REVISED
DRAFT
DETAILED
REVIEW
PAPER
FOR
AVIAN
TWO­
GENERATION
TOXICITY
TEST
EPA
CONTRACT
NUMBER
68­
W­
01­
023
WORK
ASSIGNMENT
2­
16
April
23,
2003
Prepared
for
LESLIE
TOUART,
PH.
D.
WORK
ASSIGNMENT
MANAGER
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
WASHINGTON,
D.
C.

BATTELLE
505
King
Avenue
Columbus,
Ohio
43201
Battelle
Draft
ii
April
23,
2003
TABLE
OF
CONTENTS
1.0
EXECUTIVE
SUMMARY
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1
2.0
INTRODUCTION
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4
2.1
Developing
and
Implementing
the
Endocrine
Disruptor
Screening
Program
(
EDSP)
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4
2.2
The
Validation
Process
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5
2.3
Purpose
of
the
Review
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7
2.4
Objectives
of
the
Avian
Two
Generation
Toxicity
Study
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7
2.5
Methods
Used
in
this
Analysis
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8
2.6
Acronyms
and
Abbreviations
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8
3.0
OVERVIEW
AND
SCIENTIFIC
BASIS
OF
AVIAN
TWO­
GENERATION
TESTS
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10
3.1
Avian
Endocrinology
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11
3.2
Differences
in
Avian
and
Mammalian
Endocrine
Systems
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13
3.3
Avian
Two­
Generation
Test
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14
4.0
CANDIDATE
TEST
SPECIES
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16
4.1
Japanese
Quail
(
Coturnix
japonica)
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16
4.1.1
Natural
History
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17
4.1.2
Availability,
Culture,
Handling
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17
4.1.3
Strains
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23
4.2
Bobwhite
(
Colinus
virginianus)
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25
4.2.1
Natural
History
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25
4.2.2
Availability,
Culture,
Handling
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25
4.2.3
Strains
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26
4.3
Strengths
and
Weaknesses
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26
5.0
EXPERIMENTAL
DESIGN
CONSIDERATIONS
FOR
TWO­
GENERATION
AVIAN
TESTS
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30
5.1
Exposure
Duration
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30
5.1.1
Exposure
of
the
Parental
(
P1)
Generation
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30
5.1.2
Exposure
of
the
Offspring
(
F1)
of
the
Parents
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34
5.1.3
Combined
Exposure
Scenarios
for
P1
and
F1
Generations
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34
5.1.4
Selection
of
Egg
Cohort
for
F1
Breeding
Pairs
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36
5.1.5
Selection
of
P1
and
F1
Birds
for
Pairing
and
Breeding
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37
5.1.6
Comparison
of
One­
Generation
and
Two­
Generation
Exposure
Regimens
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37
5.2
Route
of
Administration
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39
5.2.1
Food
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40
5.2.2
Water
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41
5.2.3
Bolus
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42
5.3
Dose
Selection
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43
5.3.1
Dose
Adjustment
for
Size
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44
5.3.2
Dose
Adjustment
by
Life
Stage
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45
5.4
Statistical
Considerations
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46
Battelle
Draft
iii
April
23,
2003
TABLE
OF
CONTENTS
(
Contd)

5.4.1
Sample
Size
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52
5.4.2
Hypothesis­
Testing
or
Regression
Analysis
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53
6.
ASSAY
ENDPOINTS:
FITNESS
ENDPOINTS
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55
6.1
Growth
Rate,
Food
Consumption
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57
6.2
Measures
of
Reproductive
Performance
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58
6.2.1
Fecundity
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58
6.2.2
Gamete
Viability
and
Fertilization
Rate
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59
6.2.3
Changes
in
Breeding
Behavior
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69
6.3
Neurological/
Central
Nervous
System
Impairment
Tests
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71
7.0
ASSAY
ENDPOINTS:
PHYSIOLOGICAL
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71
7.1
Organ
Growth
and
Morphological
Changes
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73
7.1.1
Development
of
Gonadal
and
Accessory
Structures
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73
7.1.2
Histopathology
in
Juveniles
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75
7.1.3
Histopathology
in
Sexually
Mature
Individuals
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76
7.1.4
Histopathology
and
Organ
Weights
of
Nonreproductive
Tissues
.
.
.
.
.
.
.
.
.
.
.
.
.
.
78
7.1.5
Japanese
Quail
Male
Cloacal
Gland
Measures
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
80
7.2
Sexual
Differentiation
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
80
7.3
Secondary
Sex
Characteristics
.
.
.
.
.
.
.
.
.
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.
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.
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.
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.
.
.
.
.
.
.
.
81
7.4
Sex
Ratio
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
82
7.5
Biochemical
Measures
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
84
7.5.1
Vitellogenin
and
Other
Biomarkers
of
Hepatic
Metabolic
Changes
.
.
.
.
.
.
.
.
.
.
.
.
85
7.5.2
Plasma
and
Renal/
Urate
Hormone
Concentrations
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
89
8.0
RESPONSE
TO
ESTROGEN
AGONISTS
AND
ANTAGONISTS
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
92
8.1
Sexually
Mature
Life
Stages
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
93
8.1.1
Sensitivity
to
17ß­
estradiol
or
Synthetic
Estrogen
Exposure
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
93
8.1.2
Antiestrogens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
94
8.1.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
94
8.2
Juvenile
Life
Stages
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
94
8.2.1
Sensitivity
to
17ß­
estradiol
or
Synthetic
Estrogen
Exposure
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
94
8.2.2
Antiestrogens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
96
8.2.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
96
9.0
RESPONSE
TO
ANDROGEN
AGONISTS
AND
ANTAGONISTS
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
97
9.1
Sexually
Mature
Life
Stages
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
97
9.1.1
Sensitivity
to
Androgenic
Steroid
Exposure
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
97
9.1.2
Antiandrogens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
98
9.1.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
99
9.2
Juvenile
Life
Stages
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
99
9.2.1
Sensitivity
to
Androgenic
Steroid
Exposure
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
99
9.2.2
Antiandrogens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
100
9.2.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
100
Battelle
Draft
iv
April
23,
2003
TABLE
OF
CONTENTS
(
Contd
)

10.0
RESPONSE
TO
THYROID
AGONISTS
AND
ANTAGONISTS
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
101
10.1
Sensitivity
to
Thyroid
Stimulation
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
101
10.2
Inhibition
of
Thyroid
Function
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
102
10.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
103
11.0
CANDIDATE
PROTOCOLS
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
103
11.1
One­
Generation
Reproduction
Tests
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
117
11.1.1
OPPTS
850.2300
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
.
117
11.1.2
OECD
206
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
118
11.1.3
ASTM
E1062­
86
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
119
11.1.4
OECD
Revised
Draft
Proposal
(
April
2000)
for
a
New
Test
Guideline,
Avian
Reproduction
Toxicity
Test
in
the
Japanese
Quail
or
Northern
Bobwhite
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
120
11.2
Two­
Generation
Life
Cycle
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
120
11.2.1
Proven­
Breeder
Design
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
121
11.2.2
Pre­
Egg­
Laying
Exposure
Design
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
122
12.0
RECOMMENDED
PROTOCOL
AND
ADDITIONAL
DATA
NEEDS
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
122
12.1
Preferred
Test
Species
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
122
12.2
Exposure
Protocol
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
123
12.3
Appropriateness
of
Reproductive
Endpoints
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
124
12.4
Preferred
Methods
for
Quantification
of
Biochemical
Endpoints
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
126
12.5
Significant
Data
Gaps
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
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.
.
.
.
.
127
13.0
IMPLEMENTATION
CONSIDERATIONS
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
128
14.0
REFERENCES
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
129
APPENDIX
A
 
Literature
Search
(
Will
be
added)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
A­
1
APPENDIX
B
 
Interviews
(
Will
be
added)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
B­
1
FIGURES
5­
1
Calculated
Dose
at
Weekly
Intervals
of
Growing
Japanese
Quail
Consuming
a
Diet
Amended
with
100
or
167
ppm
of
Test
Substance
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
46
5­
2
Power
of
a
One­
Sided
Independent­
Samples
T­
Test
as
a
Function
of
the
Percentage
Reduction
Detected
Between
the
Test
and
Reference
Means,
with
16
Replicates
per
Treatment
.
.
.
.
.
.
.
.
.
50
TABLES
2­
1
Acronyms
and
Abbreviations
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
8
4­
1
Stocks
of
Random­
Bred
or
Wild
Type
Japanese
Quail
Maintained
in
North
America
and
Europe
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
4­
2
Body
Weight
and
Reproductive
Parameters
of
Egg­
Producing
and
Meat
Producing
Strains
of
Japanese
Quail
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
.
.
.
.
.
.
21
Battelle
Draft
v
April
23,
2003
TABLES
(
Contd)

4­
3
Major
Strengths
and
Weaknesses
of
Japanese
Quail
and
Northern
Bobwhite
Related
to
Use
in
Avian
Two­
Generation
Reproduction
Toxicity
Tests
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
28
4­
4
Typical
Values
for
Reproductive
Parameters
in
Japanese
Quail
and
Northern
Bobwhite
.
.
.
.
.
.
.
29
4­
5
Comparison
of
Development
Phases
in
Japanese
Quail
and
the
Northern
Bobwhite
.
.
.
.
.
.
.
.
.
.
30
5­
1
Required
Attributes
of
Parental
and
Offspring
Exposure
Regimens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
35
5­
2
Control
Data
Coefficient
of
Variation
for
the
Raw
and
Transformed
Endpoints
Measured
from
an
Interlaboratory
Comparison
Study
on
Japanese
Quail
Exposed
to
Bis(
tri­
butyltin)
oxide
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
6­
1
Fitness
Endpoints
Specific
to
Endocrine
Active
Substances
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
56
7­
1
Physiological
Endpoints
Specific
to
Endocrine
Active
Substances
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
72
7­
2
Gross
Landmarks
of
Sexual
Differentiation
in
Japanese
Quail
and
Age
of
Appearance
.
.
.
.
.
.
.
.
75
11­
1
Comparison
of
Avian
Reproductive
Toxicity
Tests
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
105
Battelle
Draft
vi
April
23,
2003
SPECIAL
NOTE
A
substantial
technical
contribution
to
the
creation
of
this
document
was
provided
by
Dr.
Ann
Fairbrother.
Written
text
included
the
author's
own
published
work,
either
as
a
lead
or
coauthor
During
this
time,
Dr.
Fairbrother
was
working
for
a
private
company.
She
has
since
taken
a
position
with
the
EPA.
Battelle
Draft
1
April
23,
2003
1.0
EXECUTIVE
SUMMARY
Endocrine
disruptors
are
any
chemicals
that
are
known
or
suspected
to
cause
adverse
endocrine
effects
in
organisms
or
their
progeny.
Such
chemicals
have
received
increased
attention
over
the
past
decade
because
of
the
potential
harm
they
can
do
to
wild
and
domestic
animals
and
ultimately
to
humans.
Therefore,
Congress
authorized
the
United
States
Environmental
Protection
Agency
(
EPA)
to
develop
a
program
to
screen
a
wide
array
of
chemicals
found
in
drinking­
water
sources
and
food
to
determine
whether
they
possess
estrogenic
or
other
endocrine
activity
that
could
have
disruptive
endocrine
effects
in
humans.
The
aim
of
this
program
is
to
develop
a
two­
tiered
approach:
that
is,
a
combination
of
in
vitro
and
in
vivo
mammalian
and
ecotoxicological
screens
(
Tier
1),
and
a
set
of
in
vivo
tests
(
Tier
2)
for
identifying
and
characterizing
endocrine
effects
of
pesticides,
industrial
chemicals,
and
environmental
contaminants.
The
organisms
used
in
the
screening
and
testing
will
represent
a
variety
of
taxonomic
groups,
such
as
fish,
birds,
and
mammals,
for
example.

The
present
detailed
review
paper
fulfills
one
of
EPA's
objectives
in
its
validation
process,
namely,
to
summarize,
explain,
and
document
the
relevant
principles,
methods,
and
techniques
for
a
Tier
2
multigenerational
test
to
determine
the
effects
of
potential
endocrine­
disrupting
chemicals
on
birds.
Although
avian
species
are
not
part
of
the
Tier
1
screening
battery
for
assessment
of
the
effects
of
endocrine­
disrupting
chemicals,
they
are
included
in
Tier
2.
Birds
are
fundamentally
different
from
mammals
in
the
control
of
their
sexual
differentiation,
and
therefore,
mammalian
tests
provide
little
predictive
value
for
assessing
a
chemical's
potential
impact
to
birds;
separate
testing
is
required.

