDB was not detected
in control skin.

 

 

 

Day of
withdrawal Hen # Area EDB-ppb
0 3 N01 _-
6 ND --
7 ND --
8 ND --
21 9 ND __
11 ND ..
14 ND --
16 ND --

 

1 Not detected; Detection limit = 15.0 ppb EDB.

108
Appendix D (con't.)

 

 

 

VI. Fat
A. Raw data
Integrated
ppb EDB area of the
Treatment Day1 Hen # spike (X) EDB peak (Y)
EDB-contaminated diet 0 6 0 12763
25 25459
50 38188
100 593437
7 0 104505.108136
100 212294.205424
200 265485.301021
400 531618.484062
8 0 60130.47414
100 78660.93797
200 193408.202109
400 208954.341515
3 0 7621.12047
25 16439.18626
50 28191.23417
100 45019.47725

 

1 Represents the experimental day on which the hens were killed and
the fat samples were taken, i.e. it is day 0 of withdrawal.

8. Standard addition prediction equations and concentration
of EDB in fat samples of EDB hens.

 

 

 

Hen # Prediction equation r 1—bo/b111
6 1 = 465x + 13610 .99 29 ppb
7 = 995x + 102,419 .99 103 ppb
8 = 697x + 42.369 .98 61 ppb
3 Y = 368x + 8800 .99 24 ppb

 

1 Represents |X-intercept| which is the predicted concentration of
EDB in each fat sample on day 0 of withdrawal. EDB was not detected
in contro1 fat (detection limit = an area of 1705) on day 0 or
21 of withdrawal or in EDB fat on day 21 of withdrawal.

Appendix D (con't.)

C. Linear regression tables for

1119

additon equation.

each fat sample's standard

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Hen #6

Source df SS MS f Critical value
T0131 3 1,183,953.84] "" --- ----
Regression 1 1,180,841,796 1,180,841,796 759l f.005,l,2=198
Error 2 3,112,045 1,556,023 --- ——--
-NL 0 0 ---- --- ----

-P 2 3.112.045 ---- --- ----

1 Significant (P < 0.05).

Hen #7

Source df SS MS f Critical value
Total 7 1.760X 1011 --—- --- ----
Regression 1 1.733x 1011 1.773 x 1011 3791 f.001m.lm6=35.5
Error 6 2.746X 109 457,666,667 --- ----

-NL 2 953,916,254 476,809,627 1.062 f.25.2.4=2.oo
-P 4 1,792,380,746 448,077,187 --- ----

1 Significant (P < .001).

2 Not significant (P > .25).

Hen #8

Source df SS MS f Critical value
Total 7 8.8337202X 1010 ---- --- ----
Regression 1 8.5039542x 1010 8.5039542X 1010 1551 f.001.1.6=35.5
Error 6 3.29777x 109 5.4961x 1o8 --— ----

~NL 2 2.111.252.126 1,055,626,063 2.362 f.1.2.4=4.32
-P 4 1,792,380,746 448.095.187 --- -——-

1 Significant (P < .001).

2 Not significant (P > .1).

Hen #3

Source df SS HS f Critical value
Total 7 1,513,498,150 1

Regression 1 1,478,592,856 1,478,592,856 254 f.001,1,6=3.55
Error 6 34,905,294 5,817,549 "' ’ ----

-NL 2 7.662.316 3.831.158 0.56Z f.5.2.4=.83

-P 4 27,242,978 6,810,745 --- —---

1 Significant (P < .001).

2 Not significant (P > .5).

Appendix D (con't.)
VII. Muscle

A. Raw data

110

 

 

Treatment Day1

Hen #

ppb EDB
spike (X)

Integrated
area of the
EDB peak (Y)

 

EDB-contaminated diet 0

0010 0010

0010

0
5
10

1668,1868
24768.22681
43230.41139

3665.3612
21654.22868
46293.46911

3614
22258.20859
47997.46236

1123.1269
21905.20262
43033.42842

 

1 Represents the experimental day on which hens were killed and muscle
samples taken, i.e., it is day 0 of withdrawal.

8. Standard addition prediction equation and concentration
of EDB in muscle samples of EDB hens.

 

 

 

Hen # Prediction equation r l—bo/b1'1
6 i = 4042X + 2351 .99 582 ppt2
7 Y = 4296X + 2685 .99 625 ppt
8 x = 4459x + 1439 .99 323 ppt
3 Y = 4174x + 868 .99 208 ppt

 

1 Represents IX-intercept] which is the predicted concentration of

EDB in each muscle sample on day 0 of withdrawal.