The
present
report
contains
a
review
of
the
current
literature,
and
a
recommendation
of
an
initial
Tier
2
protocol
and
of
an
organism
that
will
best
meet
the
needs
for
testing.
It
identifies
issues
that
could
require
prevalidation
studies.
The
recommended
avian
test
protocol
is
designed
to
accomplish
the
following:

°
determine
whether
effects
are
a
primary
or
secondary
disturbance
of
endocrine
function
°
establish
exposure,
concentrations,
timing,
and
effects
relationships
°
be
sensitive
and
specific
°
assess
relevant
endpoints
°
include
a
dose
range
for
full
characterization
of
effects
°
be
conducted
in
accordance
with
good
laboratory
practices
(
GLP)
°
be
validated.

Two
quail
species,
the
northern
bobwhite,
Colinus
virginianus,
and
the
Japanese
quail
Coturnix
japonica,
were
considered
for
the
test
species,
because
they
are
commercially
cultured
and
available
year­
round,
are
among
the
few
avian
species
that
breed
successfully
under
laboratory
conditions,
and
unlike
most
birds,
are
able
to
produce
eggs
almost
indefinitely
under
long­
day
photoperiods.
Also,
both
species
are
accepted
models
for
assessing
acute
and
reproductive
effects
of
pesticides
and
other
chemicals
in
birds.
Although
the
Japanese
quail
is
not
indigenous
Battelle
Draft
2
April
23,
2003
to
the
United
States
and
has
undergone
extensive
domestication,
and
therefore
may
be
less
representative
of
wild
species,
it
is
recommended
here
as
the
preferred
test
species
because
of
its
small
size,
high
fecundity,
well­
characterized
reproductive
biology,
and
in
particular,
its
very
rapid
incubation
and
maturation
stages.
Pragmatically,
the
completion
of
a
mutigenerational
test
using
Japanese
quail
can
be
completed
in
less
than
half
the
time
of
a
test
using
the
bobwhite.

The
experimental
design
of
avian
reproduction
tests
has
undergone
considerable
discussion.
The
tests
can
be
designed
to
define
concentrations
of
environmental
chemicals
at
which
no
observable
adverse
effects
occur
(
NOAECs),
or
they
can
be
designed
to
develop
dose­
response
relationships
for
endpoints
of
concern
(
ECx).
Two
principal
exposure
protocols
have
also
been
considered
for
the
parental
(
P1)
generation
in
the
avian
two­
generation
reproduction
test;
a
"
proven
breeder
design,"
in
which
reproduction
is
monitored
pre­
exposure,
and
a
"
pre­
egg
laying
exposure
design,"
in
which
effects
on
maturation
are
included.
Several
first­
generation
(
F1)
exposures
have
also
been
considered,
the
two
most
likely
of
which
are
treatment
from
hatch
through
egg
laying
and
no
treatment.
Currently,
there
are
insufficient
data
to
determine
the
combination
of
exposure
protocols
for
the
P1
and
F1
generations
that
is
the
most
robust
for
documenting
changes
in
ecologically
important
fitness
endpoints,
and
that
at
the
same
time
is
most
effective
in
determining
mechanism
of
action.
A
side­
by­
side
performance
evaluation
in
prevalidation
of
the
prebreeding
and
proven­
breeder
exposure
regimens
and
the
F1
treatment
options
is
recommended
to
evaluate
the
sensitivity
and
cost­
benefit
of
these
exposure
protocols.

Because
of
the
variety
of
responses
that
test
substances
can
induce,
a
broad
set
of
reproductive
fitness
and
physiological
endpoints
was
selected
to
evaluate
reproductive
impact
and
endocrine
activity
in
an
avian
two­
generation
reproduction
toxicity
test.
Emphasis
was
given
to
endpoints
recommended
by
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
and
the
Organization
for
Economic
Cooperation
and
Development
(
Bennett,
et
al.,
2001).
Additional
endpoints
were
added
from
the
literature
review.
Fitness
endpoints
are
egg
production
and
viability,
hatching
success,
survival
of
chicks
to
14
days
of
age,
genetic
sex,
onset
of
sexual
maturation,
cloacal
gland
area,
body
weight,
and
male
copulatory
behavior.
The
physiological
endpoints
are
gross
morphology
and
histology
of
specific
organs,
levels
of
sex
hormones,
corticosterone,
and
thyroid
hormones
by
specific
methods.
Because
of
the
effects
of
handling
on
plasma
concentrations
of
many
of
these
hormones,
it
is
preferable
that
hormone
status
be
evaluated
via
the
noninvasive
sampling
of
fecal/
urate
matter.
Monitoring
of
T4/
T3
values
over
time
would
particularly
benefit
from
sampling
of
feces;
however,
methods
for
fecal
analysis
have
not
been
developed
for
these
hormones,
although
they
are
excreted
in
the
bile.
Measuring
the
sperm:
egg
interaction
is
recommended
as
an
inexpensive,
but
direct
means
of
assessing
gender­
specific
effects
on
fertility
during
egg
production
and
for
establishing
relative
fertility
of
proven­
breeder
males
prior
to
treatment.
Some
modification
of
these
endpoints
is
recommended
to
reduce
redundancy,
increase
the
cost­
effectiveness
of
the
test,
and
provide
higher­
quality
data.

To
further
optimize
two­
generation
testing
with
Japanese
quail,
several
research
areas
have
been
identified:
Battelle
Draft
3
April
23,
2003
°
Many
strains
of
Japanese
quail
have
been
developed,
largely
along
egg
production
or
body
mass
lines.
The
impact
of
strain
selection
on
the
ability
of
the
test
to
detect
endocrine
activity
and
reproductive
deficit
needs
to
be
evaluated.
°
Japanese
quail
are
recommended
over
the
northern
bobwhite
largely
for
pragmatic
reasons.
Information
that
is
not
known
at
this
time
that
could
greatly
influence
this
selection
is
the
relative
sensitivity
of
these
two
species
to
reproductive
and
endocrine
effects
of
environmental
chemicals.
°
The
potential
impact
of
natural
phytoestrogens
in
the
feed
on
the
outcome
the
test
needs
to
be
delineated
so
that
limits
of
contamination
can
be
set.
°
If
ANOVA
methods
continue
to
be
applied
to
avian
reproduction
toxicity
tests,
a
statistical
approach
for
delayed
effects
must
be
investigated.
°
Though
seemingly
a
minor
data
gap,
the
lack
of
specific
information
on
husbandry
requirements
of
the
Japanese
quail
that
will
result
in
consistent
results
in
laboratory
toxicity
tests
is
important.
°
The
number
of
replicates
(
pens)
needed
for
dose­
response
testing
is
not
known.
The
replicate
needs
for
sufficient
power
to
detect
an
effect
in
the
various
physiological
endpoints
using
ANOVA
approaches
have
not
been
established.
°
The
most
appropriate
treatment
regimen
needs
to
be
defined
for
the
P1
and
F1
generations
to
best
characterize
effects
of
potential
endocrine
disruptors
on
fitness
endpoints
with
consideration
of
endocrine
and
other
toxic
effects.
°
Baseline
data
on
the
variance,
sensitivity,
and
range
of
responses
of
many
of
the
selected
and
potential
physiological
endpoints
(
steroid
and
thyroid
hormones,
behavior,
vitellogenin,
very
low
density
lipoproteins,
aromatase)
are
needed.
°
The
source
of
the
in
ovo
concentrations
of
test
substances
has
impact
on
decisions
regarding
exposure
duration
and
interpretation
of
endpoint
responses.
However,
chemical
transfer
to
the
egg
has
been
variably
described
from
tissue
or
dietary
sources.
Elucidation
of
source
transfer
is
needed.
°
Further
study
of
estrogenic
xenobiotics
that
require
metabolic
activation
is
needed
to
determine
whether
or
not
this
occurs
in
the
embryo
and
to
understand
its
relationship
to
this
sensitive
developmental
period.

Considerations
for
a
recommended
protocol
are
discussed
in
Section
12
of
this
DRP
using
the
Japanese
Quail
as
the
test
system.
Battelle
Draft
4
April
23,
2003
2.0
INTRODUCTION
2.1
DEVELOPING
AND
IMPLEMENTING
THE
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
(
EDSP)

Chemicals
that
are
known
or
suspected
of
being
endocrine
disruptors
(
Kavlock
et
al.
1996),
also
referred
to
as
hormonally
active
agents
(
NRC
1999),
have
received
increased
attention
over
the
past
decade.
In
1996,
the
passage
of
the
two
laws,
the
Food
Quality
Protection
Act
(
FQPA)
and
Amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
mandated
the
United
States
Environmental
Protection
Agency
(
EPA)
to
screen
substances
found
in
drinking
water
sources
or
food
to
determine
whether
they
possess
estrogenic
or
other
endocrine
activity
(
Federal
Register
1998a,
1998b).
Pursuant
to
this
goal,
the
EPA
is
required
to
"
develop
a
screening
program,
using
appropriate
validated
test
systems
and
other
scientifically
relevant
information,
to
determine
whether
certain
substances
may
have
an
effect
in
humans
that
is
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen,
or
other
such
endocrine
effect..."
(
FQPA
1996).

In
21
U.
S.
C.
§
346a(
p)(
3),
the
FQPA
also
states
that
in
carrying
out
its
screening
program,
the
EPA
(
A)
shall
provide
for
the
testing
of
all
pesticide
chemicals
and
(
B)
may
provide
for
the
testing
of
any
other
substance
that
may
have
an
effect
that
is
cumulative
to
an
effect
of
a
pesticide
chemical
if
the
Administrator
determines
that
a
substantial
population
may
be
exposed
to
such
a
substance.

Additionally,
Congress
amended
the
Safe
Drinking
Water
Act
(
SDWA)
(
42
U.
S.
C.
§
300j­
17),
authorizing
the
EPA
to
provide
for
the
testing,
under
the
FFDCA
Screening
Program
.
.
.
any
other
substance
that
may
be
found
in
sources
of
drinking
water
if
the
Administrator
determines
that
a
substantial
population
may
be
exposed
to
such
substance.

Prior
to
the
passage
of
the
FQPA
and
the
SDWA,
the
EPA
initiated
several
endocrine
disruptor
investigations,
including
the
development
of
a
special
report
and
effects
assessment
(
EPA
1997a);
a
series
of
endocrine
disruptor
methods
workshops
funded
by
the
World
Wildlife
Fund,
Chemical
Manufacturers
Association
(
later
known
as
the
American
Chemistry
Council),
and
the
EPA
(
Gray
et
al.
1997;
EPA
1997b;
Ankley
et
al.
1998);
and
co­
sponsorship
(
with
the
National
Institute
of
Environmental
Health
Sciences
[
NIEHS]
and
the
Department
of
the
Interior)
of
an
independent
critical
literature
analysis
of
hormone­
active
toxicants
in
the
environment
by
the
National
Academy
of
Sciences
(
NRC
1999).

The
EPA
established
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC),
to
provide
recommendations
regarding
a
strategy
for
developing
a
testing
paradigm
for
compounds
that
may
have
activities
similar
to
naturally­
occurring
hormones.
Following
the
recommendations
made
by
EDSTAC
in
its
final
report
(
EDSTAC
1998),
the
EPA
established
the
Battelle
Draft
5
April
23,
2003
Endocrine
Disruptor
Screening
Program
(
EDSP).
The
program's
aim
is
to
develop
a
two­
tiered
approach,
e.
g.
a
combination
of
in
vitro
and
in
vivo
mammalian
and
ecotoxicological
screens
(
Tier
1)
and
a
set
of
in
vivo
tests
(
Tier
2)
for
identifying
and
characterizing
endocrine
effects
of
pesticides,
industrial
substances,
and
environmental
contaminants
(
Federal
Register
1998a,
1998b).

To
date,
the
EPA
has
implemented
the
program
on
two
fronts:
(
1)
the
development
of
the
Endocrine
Disruptor
Priority
Setting
Database,
and
the
approach
that
will
be
used
to
establish
priorities
for
screening
compounds,
and
(
2)
prevalidation
and
validation
studies
of
some
of
the
Tier
1
and
Tier
2
assays
that
are
likely
to
be
included
in
the
testing
battery.
The
Endocrine
Disruptor
Methods
Validation
Subcommittee
(
EDMVS)
has
been
set
up
to
advise
and
review
new
and
ongoing
work
in
the
validation
of
these
assays.

The
EDSP's
proposed
statement
of
policy,
including
public
comments,
was
reviewed
by
a
joint
panel
of
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA)
Scientific
Advisory
Panel
(
SAP)
and
the
EPA
Science
Advisory
Board
(
SAB)
in
May
1999.
Gray
et
al.
(
1997),
EDSTAC
(
1998),
and
the
National
Research
Council
(
NRC
1999)
concluded
that
a
tiered
approach
relying
on
a
combination
of
in
vivo
and
in
vitro
screens
for
Tier
1
was
scientifically
reasonable.
This
conclusion
was
based
upon
each
group's
assessment
of
the
current
state
of
the
science
on
the
evaluation
of
agents
affecting
the
endocrine
system.
Another
consistent
conclusion
was
the
need
to
validate
the
individual
screens
and
tests
in
the
EDSP.
Validation
and
peer
review
are
prerequisites
to
the
development
and
approval
of
test
guidelines
for
regulatory
use.
Many
of
the
documents
cited
above
and
other
EPA
EDSP­
related
information
may
be
found
at
http:\\
www.
epa.
gov/
scipoly/
oscpendo.

In
addition
to
the
EPA's
domestic
EDSP
validation
program,
a
separate
effort
to
validate
certain
screening
assays
and
tests
for
international
use
is
being
conducted
by
the
Organization
for
Economic
Cooperation
and
Development
(
OECD)
Test
Guidelines
Program.
The
EPA
actively
participates
as
a
member
of
the
OECD
test
guidelines
program
and
its
Endocrine
Disruptor
Testing
and
Assessment
Task
Force.
The
EPA
is
relying
on
the
OECD
effort
to
serve
as
the
mechanism
for
validation
of
some
of
the
components
of
its
EDSP.
Separate
domestic
and
international
activities
are
necessary
in
that
laws
and
regulatory
procedures
differ
in
various
countries.
Although
international
activities
are
distinct
from
domestic
activities,
overlapping
membership
on
various
committees
ensures
appropriate
liaison
and
communication,
eliminates
duplication
of
effort,
and
facilitates
international
harmonization.

2.2
THE
VALIDATION
PROCESS
The
EPA
(
and
EDMVS)
chose
to
follow
the
validation
process
established
by
the
Interagency
Coordinating
Committee
on
the
Validation
of
Alternative
Methods
(
ICCVAM),
of
which
the
EPA
was
a
charter
member,
for
validation
of
the
EDSP
screening
and
testing
methods.
ICCVAM
was
established
by
the
National
Institute
of
Environmental
Health
Sciences
(
NIEHS)
as
a
standing
interagency
committee
to
aid
in
the
validation,
acceptance,
and
harmonization
of
test
methods
designed
to
reduce
animal
use,
refine
procedures
involving
the
use
of
animals
so
Battelle
Draft
6
April
23,
2003
that
they
would
experience
less
stress,
and
to
replace
animal
tests
whenever
appropriate
(
ICCVAM
2000).
To
this
end,
ICCVAM
defined
a
flexible,
adaptable
framework
for
test
method
validation
that
was
applicable
to
conventional
and
alternate
methods,
and
could
be
applied
to
the
needs
of
different
agencies
and
regulatory
processes.

The
purpose
of
the
validation
is
to
establish
the
reliability
and
relevance
of
a
test
method
with
respect
to
a
specific
use.
The
process
is
science­
driven,
and
addresses
the
scientific
principles
of
objectivity
and
experimental
design
(
NIEHS
1997).
In
addition,
as
stated
in
the
ICCVAM
report,
"
A
test
is
considered
validated
when
its
performance
characteristics,
advantages,
and
limitations
have
been
adequately
determined
for
a
specific
purpose."
(
NIEHS
1997).

The
validation
process
consists
of
four
discrete
phases:
(
1)
initial
protocol
development,
(
2)
prevalidation
studies,
(
3)
validation
studies,
and
(
4)
external
scientific
peer
review.
The
initial
protocol,
developed
from
existing
information
and
experience
(
past
and
current
research),
serves
as
the
starting
point
for
initiating
the
validation
process.
Prevalidation
studies
consist
of
further
development
and
optimization
of
specific
initial
protocols
through
targeted
investigations.
Either
before
or
during
prevalidation,
a
detailed
review
paper
(
DRP)
addressing
all
critical
areas
outlined
in
Validation
and
Regulatory
Acceptance
of
Toxicological
Test
Methods
(
NIEHS
1997)
is
prepared
for
each
method
to
summarize,
explain,
and
document
decisions
regarding
the
relevant
principles,
methods,
and
techniques
recommended
for
the
initial
protocol.
Targeted
prevalidation
investigations
are
designed
to
address
questions
necessary
for
completing
an
optimized,
transferrable
protocol
suitable
for
interlaboratory
validation
studies.
Validation
studies
consist
of
comparative
interlaboratory
studies
to
establish
the
reliability
and
relevance
of
the
protocols
developed
in
the
prevalidation
stage.
Validation
requires
the
development
of
a
detailed
review
paper
to
document
what
is
known
about
the
assay
system
proposed
for
validation.

A
test
is
considered
validated
when
its
performance
characteristics,
advantages,
and
limitations
have
been
adequately
determined
for
a
specific
purpose.
The
measurement
of
a
test's
reliability
and
relevance
are
independent
stages
in
the
validation
of
a
test
method,
and
both
are
required.
Reliability
is
an
objective
measure
of
a
method's
intra­
and
interlaboratory
reproducibility.
If
the
test
is
not
sufficiently
reliable,
it
cannot
be
used
for
its
intended
purpose.
Alternatively,
if
the
test
is
not
relevant,
of
questionable
relevance
to
the
biological
effect
of
interest,
or
if
it
is
not
an
appropriate
measure
of
the
effect,
its
reliability
is
academic.
The
relevance
of
a
test
may
be
linked
to
the
mechanism
of
the
toxic
effect
it
measures
and
to
its
proposed
uses
(
NIEHS
1997).
The
studies
conducted
will
be
used
to
develop,
standardize,
and
validate
methods,
prepare
appropriate
documents
for
peer
review
of
the
methods,
and
develop
technical
guidance
and
test
guidelines
in
support
of
the
EDSP.

Following
the
validation
studies,
results
of
an
external
scientific
peer
review
of
the
study
and
the
optimized
protocols
will
be
used
to
develop
the
EPA
test
guidelines.
Battelle
Draft
7
April
23,
2003
2.3
Purpose
of
the
Review
The
purpose
of
this
detailed
review
paper
(
DRP),
prepared
as
part
of
Work
Assignment
2­
16,
is
to
define
the
basis
and
purpose
of
the
proposed
avian
two­
generation
reproductive
and
developmental
toxicity
study
for
evaluating
effects
of
potential
endocrine­
disrupting
chemicals.
The
DRP
will
summarize,
explain,
and
document
the
relevant
principles,
methods,
and
techniques;
it
will
recommend
an
initial
Tier
2
protocol
that
will
best
meet
the
needs
for
testing;
and
it
will
identify
issues
that
could
require
prevalidation
studies.

2.4
Objectives
of
the
Avian
Two­
Generation
Toxicity
Study
Tier
2
is
the
final
phase
of
the
screening
and
testing
program
and
therefore
should
provide
more
detailed
information
regarding
the
endocrine
disruption
activity
of
a
tested
chemical
or
mixture.
To
fulfill
this
purpose,
tests
are
often
longer­
term
studies
designed
to
encompass
critical
life
states
and
processes,
a
broad
range
of
doses,
and
administration
by
relevant
route
of
exposure.
In
addition,
the
effects
associated
with
endocrine
disrupting
chemicals
(
EDCs)
can
be
latent
and
not
manifested
until
later
in
life
or
may
not
be
apparent
until
reproductive
processes
occur
in
an
organism's
life
history.
Thus,
tests
for
endocrine
disruption
often
encompass
two
generations
to
address
effects
on
fertility
and
mating,
embryonic
development,
sensitive
neonatal
growth
and
development,
and
transformation
from
the
juvenile
life
state
to
sexual
maturity.
The
results
form
the
Tier
2
testing
should
be
conclusive
in
documenting
a
discernable
cause­
and­
effect
relationship
of
chemical
exposure
to
measurable
manifestation
in
the
test
organisms.

The
avian
test
protocol
that
will
be
recommended
in
this
report
(
Section
11.0)
will
be
designed
to
be
capable
of
the
following:

°
to
determine
whether
effects
are
a
primary
or
secondary
disturbance
of
endocrine
function
°
to
establish
exposure,
concentrations,
timing,
and
effects
relationships
°
to
be
sensitive
and
specific
°
to
assess
relevant
endpoints
°
to
include
a
dose
range
for
full
characterization
of
effects
to
be
conducted
in
accordance
with
good
laboratory
practices
(
GLP)
°
to
be
validated.

Although
avian
species
are
not
part
of
the
Tier
1
screening
battery,
they
are
included
in
Tier
2
and
serve
an
important
role
in
that
birds
are
fundamentally
different
from
mammals
in
the
control
of
sexual
differentiation,
and
results
from
mammalian
tests
provide
little
predictive
value
for
assessing
a
chemical's
potential
impact
on
birds.
The
following
sections
describe
the
methods
used
and
results
obtained
from
conducting
this
DRP
on
avian
multigenerational
test
methods.
Battelle
Draft
8
April
23,
2003
2.5
Methods
Used
in
This
Analysis
In
Appendix
A,
a
detailed
description
of
the
methods
employed
for
the
literature
search,
such
as
key
words,
databases,
and
results,
is
provided.
After
key
papers
were
identified,
retrieved,
and
read
for
content,
pertinent
information
was
synthesized
to
create
this
DRP.
At
the
back
of
this
report
is
a
compact
disk
read­
only
memory
(
CD
ROM)
containing
the
Reference
Manager
Database
of
all
documents
reviewed.
This
database
includes
the
reference
citation
and
abstract.

2.6
Acronyms
and
Abbreviations
Table
2­
1
lists
the
acronyms
and
abbreviations
used
in
the
DRP,
with
the
exception
of
commonly
used
units,
such
as
h
for
hour
or
L
for
liter.
Each
of
the
acronyms
and
abbreviations
also
is
introduced
at
first
use
in
the
text.

Table
2­
1.
Acronyms
and
Abbreviations
ACTH
adrenocorticotropic
hormone
ANOVA
analysis
of
variance
A&
M
(
Texas)
Agricultural
and
Mechanical
(
University)
ASTM
American
Society
for
Testing
and
Materials
apo
apolipoprotein
BSA
bovine
serum
albumin
BST
bed
nucleus
of
the
stria
terminalis,
part
of
the
quail
brain
CNS
central
nervous
system
CV
coefficient
of
variation
CyA
cyproterone
acetate
DDE
dichlorodiphenylethylene
DDT
dichlorodiphenyltricloroethane
DES
diethylstilbestrol
DEHP
diethylhexylphthalate
DHT
dihydrotestosterone
DNA
deoxyribonucleic
acid
DRP
detailed
review
paper
EB
estradiol
benzoate
EC50
median
effective
concentration
EDx
effective
dose
(
x
=
percentage
of
effect,
from
0
to
100)
EDC
endocrine­
disrupting
chemical
EDMVS
Endocrine
Disruptor
Methods
Validation
Subcommittee
EDSP
Endocrine
Disruptor
Screening
Program
EDSTAC
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
EDSTP
Endocrine
Disruptor
Screening
and
Testing
Protocols
EE2
ethinyletradiol
ELISA
enzyme­
linked
immunosorbent
assay
EPA
U.
S.
Environmental
Protection
Agency
EROD
ethoxyresorufin­
O­
deethylase
FFDCA
Federal
Food,
Drug,
and
Cosmetics
Act
FIFRA
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
FQPA
Food
Quality
Protection
Act
FSH
follicle­
stimulating
hormone
GLP
good
laboratory
practice
GnRH
gonadotropin
releasing
hormone
Battelle
Draft
9
April
23,
2003
Table
2­
1.
Acronyms
and
Abbreviations
(
Continued)