EDB was not

detected in muscle on day 21 of withdrawal or in control muscle
on day 0 or 21 of withdrawal (detection limit = an area of 696).

2 Parts per trillion.

Appendix D (con't.)

111

C. Linear regression tables for each muscle sample's standard
addition equation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Hen #6

Source df SS MS f Critical value
Total 4 1,644,952,568 ---- --- ----
Regression 1 1,633,493,472 1,633,493,472 5791 f.001,1,3=167
Error 3 8,459,096 2,819,699 --- ----

-NL 1 4,075,171 4.675.171 2.132 f.25.l.2=2.57
-P 2 4,383,925 2.191.963 --- ----

l Significnat (P < .001).

2 Not significant (P > .25).

Hen #7

Source df SS MS f Critical value
Total 5 1,857,692,011 ---- --- ----
Regression 1 1,845,862,332 1,845,862,332 6241 f.001,1,4=74.1
Error 4 11,829,679 3,975,420 --- ----

~NL 1 10,900,414 10,900,414 7.462 f.05.1.3=10.3l
-P 3 4,383,925 1.461.308 --- ----

1—5 gag; a... (P < .001).

2 Not significant (P > .05).

Hen #8

Source df 55 MS f Critical value
Total 5 1,410,887,287 ---- --- ----
Regression 1 1,391,796,588 1,391,796.588 2921 f.001.1.4=741
Error 4 19,090,699 4,772,675 --- ----

~NL 1 16,561,539 16,561,539 19.642 f o1.1.3=34 12
-P 3 2.529.160 843.053 --- ----

1 Significant (P < .001).

2 Not significant (P > .01).

Hen #3

Source df SS MS f Critical value
Total 5 1.745.020.486 ---- -- ----
Regression 1 1,742,352,822 1,742,352,822 26131 f.001,1,4=74.1
Error 4 2,667,664 666,916 -- ----
-NL 1 1.289.040 1.289.040 2.812 f.1.1.3=5.54
-P 3 1.378.624 459.541 -- ----

 

1 Significant (P < .001).
2 Not significant (P > .1).

112

Appendix D (con't.)

 

 

 

VIII. FIOur
A. Raw data
ppm EDB (X) Integrated area of the E08 peak (Y)
25 9186000.8859300
30 10211000.10917000
35 12157000.12445000

 

8. Prediction equation

A
Y = 327.835X + 794167 r

 

 

 

= .99 ru = .998 rL = 841
C. Linear regression table
Source df SS MS f Critical value
Total 5 1.1104393x 1013 ---- --- ----
Regression 1 1.0747578X 1013 1.0747578X 1013 120.51 f.001.1.4=74.1
Error 4 3.5681527X 1011 8.9203817X 101O --- ----
-NL 1 1.235887X 1010 1.235887x 1011 1.082 f.25.1.3=2.02
-P 3 3.4445640x 1011 1.148188X 1011 --- -—--

 

1 Significant (P < .001).
2 Not significant (P > .25).

0. Predicted concentration of EDB in flour.

 

 

Average
Area1 EDB-ppm2 concentrat1on-ppm

 

1 1273x 107 31.96 31.1
1.0660X 107 30.29

 

1 Integrated area of the ED8 peak.
2 Inversely predicted from Y = 327,835X + 794,167

113

Appendix D (con't.)