HAH
halogenated
aromatic
hydrocarbon
HCP
highly
carboxylated
porphyrine
hCG
human
chorionic
gonadotropin
HPG
hypothalmic­
pituitary­
gonadal
HPV
high
production
volume
ICCVAM
Interagency
Coordinating
Committee
on
the
Validation
of
Alternative
Methods
in
vitro
outside
the
body,
in
an
artificial
environment
in
ovo
in
the
egg
in
vivo
within
the
body
IPVL
inner
perivitelline
layer
LC50
median
lethal
concentration
LH
luteinizing
hormone
LOAEL
lowest
observable
adverse
effect
levels
MTD
minimum
tolerated
dose
NIEHS
National
Institute
of
Environmental
Health
Sciences
NOAEL
No
observable
adverse
effect
level
NRC
National
Research
Council
OC
organochlorine
OECD
Organization
for
Economic
Cooperation
and
Development
OP
organophosphorus
OPPT
Office
of
Pollution
Prevention
and
Toxics,
EPA
OPVL
outer
perivitelline
layer
PAH
polycyclic
aromatic
hydrocarbon
PCB
polychlorinated
biphenyl
PCR
polymerase
chain
reaction
P4
progesterone
PMSG
pregnant
mares'
serum
gonadotropin
POM
medial
preoptic
nucleus,
part
of
the
quail
brain
Prl
prolactin
Random­
bred
Random­
bred
lines
are
relatively
large
populations
of
birds
(>
100)
in
which
minimal
selection
of
breeding
stock
is
done
by
the
curator
(
Pisenti
et
al.
1998)
RH
relative
humidity
RIA
radioimmunoassay
RMD
Reference
Manager
Database
RNA
ribonucleic
acid
RTI
Research
Triangle
Institute
SAB
Science
Advisory
Board
SAP
Scientific
Advisory
Panel
SDS­
PAGE
sodium
dodecyl
sulfate­
polyacrylamide
electrophoresis
SDWA
Safe
Drinking
Water
Act
SHBG
sex
hormone
binding
globulin
T
testosterone
T1S
Tier
1
Screening
T3
triiodothyronine
T4
thyroxin
TCDD
tetrachlorodibenzodioxin
TSH
thyroid­
stimulating
hormone
UBC
University
of
British
Columbia
USC
United
States
Code
VTG
vitellogenin
Battelle
Draft
10
April
23,
2003
3.0
OVERVIEW
AND
SCIENTIFIC
BASIS
OF
AVIAN
TWO­
GENERATION
TESTS
Effects
of
chemicals
on
avian
reproduction,
development,
and
survival
have
been
studied
for
several
decades,
and
standardized
protocols
have
been
in
place
for
screening
potential
environmental
contaminants
for
such
effects.
Although
these
methods
have
some
relevance
to
the
discrimination
of
endocrine
disruptors
from
among
the
category
of
all
substances
that
causes
adverse
effects,
they
are
not
sufficiently
robust
to
determine
mode
of
action.
Because
endocrine
disruptors
are
categorized
on
the
bases
of
how
they
exert
adverse
effects
in
the
animal,
specific
endpoints
must
be
built
into
the
studies
to
differentiate
them
from
other
"
reproductive
or
developmental
toxicants"
(
DeFur
et
al.
1999).
Furthermore,
current
test
protocols
are
not
designed
to
determine
long­
term
effects
of
in
ovo
exposure,
which
may
be
the
most
relevant
responses
to
endocrine­
disrupting
compounds
in
birds
(
Fry
1995).
Therefore,
a
two­
generation
avian
reproduction
study
is
being
developed
to
provide
the
necessary
protocol
to
determine
endocrine
disruption
potential
and
ecologically
relevant
effects
of
environmental
chemicals.

Endocrine
disruptors
are
"
exogenous
agent[
s]
that
interfere
with
the
production,
release,
transport,
metabolism,
binding,
action,
or
elimination
of
natural
hormones
in
the
body
responsible
for
the
maintenance
of
homeostasis
and
the
regulation
of
developmental
processes"
(
Kavlock
et
al.
1996).
Such
effects
must
result
in
adverse
health
effects
in
an
organism
or
its
progeny,
to
be
ecologically
relevant
(
DeFur
1999).
It
is
important
to
note,
however,
that
endocrine
disruptors
are
defined
on
the
basis
of
their
mechanism
of
action.
Many
physiological
processes
that
are
hormonally
regulated,
including
reproduction
and
development,
may
be
affected
by
chemicals
through
other
toxicological
mechanisms.
Hoffman
(
1990)
reviewed
the
data
on
embryo
toxicity
and
teratogenicity
of
environmental
chemicals
to
bird
eggs.
Many
chemicals
have
shown
such
effects,
either
through
direct
application
or
via
maternal
transfer,
including
organochlorines
(
OCs),
polycyclic
aromatic
hydrocarbons
(
PAHs),
organophosphorus
compounds
(
OPs),
some
herbicides,
and
fungicides.
However,
there
are
multiple
causes
of
such
effects,
not
all
of
which
are
endocrine­
related.

For
example,
selenium
is
a
well­
known
avian
embryotoxicant,
causing
severe
embryonic
developmental
effects
following
in
ovo
exposure.
Such
effects
are
due
to
interference
with
glutathione
peroxidase
and
subsequent
oxidation
of
cell
membranes
(
Ohlendorf
1996).
Thus,
selenium
would
be
classified
as
a
reproductive
and
developmental
toxicant
but
not
as
an
endocrine
disruptor.
Endocrine
disruptors
need
to
interact
with
the
hormone
system,
not
simply
alter
a
process
that
is
under
normal
endocrine
control
(
DeFur
1999).

The
concept
that
environmental
chemicals
have
the
potential
to
disrupt
hormonally
regulated
processes
in
birds
is
not
new.
Such
mechanisms
have
been
studied
since
the
late
1960s,
and
it
was
first
discussed
as
a
generalized
class
of
chemicals
at
a
NIEHS
symposium,
"
Estrogens
in
the
Environment,"
held
in
Eugene,
Oregon,
in
1979.
Chemicals
such
as
OCs,
PAHs,
diethystilbesterol
(
DES),
and
others
(
e.
g.,
nonylphenols)
were
identified
as
having
estrogenic
properties
due
to
competitive
binding
with
cellular
estrogen
receptors
(
McLachlan
1997).
Potential
endocrine
effects
in
birds
exposed
to
environmental
pollutants
were
reviewed
further
by
Rattner
et
al.
(
1984),
who
introduced
the
concept
of
relative
potency
to
the
discussion
of
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environmental
relevance.
For
example,
whereas
dichlorodiphenyltrichloroethane
(
DDT)
and
its
derivatives
have
been
shown
to
bind
to
nuclear
estrogen
receptors
in
the
magnum
and
shell
gland
regions
of
the
oviduct,
their
relative
potency
is
only
1/
1000
of
that
of
the
natural
estrogen,
17$­
estradiol.
This
further
underscores
the
necessity
of
conducting
studies
in
a
dose­
response
fashion
and
correlating
measured
hormonal
changes
with
ecologically
relevant
fitness
endpoints,
such
as
successful
reproduction,
growth,
and
maturation.
Rattner
et
al.
(
1984)
also
broadened
the
discussion
to
include
the
potential
for
chemicals
such
as
the
OCs
to
result
in
thyroid
dysfunction.
The
current
definition
of
endocrine­
disrupting
chemicals
now
includes
those
that
act
or
have
the
potential
to
act
on
estrogens,
androgens,
and
thyroid
hormone
systems.

3.1
Avian
Endocrinology
"
The
endocrine
system
is
responsible
for
regulating
most
of
the
body's
essential
functions.
It
controls
reproduction
and
secondary
sex
characteristics,
molting
and
metamorphosis,
fluid
balances,
growth
rates,
salt
balance,
and
response
to
stress,
among
others.
These
functions
are
accomplished
by
a
diverse
set
of
small
organs
located
throughout
the
body
that
communicate
with
each
other
and
their
target
organs
by
releasing
small
amounts
of
very
potent
substances
known
as
hormones.
The
primary
endocrine
organs
are
the
adrenal
glands,
pituitary,
thyroid,
parathyroid,
ovaries/
testes,
and
pancreas.
The
kidney
produces
hormones
in
addition
to
filtering
the
blood.
In
mammals,
the
placenta
also
functions
as
an
important
endocrine
organ
Although
many
of
the
processes
and
hormones
involved
with
reproduction,
development,
and
homeostasis
are
well
conserved
across
species,
there
are
sufficient
differences
between
mammalian
and
avian
systems
to
warrant
the
development
of
avian­
specific
tests
(
Dawson
2000).

Control
of
the
endocrine
system
is
by
the
neurosecretory
neurons
within
the
hypothalamus.
These
receive
input
from
internal
and
external
cues
and
also
control
the
rate
at
which
the
hypothalamus
synthesizes
and
secretes
various
peptides
or
glycoproteins
known
as
releasing
hormones
(
e.
g.,
gonadotrophic
releasing
hormone
[
GnRH]).
These
hormones
then
stimulate
the
pituitary
to
synthesize
and
secrete
other
peptide
hormones
(
e.
g.,
follicle
stimulating
hormone
[
FSH];
thyroid
stimulating
hormone
[
TSH]),
which
pass
into
the
circulation
and
go
to
the
target
endocrine
glands.
Glands
such
as
the
ovaries,
testes,
and
oviduct
are
stimulated
to
secrete
the
active
steroid
sex
hormones,
including
estrogens
and
androgens.
"
Estrogen,
testosterone,
and
other
steroid
hormones
are
distributed
around
the
body
bound
to
another
protein,
the
sex
hormone­
binding
globulin
(
SHBG).
The
relative
affinity
of
the
hormones
to
bind
to
SHBG
versus
to
cell
receptors
influences
the
potency
of
hormones
within
and
across
species.
Interaction
of
chemicals
with
the
SHBG­
hormone
binding
may
be
a
cause
of
species­
specific
differences
in
hormone­
mediated
effects
of
xenobiotics.
There
are
a
number
of
interactions
among
the
various
endocrine
axes
and
between
the
endocrine
and
nervous
systems.
In
the
case
of
the
reproductive,
hypothalamic­
pituitary­
gonadal
(
HPG)
axis,
steroid
hormones
produced
by
the
gonads
feed
back
to
the
hypothalamus
and
pituitary
gland
to
alter
HPG
axis
function.
This
is
termed
a
negative
feedback
loop
and
is
a
mechanism
by
which
many
of
the
endocrine
axes
are
regulated.
Blood
calcium
levels
are
regulated
in
a
manner
similar
by
the
parathyroid
gland.
Estrogen
interacts
with
parathormone,
which
is
produced
by
the
parathyroid
gland,
regulating
calcium
levels
by
causing
an
increased
deposition
of
calcium
in
the
long
bones.
Hormones
Battelle
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produced
by
the
kidney
also
participate
in
the
regulation
of
calcium
as
well
as
in
the
maintenance
of
blood
pressure
and
proper
balance
in
the
body
of
ions
such
as
potassium
and
sodium.
The
adrenal
glands,
located
just
above
the
kidneys,
produce
corticosteroids,
which
are
important
under
conditions
of
chronic
stress"
(
Bennett,
et
al.,
2001).

"
The
corticosteroids
also
influence
the
immune
system
and
estrogen
production.
Less
well
known
is
their
production
of
hormones
that
interact
with
the
kidney
to
maintain
ionic
balance"
(
Bennett,
et
al.,
2001).

"
In
birds,
sex
steroid
hormones
are
responsible
for
a
variety
of
reproductive
functions.
These
include
development
of
secondary
sexual
characteristics
such
as
plumage
coloration
and
mating
songs,
phenotypic
gender
determination
including
oviduct
development
and
maturation,
shell
gland
function,
oviduct
development
and
maturation,
and
cornification
of
the
cloacal
epithelium.
Progesterone
causes
development
of
the
brood
patch,
which
is
further
vascularized
under
the
influence
of
estrogen.
Progesterone
also
is
responsible
for
broody
behavior,
such
as
nest
attentiveness
during
incubation
and
during
posthatch
in
altricial
species.
Estrogen
also
regulates
calcium
deposition
and
release
from
bones,
as
well
as
brain
development.
In
birds
and
other
egg­
laying
animals,
estrogen
is
responsible
for
the
stimulation
of
production
of
vitellogenin
(
VTG)
by
the
liver.
VTG
is
the
precursor
for
the
primary
proteins
that
are
incorporated
into
the
egg
for
nourishment
of
the
embryo
during
prehatch
development"
(
Bennett,
et
al.,
2001).