 

 

 

IX. Diet
A. Raw data
ppm EDB (X) Integrated area of the E08 peak (Y)
5 3082200.3037900
10 4999200.4833200
15 6640100

 

8. Prediction equation
1 = 359,894X + 1,279,471 r = .999998 ru = .999999 rL = 999747

C. Linear regression table

 

 

 

Source df SS MS f Critical value
Total 4 9.0864291X 1012 --_— -- ----
Regression 1 9.0666727X 1012 9.0666727X 1012 13771 f.001.1.3=167
Error 3 1 975640x 1010 6585468567 —- ----
-NL 1 4.9971607X 109 4.9971607X 109 0.682 f.25.1.12=2.57
-P 2 1.4759245x 1010 7,379,622,500 -- ----

 

1 Significant (P < .001).
2 Not significant (P > .25).

0. Predicted concentration of EDB in diet.

 

 

 

Average
Area1 EDB—ppm2 concentration-ppm
3.801.900 7.01 6.7

3.562.300 6.34

 

1 Integrated area of the E08 peak.
2 Inversely predicted from Y = 359,894X + 1.279.471.

114

Appendix E. Preparation of microsomal isolation, Biuret, and MFO reagents.

I.

II.

III.

Microsomal isolation reagents

1)

2)

Potassium chloride (kcl) - 150 mM

Dissolve 11.2 g of kcl (Mallinckrodt)1 in 900 m1 double-distilled
water (00 H20). Bring to one liter with DD H20. Store at 4°C.

Homogenization buffer - pH 7.4

Dissolve 2.4 g Trizma® base (Sigma, 1-1503) and 15 g kcl in 800
m1 DD H20. Adjust the pH to 7.4 with 0.1 N hydrochloric acid.
Bring to one liter with DD H20. Store at 4°C.

Tris-Hcl - pH 7.4, 200 mM

Dissolve 24.22 g Trizma® hydrochloride (Sigma, T-3253) in 800 ml

00 H20. Adjust pH to 7.4 with 2.5 N sodium hydroxide (NaOH).
Bring to one liter with DD H20. Store at 4°C.

Biuret crude protein determination reagents

1)

NaOH - 6%

Dissolve 60.0 g NaOH (Mallinckrodt) in 900 ml DD H20. Bring to
one liter with 00 H20. Store at room temperature.

Bovine serum albumin (BSA) standard - 5 mg BSA/ml 150 kacl

Dissolve 125 mg BSA (Sigma, A-9647) in 10 ml 150 kacl. Mix gently
to avoid foaming. Bring to 25 ml with 150 kacl. Divide into
5 ml aliquots. Store at -25°C.

Biuret reagent

Heat 250 ml 00 H20 to 60°C. Add 50.0 g sodium carbonate
(Mallinckrodt). Stir vigorously to dissolve the sodium carbonate.
Slowly add 86.5 g sodium citrate (Mallinckrodt). Allow the solution
to cool. Dissolve 8.6 9 copper sulfite -5 H20 in 50 ml DD H20.
Combine the 2 solutions in a 500 m1 volumetric flask. Bring to
500 ml with DD H20. Mix well. Store at room temperature.

MFO reagents

1)

Glucose-6-phosphate(G6P)-pH 7.0, 100 mg G6P/m1 DD H20

6)

7)

115

Dissolve 1.0 g G6P monosodium salt (Sigma, G-7879) in 5 ml 00 H20.
Adjust pH to 20 with l N NaOH. Bring to 10 ml with 00 H20. Store
at -25°C.

Magnesium chloride (MgC12)-200 mM

Dissolve 5.0 g MgC12-6H20 in 100 ml DD H20. Bring to 125 ml with
00 H20. Store at 4°C.

Glucose-6-phosphate dehydrogenase (G6PD) - 0.5 units/D1 200 mM
Tris-HCl

Add 1 m1 200 mM Tris-HCl to 500 units of G6PD (Sigma, G-6378).
Store at -25°C.

Formaldehyde - 30 mM

Bring 500 pl of reagent grade formaldehyde (Mallinckrodt, 12m)
to 100 ml with 00 H20. Take 50 ml of this solution and bring to
100 ml with DD H20. Store at 4°C.