The
steroid
sex
hormones
are
synthesized
from
cholesterol
through
the
hydrolysis
of
side
chains
by
cytochrome
P450
enzymes
in
families
11,
17,
19,
21,
and
27,
to
form
progesterone.
These
enzymes
are
not
inducible
by
xenobiotics,
but
they
can
be
inhibited.
Progesterone
is
further
modified
to
form
testosterone
and
its
active
derivative,
androstenedione.
Another
P450
enzyme,
aromatase,
takes
these
androgens
to
estrogens,
estradiol
and
estrone,
respectively
(
Fairbrother
2000).
Cytochrome
P450
enzymes
also
play
a
role
in
the
degradative
metabolism
of
the
steroid
hormones.
Cytochrome
P450
enzymes
in
families
1
through
4
metabolize
natural
steroids
to
more
polar
forms
for
excretion
in
the
urine
and
are
inducible
by
a
variety
of
chemicals
(
Dawson
2000).

"
The
thyroid
gland
is
another
major
endocrine
organ.
It
is
a
butterfly­
shaped
gland
located
at
the
base
of
the
neck
of
most
vertebrates
and
depends
upon
an
adequate
supply
of
iodine
for
normal
functioning"
(
Bennett,
et
al.,
2001).
Thyroid
hormones
are
peptide
hormones
and
are
synthesized
in
the
follicles
of
the
thyroid
gland;
this
synthesis
produces
a
large
globular
glycoprotein
called
thyroglobulin.
This
contains
iodinated
tyrosine
residues,
which
are
coupled
to
form
iodinated
thyronine,
the
major
form
of
which
contains
four
iodides
and
is
known
as
thyroxin,
also
known
as
tetraiodothyronine
(
T4).
T4
is
the
major
circulating
hormone,
but
is
not
the
active
form.
T4
is
converted
into
tri­
iodothyronine
(
T3),
which
is
the
physiologically
active
thyroid
hormone.
Conversion
occurs
in
the
liver
and
is
controlled
by
the
enzyme
Type
I
deiodinase.
Only
about
30%
to
40%
of
the
circulating
T4
is
converted
to
T3;
another
15%
to
20%
is
deaminated
to
form
tetraiodothyroacetate,
which
is
rapidly
excreted.
Other
glucuronides
also
are
formed
and
excreted.
Battelle
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23,
2003
"
Production
of
thyroid
hormone
is
regulated
very
closely
by
the
hypothalamus
and
pituitary,
through
a
negative
feedback
cycle.
The
corticosteroids,
testosterone,
and
estrogen
can
depress
production
of
thyroid
hormone,
and
some
of
the
soluble
mediators
of
immune
system
function
(
interferon
and
interleukins)
have
slight
effects
as
well.
T3
is
responsible
for
controlling
the
metabolic
rate
of
an
animal,
regulating
body
temperature,
interacting
with
growth
hormone
to
determine
body
size,
and
playing
an
integral
role
in
molting,
metamorphosis,
and
smoltification
of
various
species
of
invertebrates,
amphibians,
and
fish"
(
Bennett,
et
al.,
2001).

3.2
Differences
in
Avian
and
Mammalian
Endocrine
Systems
"
There
are
many
similarities
among
birds,
mammals,
and
other
vertebrate
classes
regarding
endocrine
systems.
These
vertebrate
classes
share
similar
hormones
and
hormone
receptors,
and
fundamental
feedback
mechanisms
are
similar.
However,
important
differences
between
birds
and
mammals
and
other
vertebrate
classes
do
exist.
Birds
offer
unique
morphological,
physiological,
and
behavioral
adaptations
that
are
not
widely
found
in
other
vertebrates,
including
flight,
which
requires
adaptations
for
high
metabolic
rate
and
reduced
body
mass,
oviparity
with
hard­
shelled
eggs,
a
different
physiological
basis
for
gender
development,
and
complex
mate
attraction
behaviors,
especially
in
songbirds"
(
Bennett
et
al.
2001).
These
differences
were
reviewed
by
Fry
(
1995)
and
Fairbrother
(
2000),
and
they
are
summarized
here.

Sex
determination
and
control
of
differentiation
is
linked
to
the
heterogametic
sex.
In
mammals,
this
is
the
male
(
XY);
in
birds,
it
is
the
female
(
ZW).
The
so­
called
default
sex,
which
is
the
phenotype
to
which
the
embryo
will
develop
in
the
absence
of
sex­
specific
hormones,
is
the
homogametic
sex.
Mammals
will
develop
as
female
(
XX)
and
birds
as
male
(
ZZ)
if
no
sex
differentiation
hormones
are
present.
Therefore,
birds
require
that
estradiol
be
synthesized
to
cause
differentiation
of
the
gonad
into
an
ovary.
Lack
of
estrogen,
regardless
of
androgen
levels,
will
result
in
development
of
phenotypic
males.
In
mammals,
the
reverse
is
true:
embryos
will
develop
into
phenotypic
females
unless
sufficient
levels
of
androgens
are
present
to
induce
gonadal
differentiation
into
testicular
tissue.
In
mammals,
normal
maternal
estrogens
are
sequestered
by
fetoprotein,
which
has
a
high
estrogen
binding
affinity
and
protects
fetal
tissues
from
estrogen
exposure.
Exogenous
estrogenic
substances
also
may
bind
to
this
protein,
reducing
the
potential
for
effect
on
the
developing
embryo.
Birds
do
not
have
this
protein,
and
embryos
are
exposed
in
ovo
to
the
same
level
of
estrogen
as
was
in
maternal
circulation
during
egg
formation.

Because
of
the
differences
in
steroid
hormonal
control
of
sexual
differentiation,
xenobiotic
estrogens
have
different
effects
in
birds
and
mammals
during
embryonic
development.
In
male
birds,
excess
estrogens
will
stimulate
the
primordial
germ
cells
to
become
localized
in
the
cortex
of
the
gonad
as
well
as
in
the
more
normal
location
in
the
medulla
in
a
dose­
dependent
manner.
These
cortical
cells
differentiate
into
primordial
follicles,
and
the
gonad
begins
to
resemble
an
ovary.
The
seminiferous
tubules
are
retained,
but
the
number
of
primordial
germ
cells
will
be
lower
than
normal,
resulting
in
low
to
no
spermatogenesis.
Genetic
males
also
may
develop
an
oviduct.
Estrogenization
of
female
birds
does
not
change
the
ovary,
but
does
change
the
oviduct
and
causes
retention
of
the
right
oviduct,
which
in
birds
normally
regresses
prior
to
maturation.
Battelle
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Some
of
the
OC
pesticides
and
other
xenobiotics
need
to
be
metabolized
into
active
products
before
the
estrogenizing
effects
are
expressed.
Most
of
the
metabolic
products
of
OCs
are
hydroxylated
forms
that
are
water­
soluble
and
readily
excreted
in
the
urine/
urates.
However,
in
bird
embryos,
the
metabolized
products
cannot
be
excreted
(
i.
e.,
they
remain
in
the
egg),
and
so
the
embryo
can
be
exposed
throughout
the
length
of
gestation.
In
mammals,
maternal
excretion
can
reduce
the
effective
dose
and/
or
completely
eliminate
the
product
before
the
end
of
gestation.
Because
these
products
have
100
to
1000
times
less
potency
or
binding
efficiency
than
natural
estrogens,
direct
reproductive
effects
on
adults
are
expected
to
be
negligible.
However,
avian
embryos
are
at
significant
risk
because
of
retention
in
the
egg
and
estrogen­
dependency
of
sex
expression.

3.3
Avian
Two­
Generation
Test
The
proposed
avian
two­
generation
reproductive
study
for
determination
of
toxicants
with
endocrine­
disrupting
effects
is
based
on
a
premise
similar
to
that
of
the
mammalian
study
(
OECD
1999).
The
test
must
be
sufficiently
robust
to
document
changes
in
fitness
endpoints,
such
as
reproductive
output,
developmental
adequacy,
and
appropriate
behaviors,
but
equally
specific
to
determine
mechanism
of
action.
It
is
essential
that
a
two­
generation
test
be
able
to
assess
the
impact
of
endocrine­
disrupting
chemicals
on
endocrine­
mediated
processes
as
systems
organize
during
embryonic
development
and
as
they
are
activated
in
adult
birds.
There
are
four
critical
life
stages
of
birds
during
which
endocrine­
mediated
processes
take
place
and
that
therefore
could
be
sensitive
to
endocrine
disruption:
1)
in
ovo,
2)
offspring
(
F1)
generation
chick
growth,
3)
parental
(
P1)
generation
and
F1
sexual
maturation,
and
4)
P1
and
F1
egg­
laying.
Therefore,
the
study
must
include
two
egg­
laying
cycles
to
assess
effects
on
ecologically
relevant
fitness
endpoints
of
endocrine
dysfunction
at
each
of
these
stages.

In
brief,
the
test
is
designed
to
expose
birds
to
environmental
chemicals
suspected
as
having
endocrine­
disrupting
effects
from
examination
of
structure­
activity
relationships
or
test
data
from
other
species
or
in
vitro
studies.
The
P1generation
is
exposed,
and
reproductive
endpoints
such
as
egg
production
are
measured,
along
with
appropriate
measures
of
estrogen,
testosterone,
and
thyroid
hormone
synthesis,
metabolism,
and
activity.
The
F1
generation
is
hatched
and
evaluated
to
2
weeks
of
age,
at
which
time
a
subset
is
selected
for
pairing
and
further
evaluation
of
reproduction,
development,
and
endocrine
function.
This
generation
may
be
exposed
to
the
chemical
throughout
its
lifetime,
or
a
subset
may
be
exposed
while
another
cohort
is
not.
The
F2
generation
also
is
hatched
and
observed
to
2
weeks
of
age
but
is
not
exposed
to
the
chemical.
Test
species
will
be
either
Japanese
quail
(
Coturnix
japonica)
or
bobwhite
quail
(
Colinus
virginianus)
because
of
their
small
size,
short
time
to
sexual
maturation,
ease
of
handling,
and
an
existing
large
database
of
information
regarding
reproductive
effects
of
environmental
contaminants.

The
identification
of
endocrine­
mediated
effects
on
avian
reproduction
can
occur
at
the
test­
individual
or
test­
population
level
of
analysis.
This
needs
to
be
considered
in
determining
the
ultimate
objective
of
the
test.
If
the
emphasis
in
a
two­
generation
study
is
to
determine
the
impact
of
a
potential
endocrine
disruptor
at
the
test­
population
level,
then
an
integrative
endpoint
Battelle
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15
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2003
that
expresses
the
outcome
of
the
study
in
terms
of
effect
on
overall
productivity
of
a
population
must
be
identified.
This
type
of
study
requires
determination
of
dose­
response
curves
for
the
integrative
endpoint.
Other,
specific
endocrine­
related
endpoints
would
then
be
evaluated
alongside
the
primary
productivity
endpoint
to
identify
the
chemical
as
an
endocrine
disruptor
rather
than
simply
a
reproductive
toxicant,
and
to
identify
sensitive
life
stages.
Endocrine­
specific
endpoints
also
may
identify
exposure
levels
at
which
endocrine­
mediated
effects
occur
in
individual
birds
but
not
in
the
treatment
group
as
a
whole,
or
cause
measurable
hormonal
changes
without
resulting
in
gross
physiological
differences.
For
example,
because
only
30%
to
40%
of
the
naturally
circulating
levels
of
T4
is
converted
into
the
active
T3
form,
a
change
in
production
of
T4
may
not
result
in
significant
effects
to
T3
levels.
Thus,
incorporation
of
sensitive
measures
of
endocrine­
related
endpoints
will
allow
a
hazard
assessment
 
that
is,
a
determination
of
whether
chemicals
are
potentially
endocrine­
disrupting
 
without
resulting
in
ecologically
relevant
risk
estimates
(
Bennett
et
al.
2001).

Treatment
of
the
P1
generation
can
begin
either
prior
to
sexual
maturation
or
after
the
onset
of
egg­
laying.
The
advantages
of
starting
dietary
treatment
prior
to
sexual
maturation
are
".
.
.
the
ability
to
measure
changes
in
the
onset
of
laying
and
effects
resulting
from
inhibition
or
delay
of
gonadal
development.
The
disadvantages
are
that
birds
that
are
incompatible
or
infertile
for
reasons
other
than
the
test
substance
cannot
be
removed,
which
may
reduce
the
statistical
power
of
the
test
and
the
ability
to
detect
treatment
effects
if
they
exist.
The
advantages
of
starting
treatment
during
egg­
laying
include
the
ability
to
remove
nonlaying
and
infertile
pairs
prior
to
the
initiation
of
treatment
and
the
option
to
use
pretreatment
measurements
as
covariates
in
statistical
analyses
to
remove
nontreatment
sources
of
variation.
Starting
treatment
during
egg­
laying
also
provides
information
on
rapidity
with
which
reproductive
effects
can
be
observed,
which
can
be
useful
in
risk
assessments
of
test
substances
that
degrade
rapidly
in
the
environment.
Because
treatment
effects
on
reproductive
endpoints
may
increase
during
the
course
of
the
treatment
period,
statistical
analyses
need
to
be
sensitive
to
the
potential
temporal
patterns
in
the
endpoint
measurements"
(
Bennett,
et
al.,
2001).
Disadvantages
include
the
inability
to
measure
changes
in
the
onset
of
laying
and
effects
resulting
from
inhibition
or
delay
of
gonadal
development.
These
two
options
are
discussed
in
greater
detail
in
Section
5.1.1.

Treatment
of
the
F1
chicks
ensures
that
endocrine­
mediated
effects
occurring
during
growth
and
development
of
chicks
as
a
result
of
direct
exposure
as
well
as
from
in
ovo
exposure
will
be
assessed.
Treatment
of
the
F1
chicks
also
is
considered
to
more
closely
represent
a
continuous­
exposure
scenario
(
Bennett,
et
al.,
2001).
"
However,
analysis
and
interpretation
of
the
results
will
have
to
consider
how
effects
occurring
at
various
life
stages
may
be
confounded"
(
Bennett,
et
al.,
2001).
Additionally,
effects
from
such
full­
life­
cycle
exposure
scenarios
need
to
be
interpreted
with
caution
for
those
test
substances
that
degrade
rapidly
in
the
environment
that
could
be
present
at
only
certain
life
stages.
"
Ending
treatment
of
the
F1
generation
prior
to
egg­
laying
removes
some
of
the
potentially
confounding
effects
of
endocrine­
mediated
effects
on
F1
reproductive
potential
occurring
from
in
ovo
and/
or
early­
life
exposures.
It
can
be
argued
that
the
effects
of
exposure
during
egg­
laying
can
be
observed
in
the
P1
generation
and
may
not
need
to
be
repeated
in
the
F1
generation.
However,
by
ending
the
treatment
period
just
prior
to
egg­
laying,
there
may
be
a
temporal
pattern
of
effects
observed
in
the
production
of
the
F2
Battelle
Draft
16
April
23,
2003
generation,
depending
on
the
clearance
rate
of
the
test
substance
and
related
deposition
rate
into
eggs,
and
the
rate
of
recovery
of
the
F1
birds
from
any
residual
toxic
effects"
(
Bennett,
et
al.,
2001).
Several
exposure
options
have
been
proposed
to
cover
these
various
arguments.
They
are
discussed
in
more
detail
in
Section
5.1.2.

The
experimental
design
of
avian
reproduction
tests
has
undergone
considerable
discussion.
The
tests
can
be
designed
to
define
concentrations
of
environmental
chemicals
at
which
no
observable
adverse
effects
occur,
or
they
can
be
designed
to
develop
dose­
response
relationships
for
endpoints
of
concern.
Determination
of
no
observable
adverse
effects
levels
(
NOAELs)
requires
use
of
analysis
of
variance
(
ANOVA)
designs,
whereas
development
of
dose­
response
curves
uses
regression
techniques
(
Bennett
et
al.
2001).
The
issue
of
appropriate
statistical
design
is
examined
in
further
detail
in
Section
5.4.2.

4.0
CANDIDATE
TEST
SPECIES
Two
quail
species,
the
northern
bobwhite
and
the
Japanese
quail,
are
the
most
likely
candidates
for
use
in
a
two­
generation
reproductive
screening
assay
and
are
the
focus
of
this
review.
Because
of
their
terrestrial
habit,
both
species
are
considered
to
be
representative
of
terrestrial
birds
and
are
accepted
models
for
assessing
both
acute
and
reproductive
effects
of
pesticides
and
other
chemicals
in
wild
birds
(
Office
of
Pollution
Prevention
and
Toxics
[
OPPT],
EPA,
OPPT
Guideline
850.230
[
Federal
Register
1978];
OECD
Guideline
206
[
OECD
1984]).
Although
both
species,
but
particularly
the
Japanese
quail,
have
undergone
domestication
and
therefore
may
be
less
representative
of
wild
birds,
they
are
used
in
reproductive
assays,
because
few
wild
species
adapt
well
to
laboratory
conditions
and
breed
successfully
in
the
laboratory.
Also,
unlike
most
birds,
the
breeding
cycle
of
quail
is
not
restricted
with
a
photorefractory
period,
so
they
are
able
to
produce
eggs
almost
indefinitely
under
photoperiods
longer
than
about
12
h.
In
addition,
as
precocial
species,
they
are
likely
to
be
more
sensitive
to
changes
in
steroid
concentrations
during
all
life
stages.

4.1
Japanese
Quail
(
Coturnix
japonica)

This
Old
World
quail
has
been
intensively
domesticated
for
more
than
800
years
in
Japan.
Since
the
beginning
of
the
twentieth
century,
breeding
stock
has
been
selected
for
body
size
and
egg
production
throughout
the
world
(
Cooper
1976).
Use
of
Japanese
quail
as
a
laboratory
animal
began
in
the
late
1950s,
and
because
of
its
adaptability
to
battery
breeding
cages,
small
size,
and
high
fecundity,
it
has
been
used
extensively
in
research.
As
a
result,
numerous
strains
of
Japanese
quail
have
been
developed
and
conserved
in
government
and
academic
institutions
and
by
commercial
suppliers.
Among
these
strains,
body
size
characteristics,
rates
of
sexual
maturation,
and
reproductive
capabilities
can
differ
significantly.
The
Japanese
quail
has
been
used
extensively
in
reproductive
toxicity
testing
in
the
European
community
and
to
a
lesser
extent
in
the
United
States.
Some
testing
guidelines
include
detailed
information
on
their
laboratory
husbandry
(
OECD
Guideline
206;
OPPT
Guideline
850.2300;
ASTM
Method
E1062­
86).
Battelle
Draft
17
April
23,
2003
4.1.1
Natural
History
The
Japanese
quail,
once
classified
as
a
race
of
the
common
quail
(
Coturnix
coturnix),
is
now
considered
to
form
a
superspecies
with
C.
coturnix
and
possibly
also
with
C.
pectoralis
(
Cheng
and
Kimura
1990).
It
belongs
to
the
largest
family
(
Phasianidae)
of
the
galliformes,
and
based
on
deoxyribonucleic
acid
(
DNA)­
DNA
hybridization
evidence
is
only
distantly
related
to
New
World
quail,
such
as
the
northern
bobwhite
(
Hoyo
et
al.
1994).
The
Japanese
quail
is
a
migratory
species
widely
distributed
throughout
eastern
Asia.
Feral
populations
of
Japanese
quail
have
been
established
in
the
Hawaiian
Islands
and
in
Great
Britain,
but
three
attempts
to
introduce
the
species
as
a
game
bird
into
the
United
States
have
been
unsuccessful
(
Cooper
1976).
Little
habitat
information
is
available
for
the
Japanese
quail;
however,
this
ground­
dwelling
species
has
been
observed
using
a
wide
range
of
open
habitats,
including
cultivated
areas.
It
feeds
on
a
variety
of
plant
materials
and
terrestrial
invertebrates
and
nests
in
grassland
areas.
Clutch
sizes
vary
from
5
to
8
eggs
in
Japan
to
9
to
10
eggs
in
Russia.
Incubation
is
typically
18
days
in
the
wild
(
Hoyo
et
al.
1994).
Siblings
form
coveys
through
their
first
winter
and
disperse
the
following
spring.
In
the
wild,
their
average
body
weight
is
about
90
g
(
Hoyo
et
al.
1994).

4.1.2
Availability,
Culture,
Handling
Several
academic
and
governmental
institutions
in
the
United
States
and
Canada
maintain
random­
bred
stocks
of
Japanese
quail
(
Pisenti
et
al.
1999).
The
largest
quail
collection
in
North
America
is
maintained
at
the
Quail
Genetic
Stock
Center
at
the
University
of
British
Columbia
(
UBC),
Vancouver,
British
Columbia,
through
support
by
the
Natural
Sciences
and
Engineering
Research
Council
of
Canada.
Among
its
conserved
strains
of
Japanese
quail
are
several
outbred
stocks
including
a
wild
type
from
Japan.
In
the
United
States,
some
of
the
oldest
established
quail
lines
for
research
are
at
the
Universities
of
California,
Georgia,
and
Maryland
(
Table
4­
1).
In
addition,
some
commercial
sources
maintain
large
colonies
of
Japanese
quail
to
supply
research
and
toxicity
testing
organizations.
Most
of
the
stocks
available
in
North
America
and
Japan
are
derived
from
stocks
originally
selected
for
egg
production
and
are
smaller,
faster­
maturing,
less­
docile
strains
than
those
commonly
used
in
the
European
community
for
reproductive
toxicity
testing.
However,
as
the
quail
meat
industry
grows,
more
strains
of
the
higher­
body
weight
birds
are
being
developed
in
the
United
States.
As
addressed
in
Section
4.1.3,
the
growth
and
reproductive
differences
between
the
strains
can
be
substantial.

The
care
and
handling
of
Japanese
quail
for
laboratory
use
have
been
well
documented.
(
NRC
1969;
Cooper
1976;
CCAC
1984;
Ottinger
and
Rattner
1999),
and
testing
guidelines
include
detailed
information
on
their
husbandry
for
reproductive
toxicity
tests
(
OPPT
Guideline
850.230;
OECD
206).
Coturnix
grow
very
rapidly;
females
reach
sexual
maturity
in
about
6
weeks,
depending
on
strain.
Day­
old
quail
weigh
about
7
g,
and
at
maturity,
they
weigh
between
120
g
to
more
than
200
g.
As
a
consequence,
provision
of
adequate
space
per
bird
during
this
short
growth
period
is
crucial.
During
the
growth
period,
temperature
requirements
that
are
38
º
C
at
hatch
decrease
by
about
3
º
C
per
week
until
the
young
reach
4
weeks
of
age.
Young
are
very
susceptible
to
drafts
during
this
period.
At
4
weeks
of
age,
they
are
fully
feathered
and
often
are
Battelle
Draft
18
April
23,
2003
moved
to
breeder
cages.
Pairing
birds
before
sexual
maturity
aids
in
reducing
aggressive
behavior
between
pairs
(
NRC
1969).
Early
pairing
can
be
accomplished,
because
gender
is
discernable
by
plumage
color
and
pattern
as
early
as
3
weeks
of
age.
The
cloacal
gland,
a
bulbous
structure
located
at
the
upper
edge
of
the
cloaca
in
males,
also
can
be
used
to
discriminate
between
sexes,
but
it
is
affected
by
photoperiod
(
Sachs
1967).
Japanese
quail
are
very
hardy
once
the
brooding
stage
is
past.

Wild­
type
hens
and
hens
from
strains
selected
for
egg
production
will
lay
about
1
egg
per
day
and
will
produce
about
300
eggs
per
year.
Heavier
strains
lay
fewer
eggs
(
Table
4­
2).
The
average
weight
of
eggs
laid
by
mature
Japanese
quail
is
about
10
g.
However,
older
birds
usually
produce
larger
eggs,
larger
embryos,
and
larger
chicks
(
Cooper
1976).
A
mating
ratio
of
one
male
to
one
female
results
in
the
highest
fertility
rate.
Duration
of
fertility
after
removal
of
the
male
is
about
6
days.
After
remating
the
pair,
fertile
eggs
are
produced
on
the
third
day
(
Woodard
and
Abplanalp
1967).
Full
egg
production
is
reached
about
2
to
3
weeks
after
egg­
laying
begins.
Optimal
reproduction
typically
spans
5
to
6
months
(
CCAC
1993);
maximum
hatchability
occurs
when
parents
are
between
8
and
20
weeks
of
age
(
CCAC
1984).
Fertility
in
Japanese
quail
progressively
decreases
after
about
56
weeks
of
age
under
stimulatory
photoperiod
(
16L:
8
D).
By
this
time,
courtship
and
mating
behavior
are
greatly
reduced
and
plasma
testosterone
levels
also
are
decreased
(
Ottinger
et
al.
1983).
Also,
eggshells
of
older
birds
are
usually
thinner
and
result
in
a
lower
hatch
(
Cooper
1976).