Dimethyl sulfoxide - potassium hydroxide (DMSO-KOH)

Prepare a l M KOH solution by dissolving 5.6 g KOH (Mallinckrodt)
in 70 ml 00 H20. Bring to 100 ml with DD H20. Prepare the DMSO-KOH
solution by combining 85 ml DMSO (Mallinckrodt) with 15 m1 1M KOH.
Store at room temperature.

BenzoGK)pyrene substrate2 (BP)-6.4 mM

In a separatory funnel, add 123 pmoles (32.29 mg) cold BP (Sigma.
B-3500) and 1600 uCi (128 moles) of tritium—labeled BP (Amersham,
TRK.66, 12.5 uCi/umole) to 50 ml glass-distilled hexane
(Mallinckrodt). Add 25 nfl DMSO-KOHzDD H20 (1:1) ix) the ‘funnel.
Mix, allow the layers to separate, and discard the DMSO-KOHzDD
H20 (1:1) layer. Repeat the extraction 2 more times. Transfer
the hexane layer to a 50 ml glass-stoppered tube. Dry under
nitrogen. After the hexane has evaporated. fill the tube with
nitrogen, st0pper the tube, and store at -25°C. When the substrate
is to be used, add 40 ml acetonitrile (Burdick and Jackson) and
mix. If the substrate is stored dissolved in acetonitrile, it
must be cleaned befure use. Dry 10 ml of the BP-acetonitrile
solution under nitrogen. Suspend the BP in 10 m1 glass—distilled
hexane. Extract 2 times with 6 ml of 2.5 N HaOH in 40% ethanol.
Dry under nitrogen. Resuspend in 10 ml acetonitrile.

Aminopyrine substrate3 (AP) - 230 mg AP/ml methanol

116

Dissolve 2.3 9 AP (Aldrich, 013910-6) in 5 ml reagent grade methanol
(MCB). Bring to 10 ml with methanol. Store at -25°C.

8) Nash reagent

Dissolve 75 g ammonium acetate (Mallinckrodt) in 125 mg 00 H20.
Add 2 DH acetylacetone (Mallinckrodt) and 1.5 nfl glacial acetic
acid (Mallinckrodt). Bring to 500 ml with DD H20. Store at 4°C.
Discard when it becomes yellow.

9) Zinc sulfate (ZnSO4) - 20%

Dissolve 100 g ZnSO4 in 400 ml 00 H20. Bring to 500 ml with 00
H20. Store at room temperature.

10) Barium hydroxide (BaOH) - saturated

Bring 250 ml 00 H20 to a boil. Slowly add BaOH (Mallinckrodt)
until it will no longer go into solution. Filter the solution
while hot through Whatman #1 filter paper into a bottle. Allow
the solution to cool before capping the bottle. Store at room
temperature.

11) Benzo( )pyrene cocktail

In a one gallon brown bottle, combine 1800 mg universal LCS cocktail
(Fisher, ScintiverseTMI), 360 m1 gold label ethyl alcohol
(Mallinckrodt), 150 m1 dimethyl sulfoxide and 15 ml 1M acetic acid.
Store at room temperature.

 

1 The companies from which chemicals were purchased for this study are listed
in parentheses.

2 3,4-benzopyrene
3 4-(dimethylamine)-l,2-dihydro-l,5—dimethyl-2-pheny1-3H-pyrazol=3-one

Appendix F. MFO assay raw data.

I. Day 0 of withdrawal

A. Biuret protein determination

117

 

 

 

1) Codes

Treatment Band # Liver weight-g Sample #

EDB-contaminated 24563 53.23 1

24569 38.45 2

24561 38.29 3

24565 53.23 4

Non-contaminated 24576 39.25 5\

6

24580 36.88

 

2) Determination of liver microsomal protein content.