Table
4­
1.
Stocks
of
Random­
Bred
or
Wild
Type
Japanese
Quail
Maintained
in
North
America
and
Europe(
a)

Stock
Name
and
Description
History
of
Origin
Number
of
Birds
Curator
Quail
Random­
bred,
Arkansas
RBS
random­
bred
control
from
Eastern
Shore
random­
bred
Acquired
from
Eastern
Shore
as
random­
bred
control,
1990
36M/
36F
Anthony
Athens
control
quail
random­
bred
control
w/
white
eggshell
mutation
in
gene
pool,
propagated
by
random­
pair
matings
Kept
as
closed
flock
since
1963
120M/
120F
Burke
Louisiana
random­
bred
quail
unselected,
random­
bred
population,
some
color
mutations
(
tuxedo,
redhead,
white
egg)
Kept
at
Louisiana
State
as
closed
flock
for
>
20
years
60M/
120F
Satterlee
Ohio
R1
propagated
using
36
pairs/
generation
Derived
from
cross
of
Athens
random­
bred,
Athens
white
egg,
and
Wisconsin
stock;
closed
flock
38
generations
36M/
36F
Nestor
UBC
A
random­
bred
flock
Derived
from
cross
of
UCD
randombred
quail
and
quail
stock
from
Korea;
closed
flock
70
generations
48M/
96F
Cheng
UBC
B
exceptionally
nervous
random­
bred;
spade
homozygotes
have
defective
feathers
Acquired
from
U
Alberta
1977,
combined
with
UBC
SP
(
spade
mutation,
affects
feathers)
by
1998
34M/
48F
Cheng
Stock
Name
and
Description
History
of
Origin
Number
of
Birds
Curator
Battelle
Draft
19
April
23,
2003
UBC
M
rough­
textured
homozygotes
have
feathers
that
appear
matted
and
rough;
females
produce
fewer
viable
embryos
(
RT
mutation
not
yet
reported
in
literature);
have
extended
brown
allele;
slightly
heavier
than
UBC
A
UBC
M
from
commercial
(
Marsh
Farms)
strain
in
1975;
combined
with
UBC
RT
in
1989
24M/
48F
Cheng
UBC
N
very
docile
random­
bred
Acquired
from
U
Nagoya
(
Japan),
1988
24M/
48F
Cheng
UBC
NC
random­
bred,
sensitive
to
changes
in
photoperiod
Acquired
from
NCSU
random­
bred
quail,
1990
34M/
48F
Cheng
UBC
S
Acquired
from
U
Saskatchewan,
1983
24M/
48F
Cheng
UBC
WILD
Foundation
stock
was
12
feral
birds
caught
in
Hawaii,
1985
48M/
96F
Cheng
UCD
Random­
bred
quail
wild­
type
feather
color
pattern,
unselected,
randomly
grouped
in
colony
cages
(
2M/
4F);
reproduce
every
6
to
8
months
Derived
from
stock
imported
from
Japan
and
Taiwan
(
1950s
and
1972)
200+
Wilson
U
Maryland
random­
bred
quail
Acquired
from
U
Wisconsin/
US
Department
of
Agriculture,
1970s;
kept
as
closed
flock
for
~
20
years
100
Ottinger
UNL
Wild­
type
Coturnix
Acquired
from
U
Georgia­
Athens
30­
60
pairs
Beck
Purdue
Coturnix
KGB
 
selected
behavioral
trait
selected
18
generations
for
nonaggressive
behavior,
starting
1988
Derived
from
Athens
control
quail
(
U
Georgia­
Athens)
10,000
birds
Muir
Arkansas
H10
 
selected
growth
trait
selected
18
generations
for
high
10­
day
body
weight
Derived
from
Arkansas
RBC
36M/
36F
Anthony
Arkansas
H17
 
selected
growth
trait
selected
18
generations
for
high
17­
day
body
weight
Derived
from
Arkansas
RBC
36M/
36F
Anthony
Arkansas
H28
 
selected
growth
trait
selected
18
generations
for
high
28­
day
body
weight
Derived
from
Arkansas
RBC
36M/
36F
Anthony
Arkansas
H40
 
selected
growth
trait
selected
18
generations
for
high
40­
day
body
weight
Derived
from
Arkansas
RBC
36M/
36F
Anthony
Arkansas
HL
 
selected
growth
trait
selected
for
high
early
body
weight
gain
(
10­
17
days),
low
late
body
weight
gain
(
17­
28
days)
36M/
36F
Anthony
Arkansas
LH
­
selected
growth
trait
selected
for
low
early
body
weight
gain
(
10­
17
days),
high
late
body
weight
gain
(
17­
28
days)
36M/
36F
Anthony
Athens
52
high
body
weight
selected
Derived
from
cross
Athens
51
and
53
(
both
selected
38
generations
for
high
4­
week
body
weight)
36M/
36F
Burke
Stock
Name
and
Description
History
of
Origin
Number
of
Birds
Curator
Battelle
Draft
20
April
23,
2003
Athens
54
low
body
weight
selected
Derived
from
cross
of
two
stocks,
both
selected
38
generations
for
low
4­
week
body
weight
36M/
36F
Burke
Athens
56
 
selected
growth
trait
Intermediate
body
weight
stock,
cross
of
longterm
selected
high
and
low
body
weight
stocks
Derived
from
cross
of
Athens
P­,
T­,
and
S­
lines
36M/
36F
Burke
Athens
P­
line
selected
100
generations
for
high
4­
week
body
weight,
28%
protein
diet;
at
70
generations,
adult
size
was
>
150%
above
that
of
controlled
standard
population
Derived
from
Athens
control
quail
60M/
60F
Burke
Athens
T­
line
selected
100
generations
for
high
4­
week
body
weight,
low­
protein,
thioruacil
stress
diet;
resists
growth
depression
on
diets
with
up
to
0.2%
thiouracil
Derived
from
Athens
control
quail
60M/
60F
Burke
Ohio
HW
inbreeding
coefficient
(
F)
=
0.417;
selected
30
generations
generations
for
increased
4­
week
body
weight
Derived
from
Ohio
R1
48M/
48F
Nestor
Ohio
HW­
HP
inbreeding
coefficient
(
F)=
0.375;
selected
for
male
increased
4­
week
body
weight,
for
female
increased
plasma
phosphorus
(
indicator
of
yolk
precursors)
Derived
from
Ohio
HW
36M/
36F
Nestor
Ohio
HW­
LP
inbreeding
coefficient
(
F)=
0.357;
selected
for
male
increased
4­
week
body
weight,
for
female
decreased
plasma
phosphorus
2
weeks
after
start
of
lay
Derived
from
Ohio
HW
36M/
36F
Nestor
Ohio
LW
inbreeding
coefficient
(
F)=
0.357;
selected
30
generations
for
decreased
4­
week
body
weight
Derived
from
Ohio
R1
48M/
48F
Nestor
UBC
G­
QM
selected
for
average
female
body
weight
280g
at
6
weeks
UBC
G
derived
from
a
commercial
strain
(
Marsh
Farms)
combined
with
UBC
QM
in
1992
24M/
48F
Cheng
UBC
QF
selected
for
average
female
body
weight
280g
at
6
weeks
Acquired
from
Deschambault
in
1990
48M/
96F
Cheng
UBC
QM
selected
for
average
male
body
weight
280g
at
6
weeks
Acquired
from
Deschambault
in
1990,
combined
with
UBC
G
in
1992
see
UBC
G­
QM
Cheng
Bobwhite
NIU
bobwhites,
blood
type
 
gene
pool
various
erythrocyte
alloantigens
in
quail
Acquired
from
Mississippi
State
U
in
1992
50+
Briles
a)
Adapted
from
data
in
Pisenti
et
al.
(
1999).
Battelle
Draft
21
April
23,
2003
Table
4­
2.
Body
Weight
and
Reproductive
Parameters
of
Egg­
Producing
and
Meat
Production
Strains
of
Japanese
Quail
Parameter
UBC
Wild
Type
Northwest
Game
Birds
UBC­
QO
Quail
Internt
Georgia,
USA
Texas
A&
M
Body
weight
(
g)
Male
Female
144
170
232
297
218
243
312
331
Age
at
sexual
maturity
(
days)
32
28­
35
63
49­
70
42
53
49­
56
Number
eggs/
year
288
300
183
230
234
Number
eggs/
hen/
day
0.79
0.82
0.5
0.64
Percent
Fertility
87
90
Percent
Hatchability
90
85
Reproduction
is
greatly
affected
by
inbreeding
in
Japanese
quail,
and
mating
systems
need
to
be
selected
to
avoid
pairing
of
closely
related
individuals.
Sittmann
et
al.
(
1966)
demonstrated
a
66%
decrease
in
hatch
from
one
generation
of
full­
sibling
mating
and
nearly
complete
loss
of
hatch
by
the
third
generation.
Fertility
and
hatchability
decline
by
about
1%
for
each
1%
additional
inbreeding.
Brooder
mortality
also
increases
substantially
with
each
increase
in
inbreeding.
Bateson
(
1983)
demonstrated
a
mating
preference
for
unfamiliar
first
cousins
among
Japanese
quail;
therefore,
single­
enclosure
mating
systems
may
be
less
successful
than
the
more
controlled
mating
systems
in
preventing
inbreeding.
To
prevent
inbreeding
depression
in
resource
stocks
bred
in
single­
enclosure
systems
where
all
males
have
access
to
all
females,
a
minimum
of
100
birds
are
needed.
Japanese
quail
stocks
are
often
maintained
by
random
pairing
or
grouping
in
cages,
in
which
case
25
pairs
but
usually
more
than
150
birds,
are
needed
to
keep
inbreeding
at
a
minimum
(
Pisenti
et
al.
1999).
Typically,
random­
bred
stocks
are
kept
as
closed
populations;
however,
new
bloodlines
are
often
introduced
to
the
flock
if
inbreeding
depression
is
observed.

Japanese
quail
lay
large
eggs
relative
to
their
body
size
 
7%
to
8%
of
their
body
weight
 
with
large
yolk
volume,
providing
a
relatively
large
potential
depot
for
xenobiotic
transfer
from
hen
to
developing
embryo.
As
is
typical
for
nidifugous
young,
a
reserve
of
yolk
is
gradually
discharged
into
the
blood
plasma
for
several
days
post­
hatch.
Most
strains
of
Japanese
quail
produce
colored,
often
mottled
eggshells
that
can
make
detection
of
cracks
difficult.
However,
some
strains
produce
light­
colored
or
white
shells
(
Poole
1964).
Incubating
eggs
require
a
temperature
of
37.5
±
0.3
º
C
and
relative
humidity
(
RH)
of
at
least
60%.
Egg
incubations
systems
are
available
to
automatically
rotate
the
eggs
every
2
to
4
h.
Pedigree
baskets
are
routinely
used
to
track
parentage
of
the
young
at
hatch.
Fertility
of
eggs
stored
at
14
±
0.3
º
C
and
70%
RH
is
high
for
the
first
7
days,
after
which
it
falls
off
dramatically.
Rapid
decline
in
hatchability
occurs
if
the
eggs
are
held
longer
than
7
days.
Storage
can
be
extended
to
10
to
14
days,
if
the
Battelle
Draft
22
April
23,
2003
eggs
are
covered
in
plastic
to
prevent
desiccation
(
NRC
1969;
Ottinger
and
Rattner
1999).
The
incubation
period
for
Japanese
quail
is
14
to
18
days
depending
on
strain.
Humidity
requirements
during
hatch
are
higher
(
70%
RH
minimum),
and
eggs
can
be
sprayed
with
distilled
water
to
help
prevent
embryonic
membranes
from
drying
after
pipping
(
Olsen
1992;
Ottinger
and
Rattner
1999).
Hatchlings
can
be
sexed
by
cloacal
examination
(
Homma
et
al.
1966).

Japanese
quail
respond
to
sudden
disturbance
by
flushing,
and
injury
can
result
during
capture,
if
the
bird
strikes
against
the
ceiling
of
the
cage.
Cage
height
is
therefore
kept
at
about
25.5
cm
to
reduce
the
potential
for
injury.
Some
species,
such
as
the
feral
Hawaiian
strain,
are
considered
flighty,
whereas
larger
strains
appear
to
be
more
docile.
In
all,
they
are
relatively
easy
to
handle,
although
manual
restraint
can
occasionally
induce
tonic
immobility,
an
instinctive
fear
response
characterized
by
motor
inhibition
and
loss
of
righting
response
for
a
period
of
time
(
Satterlee
et
al.
1993).
Disturbing
laying
hens
may
cause
a
pause
in
egg­
laying.
Moving
layers
to
new
caging
may
result
in
a
cessation
of
egg
production
for
2
to
3
weeks
(
NRC
1969).
Interruption
of
egg­
laying
will
have
to
be
considered
in
selection
of
endpoints
and/
or
sampling
schedule.