Calibration curve

 

 

Equation for

mg protein (x) Absorbance (Y) calibration curve

 

 

 

 

 

0 .063 Y = .042x + .055 r = .99
0 .057
3 .170
3 .165
5 .285
5 .255
Samples
mg protein mg protein/ X mg protein/
100 ul
Sample # Absorbance microsomes1 ml microsomes ml microsomes
1 .118 1.5 15 - 15.5
1 .121 1.6 16
2 .110 1.3 13 13.5
2 .116 1.4 14
3 .101 1.1 11 12.0
3 .110 1.3 13
4 .122 1.6 16 16.0
4 .121 1.6 16
5 .160 2.5 25 25.5
5 .162 2.6 26
6 .112 1.4 14 14.0
6 .113 1.4 14

 

1 Inversely predicted from the calibration curve.

118

Appendix F (con't.)

3) Microsomal dilutions1

 

 

mg protein/

 

200 mM Tris 200 pl
Sample # microsomes-ml HCl-ml microsomes
1 1.0 2.1 1.0
2 1.0 1.7 1.0
3 1.0 1.4 1.0
4 1.0 2.2 1.0
5 1.0 4.1 1.0
6 1.0 1.8 1.0

 

1 1.0 ml of microsomes was diluted with Tris.HCl to obtain 1 mg protein/200
ul microsomes.

Appendix F (con't.)

8. Calculation of activity of aminopyrine N-demethylase.

Calibration curve

 

 

Equation for

 

 

 

 

 

nmoles CHZO Absorbance calibration curve
A
0 .033 Y = .001X + .034 r = .99
0 .045
60 .111
60 .107
120 .193
120 .183
240 .370
240 .333
Samples
Net Net
‘ nmoles CH20/ nmoles CH20/
nmoles X nmoles mg protein/ mg protein/
Sample # Absorbance CH202 CHZO 30 min. minute
131 .122 67.1 64.4 42.3 1.41
15 .115 61.7
lb .063 22.1 22.1
1b .063 22.1
2S .123 67.8 64.0 44.2 1.47
25 .111 60.8
25 .060 19.8 19.8
2b .060 19.8
3S .084 38.1 38.1 18.3 0.61
35 .084 38.1
3b .062 21.3 19.8
3b .058 18.2
4s .107 55.6 45.3 23.6 0.79
45 .080 35.0
4b .070 27.4 21.7
4b .055 15.9
55 .095 46.5 48.4 30.5 1.02
55 .100 50.3
5b .060 19.8 17.9
5b .055 15.9
65 .095 46.5 48.0 28.2 0.94
65 .099 49.5
6b .062 21.3 19.8
6b .058 18.2

 

1 S = sample. b = blank

2 Inversely predicted from the calibration curve.
3 Calculated by substracting blank values from sample values.

Appendix F (con't.) 120

C. Calculation of activity of aryl hydrocarbon hydroxylase.

 

 

X Net

 

Effi- pmoles pmoles pmoles

Sample # 0PM2 CPM3 ciency4 dpm BP-0H5 BP-on6 BP-0H595
Total count 287289 339169 .251 1144577 --- --- --
Tota1 count 297011 354691 .279 1064556 --- --- --
Total count 317051 372260 .267 1187457 --- --- --

151 5225 51975 .226 23119 131 126 65

1S 4853 51478 .225 21569 122

1o 2559 49195 .225 11373 64 61

1o 2311 49290 .227 10181 58

2S 5696 53069 .229 24873 141 142 80

2S 5909 54194 .233 25361 143

2b 2571 50158 .230 11178 63 62

25 2480 50446 .232 10690 60

3S 5222 51729 .225 23209 131 123 57

3S 4607 51505 .227 20295 115

3b 2628 50223 .230 11426 65 66

3o 2682 48783 .223 12027 67

4s 3493 52134 .235 14864 84 91 23

4s 4017 52133 .232 17315 98

4o 2621 50088 .231 11346 64 68

4o 2880 50192 .229 12576 71

5S 4087 52850 .235 17391 98 103 35

5S 4405 52082 .230 19152 108

5b 2782 49663 .226 12310 70 68

5b 2662 49709 .227 11727 66

6S 5272 51355 .223 23641 134 125 48

6S 4754 53073 .233 20403 115

6b 2782 49139 .224 12420 70 77

6b 3306 49212 .222 14892 84

 

= sample; b = blank

(JUN-4

S
8 channel cpm before 3H toluene spike.
8 channel cpm after 3H toluene spike.

4 Efficiency = (cpm after 3H toulene spike-cpm before 3H toluene

spike)/2.07 x 105 dpm.

5 Represents pmoles hydroxylated benzo(a)pyrene produced/mg protein/

minute = dpm X 3 X 64,000

 

1132197 X 30

5 Calculated by subtracting blank values from sample values.

1121
Appendix F (con't.)

II. Day 21 of withdrawal

A. Biuret protein determination

 

 

 

1) Codes

Treatment Band # Liver weight-g Sample #

Non-contaminated 24581 51.17 1

24577 42.55 2

EDB-contaminated 24556 38.88 3

24566 32.73 4

24562 41.51 5

24552 31.37 6

Control broilers1 20 --- 7

21 --- 8

P88 broilers1 22 --- 9

23 --- 10

 

1 Broilers were carried as positive controls. Control broilers received
non-contaminated broiler starter for 7 days prior to the assay.
PBB broilers received diet at 80 ppm PBBS for 7 days prior to
the assay.

2) Determination of liver microsomal protein content.

Calibration curve

 

 

Equation for
mg protein (X) Absorbance (Y) calibration curve

 

.A
.055 Y = .050X + .058 r = .99
.057
.212
.212
.301
.307

mmwwoo

 

Samples

 

mg protein/

 

100 pl mg protein/ X mg protein/
Sample # Absorbance microsomes m1 microsomes m1 microsomes
l .164 2.13 21.3 21.6
1 .167 2.19 21.9
2 .119 1.23 12.3 12.2
2 .118 1.21 12.1
3 .091 .66 6.6 6.6
3 .091 .66 6.6
4 .071 .26 2.6 6.3
4 .107 .99 9.9
5 .112 1.09 10.9 10.3
5 .106 .97 9.7
6 .096 .77 7.7 8 l
6 .100 .85 8.5
7 .091 .66 6.6 6.0
7 .085 .54 5.4
B .111 1.07 10.7 10.8
8 .112 1.09 10.9
9 .111 1.07 10.7 9.7
9 .101 .87 8.7
10 .083 .50 5.0 5.1
10 .084 .52 5.2

 

1 Inversely predicted from the calibration curve.
2 P88 = polybrominated biphenyl.

122
Appendix F (con't.)

3) Microsomal dilutions1

 

 

 

200 mM mg protein/
Sample # microsomes-m1 Tris-HCl 200 pl microsomes
l 2.0 6.52 1.0
2 2.0 2.88 1.0
3 .0 .64 1.0
4 .0 .52 1.0
5 2.0 2.12 1.0
6 2.0 1.24 1.0
7 2.0 .40 1.0
8 2.0 2.32 1.0
9 2.0 1.88 1.0
10 2.0 .04 1.0

 

1 2.0 ml of microsomes were diluted with Tris-HCl to obtain 1 mg
protein/200 ul microsomes.

.1223
Appendix F (con't.)

8. Calculation of activity of aminOpyrine N-demethylase.

Calibration curve

 

 

Equation for

 

 

 

 

 

nmoles CH20 Absorbance calibration curve
A
0 .040 Y = .002X + .034 r = .99
0 .040
60 .129
60 .130
120 .230
120 .225
240 .440
240 .430
Samples
__ Net Net
X nmoles CHZO nmoles CH20
nmoles nmoles mg protein/ mg protein/
Sample # Absorbance 611202 61120 30 min.3 minute
151 .112 47.0 61.5 18.9 0.63
15 .160 76.0
1b .065 18.6 42.6
1o .074 24.0
25 .122 53.0 49.1 41.3 1.38
25 .109 45.2
2b .040 3.4 7.8
2b .055 12.5
3S .122 53.0 41.5 24.8 0.83
3s .084 30.0
3b .062 16.7 16.7
3b .062 16.7
45 .103 41.5 42.1 20.2 0.67
4S .105 42.7
4b .070 21.6 21.9
4b .071 22.2
55 .081 28.2 25.8 6.6 0.22
55 .073 23.4
5b .070 21.6 19.2
5b .062 16.7
65 .205 103.2 101.7 63.5 2.12
65 .200 100.2
6b .110 45.8 38.2
5b .085 30.6
7s .100 39.7 39.1 15.4 0.51
7S .098 38.5
7b .072 22.8 23.7
7b .075 24.6
85 .131 58.5 57.9 39.9 1.33
85 .129 57.2
8b .072 22.8 18.0
8b .056 13.1
9S .174 84.4 86.6 77.1 2.57
95 .181 88.7
9b .050 9.5 9.5
9o .050 9.5
105 .131 58.5 66.1 50.6 1.69
105 .156 73.6 ~
100 .060 15.5 15.5
10b .060 15.5

 

1 S = sample; b = blank.
2 Inversely predicted from the calibration curve.
3 Calculated by subtracting blank values from sample values.

Appendix F (con't.)

C. Calculation of activity of aryl hydrocarbon hydroxylase.

1124

 

 

 

X Net
Effi- pmoles pmoles pmoles
Sample # cpm2 cpm3 ciency4 dpm BP-0H5 BP-0H5 BP-0H546
Tota1 count 228457 290545 .300 761523 --- --- ---
Total count 280730 346417 .317 885584 --- --- ---
Total count 237803 302349 .312 762189 --- —-— ---
15* 6046 55674 .240 25192 201 177 143
15 4592 53971 .239 19213 153
1b 1019 49610 .235 4336 35 34
lb 1016 49915 .236 4305 34
25 4638 53908 .238 19487 155 155 120
25 4665 54489 .241 19357 154
2b 1106 50709 .240 4608 37 35
2b 993 50637 .240 4138 33
3s 3078 53486 .244 12615 101 95 61
3s 2738 52839 .242 11314 90
3b 1065 50474 .239 4456 36 34
3b 967 50588 .240 4029 32
4s 4362 53515 .237 18405 147 139 107
45 3967 53558 .240 16529 132
4b 1028 50994 .241 4266 34 32
4b 906 51757 .246 3683 29
55 1718 51135 .239 7188 57 58 23
55 1757 51399 .240 7321 58
5b 1115 50977 .241 4627 37 35
5b 985 50857 .241 4087 33
65 7478 57040 .239 31289 249 264 231
65 8097 55939 .231 35052 279
6b 989 50608 .240 4121 33 33
6b 1000 51345 .243 4115 33
7S 4006 54066 -.242 16554 132 136 104
7S 4345 55384 .247 17591 140
7b 1029 51505 .244 4217 34 32
7b 940 51956 .246 3821 30
85 4141 54644 .244 16971 135 131 96
85 3895 54212 .243 16029 128
8b 1073 50905 .241 4452 35 35
8b 1056 50405 .238 4437 35
9s 23299 73640 .243 95881 764 781 745
9S 24151 74042 .241 100212 799
9b 1013 51922 .246 4118 33 36
95 1175 51493 .243 4835 39
105 15146 64315 .238 63639 507 493 459
105 14165 62984 .236 60021 478
100 1047 51528 .244 4291 34 34
10b 1055 51633 .244 4324 34

 

S = sample; 6 = blank.
B

”Nd

spike)/2.07 x 105 dpm.

channel cpm before 3H toluene spike.
8 channel cpm after 3H toluene spike.
4 Efficiency = (Cpm after 3H toluene Spike — cpm before 3H toluene

5 Represents pmoles hydroxylated benzokx)pyrene produced/mg protein/minute

= dpm x 3 x 64.000
803.099 x 30

5 Calculated by subtracting blank values from sample values.

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