The
threshold
for
reproductive
system
stimulation
in
Japanese
quail
is
11
h
of
light
and
13
h
of
darkness
per
day
(
11L:
13D).
Maximal
photoresponse
is
obtained
at
14L:
10D
and
maintained
at
maximum
up
to
18L:
6D.
However,
captive
quail
populations
often
display
marked
photoperiodic
drift
and
large
variability
in
reproductive
response.
This
appears
to
be
a
result
of
the
increasing
population
of
individuals
in
stocks
of
Coturnix
that
reach
sexual
maturity
on
shorter
day
lengths.
Dobson
et
al.
(
1992)
reported
up
to
10%
of
a
population
may
breed
on
8L:
16D
photoperiods
if
selective
breeding
against
early
photoresponses
is
not
maintained.
Others
have
reported
even
greater
percentages
of
test
populations
that
enter
into
reproduction
when
raised
under
the
recommended
photoperiod
for
gonadal
quiescence
in
avian
reproduction
tests
(
OECD
Guideline
206).
For
example,
Yamamoto
et
al.
(
1996)
found
that
60%
of
the
Japanese
quail
used
for
a
reproductive
toxicity
study
entered
into
reproduction
when
maintained
under
the
recommended
short
day
length
of
7
h
to
8
h
of
light.
When
the
day
length
was
shortened
to
6
h
of
light,
20%
of
the
birds
became
reproductive.
Decreasing
day
length
to
5
h
of
light
reduced
the
incidence
of
reproduction
to
less
than
5%
of
the
population.
However,
the
reduced
day
length
(
6L:
18D)
resulted
in
lower
food
intake,
and
thus,
reduced
uptake
of
the
test
substance
(
fenthion),
which
in
turn
resulted
in
a
much
reduced
response
to
the
chemical
exposure
than
was
previously
observed
in
their
laboratory.
Some
strains
of
Japanese
quail
have
been
developed
that
do
not
require
photostimulation
of
reproduction
(
Dobson
et
al.
1992).
Photoperiodic
drift
within
a
strain
of
quail
could
compromise
attempts
to
measure
reproductive
endpoints
associated
with
sexual
maturation
(
gonadal
development,
cloacal
gland
size,
time
to
first
egg
laid,
level
of
circulating
hormones).
Therefore,
careful
consideration
should
be
given
to
strain
selection
prior
to
use
in
avian
reproduction
tests.

Light
intensity
is
also
important
in
maintaining
quail.
Light
intensity
that
is
too
high
will
encourage
aggressive
behavior.
The
quail
should
be
exposed
to
about
10
lux
artificial
light,
measured
at
the
level
of
the
feeder,
which
approximates
the
daylight
visual
spectrum
to
initiate
and
maintain
reproduction.
However,
to
reduce
aggression
or
maintain
birds
in
nonreproductive
Battelle
Draft
23
April
23,
2003
condition,
lower
levels
of
light
may
need
to
be
provided.
To
ensure
a
stimulatory
photoperiod
as
well
as
a
long
feeding
period
to
maximize
consumption
of
the
test
substance,
a
photoperiod
of
16
h
or
17
h
of
light
and
7
h
or
8
h
of
dark
is
commonly
used.
If
the
breeding
colony
is
maintained
on
this
light
cycle
throughout
the
year,
the
hens
may
experience
premature
aging
from
the
calcium
drain
associated
with
constant
egg­
laying
(
Ottinger
and
Rattner
1999).

Coturnix
can
be
maintained
from
hatchling
through
reproduction
on
a
diet
containing
26%
to
28%
protein
(
Howes
1965)
and
an
energy
content
of
2000
productive
energy
calories
per
kilogram.
High­
energy
diets
should
be
avoided,
because
they
cause
steatosis
(
Howes
and
Fitzgerald
1966).
Supplementary
calcium
(
3.0%)
is
required
for
egg­
laying
and
should
be
supplied
just
prior
to
maturity
(
Nelson
et
al.
1964).
Commercial
game
bird
and
turkey
starter
diets
are
available
and
are
commonly
used
for
Japanese
quail
production.
Most
commercial
and
custom
diets
are
formulated
with
soy
and
corn,
which
contain
phytoestrogens
in
widely
varying
amounts.
The
impact
of
soy
and
corn
diet
on
endocrine
disruptor
testing
has
not
been
studied.
However,
commercial
turkey
starter
amended
with
extract
from
forage
plants
containing
elevated
levels
of
phytoestrogens
(
biochenin
A
and
genistein)
was
shown
to
cause
a
delay
in
the
onset
of
egg
production
in
California
qual
(
Lophortyx
californicus)
and
to
reduce
egg
production
overall
in
this
species
(
Leopold
et
al.
1977).
Unfortunately,
only
relative
concentrations
 
high,
medium,
and
low
 
of
the
amended
phytoestrogens
were
reported.
A
later
study
by
Lien
et
al.
(
1987)
showed
that
bobwhite
would
have
to
consume
in
excess
of
1
mg/
bird/
day
of
biochenin
A
to
adversely
impact
reproduction.

Stainless
steel
construction
is
recommended
for
caging
used
in
avian
toxicity
tests,
although
galvanized
construction
is
acceptable.
Quail
cages
and
brooders
are
commercially
available
in
galvanized
steel
from
production
factories.
However,
stainless
steel
caging
must
be
customconstructed
There
is
an
increasing
interest
in
cages
constructed
of
metal
wire
coated
with
perflourocarbon
plastics
to
provide
thermoneutral
surfaces,
ease
of
cleaning,
and
reduced
cost.
Some
also
feel
that
the
coatings
reduce
egg
breakage
and
may
provide
a
more
comfortable
flooring
for
the
birds.
However,
use
of
plastic
water
providers
and
food
containers
may
contain
plasticizers
that
should
be
avoided
in
tests
measuring
endocrine
endpoints.
Wire
pens
with
slanting
floors
and
egg­
catchers
or
other
measures
to
prevent
breakage
of
eggs
are
recommended
for
adults.

4.1.3
Strains
A
review
of
available
documentation
on
genetic
stocks
of
the
Japanese
quail
sponsored
by
the
University
of
California
Genetic
Resources
Conservation
Program
indicated
that
all
the
strains
of
Japanese
quail
in
North
America
and
the
European
community
developed
through
selective
breeding
for
food
production
and/
or
biological
research
are
descended
from
C.
japonica
(
NRC
1985;
Pisenti
et
al.
1999).
Small
wild
populations
of
C.
japonica
still
are
found
in
Japan,
and
a
random­
bred
population
of
the
wild
type
is
maintained
at
the
Quail
Genetic
Resource
Centre
at
UBC.
A
strain
originating
from
feral
quail
captured
on
the
island
of
Hawaii
also
is
maintained
at
the
center.
Battelle
Draft
24
April
23,
2003
Many
specialized
genetic
stocks
of
morphological
and
physiological
mutations
have
been
developed
by
universities
and
government
research
organizations
(
Pisenti
et
al.
1999).
Likewise,
research
institutions
and
some
commercial
suppliers
have
developed
and
continue
to
conserve
through
randomly
breeding
populations
a
number
of
strains
in
which
individual
birds
have
many
traits
in
common
(
Table
4­
1).
These
random­
bred
strains
are
the
sources
of
test
organisms
for
current
avian
endocrine
and
reproductive
toxicity
studies
conducted
with
Japanese
quail
(
Pisenti
et
al.
1999).
In
the
United
States
and
Japan,
quail
currently
used
for
toxicity
testing
and
endocrine
studies
are
derived
from
strains
selected
for
their
egg­
producing
traits.
European
studies
often
make
use
of
strains
of
Japanese
quail
that
have
been
selected
for
meat
production.
These
strains
may
differ
not
only
in
growth
rate
and
body
size,
but
also
in
carcass
composition
(
Barbato
et
al.
1984),
sexual
maturation,
and
egg
production
(
Table
4­
2).
Increased
lipid
deposition
in
the
form
of
body
fat
and
yolk
material
is
often
observed
in
meat
production
strains
fed
ad
libitum
(
Reddy
and
Siegel
1976;
Barbato
et
al.
1984).
Increased
food
consumption
and
lipid
deposition
in
yolk
could
result
in
greater
exposure
of
heavier
birds
to
test
substance
in
the
feed
and
in
ovo.
To
determine
whether
there
are
significant
differences
in
growth
and
reproductive
characteristics
for
the
various
strains
of
Japanese
quail
currently
in
use,
researchers
and
commercial
quail
facilities
in
the
United
States,
Japan,
and
the
European
community
were
canvassed
to
obtain
data
on
strain
maturation
rate,
productivity,
length
of
peak
production
period,
eggshell
properties,
aggression
between
members
of
a
pair,
reliance
on
photostimulation
to
induce
reproduction,
and
sexual
aggressiveness.
This
information
is
reported
in
Table
4­
2.

In
general,
the
smaller,
egg­
producing
strains
appear
to
mature
more
rapidly,
have
higher
productivity,
and
retain
peak
fertility
and
productivity
for
a
longer
period
than
the
larger,
meatproduction
strains.
However,
there
is
some
evidence
in
the
literature
that
strains
selected
for
rapid
growth
have
more
ovarian
follicles
in
rapid
development
during
the
egg­
laying
cycle
than
do
random­
bred
lines
(
Ye
et
al.
1999).
This
apparent
contradiction
may
be
explained
by
the
higher
incidence
of
follicles
lost
into
the
body
cavity,
follicular
atresion,
and
production
of
abnormal
eggs
in
strains
selected
for
increased
growth
rate
(
Bacon
et
al.
1973;
Nestor
et
al.
1982).
Egg
production
of
Japanese
quail
lines
selected
for
high
body
weight
at
4
weeks
of
age
declined
over
generations
(
Nestor
et
al.
1996).
Notably,
in
a
random­
bred
line
maintained
using
a
paired
mating
system,
nine
reproduction
traits
declined
over
30
generations
(
Nestor
1977).
Many
of
these
traits
are
related
in
the
Japanese
quail;
therefore,
changes
in
a
single
trait
over
the
generations
could
be
correlated
with
other
traits
(
Nestor
et
al.
1995).
The
declining
traits
were
egg
production,
hatch
of
eggs
set,
egg
weight,
albumen
weight,
shell
weight,
yolk
weight,
body
weight
at
end
of
lay,
and
change
in
body
weight
during
laying,
and
percentage
liver
dry
matter.
Selection
for
high
body
weight
also
appears
to
reduce
the
minimal
requirements
for
males
to
advance
to
sexual
maturity
(
Anthony
et
al.
1993).
Selection
is
also
made
for
docility,
both
for
its
effects
on
poultry
welfare
and
on
egg
quality
(
Jones
1996).
These
quail
tend
to
have
reduced
corticosterone
responses
(
Satterlee
and
Johnson
1988).

How
the
various
types
of
strains
differ
in
sensitivity
to
EDCs
is
not
known
and
should
be
evaluated
to
determine
whether
the
differences
would
affect
interlaboratory
comparisons
and
hazard
evaluation.
An
option
currently
being
considered
in
Japan
to
address
this
issue
of
comparable
test
birds
is
to
restrict
test
populations
to
those
arising
from
a
specific
Battelle
Draft
25
April
23,
2003
egg­
production
strain
and
source
for
all
endocrine­
disruptor
tests
(
Ito,
Yoshihiko,
Research
Institute
for
Animal
Science
in
Biochemistry
and
Toxicology,
Kanagawa,
Japan,
currently
Chair
of
the
Endocrine
Disruptor
Task
Force
for
Japanese
Environmental
Protection
Agency,
personal
communication,
2002).
However,
criteria
for
selecting
a
standard
strain
or
standard
type
strains
must
be
carefully
established
and
should
take
into
consideration
limits
on
body
size,
eggshell
quality,
fertility,
photoperiod
drift,
behavior,
and
EDC
sensitivity.

4.2
Bobwhite
(
Colinus
virginianus)

The
northern
bobwhite
is
a
New
World
quail
only
distantly
related
to
quail
of
Old
World
origins
(
Hoyo
et
al.
1994).
As
a
native
species
of
the
United
States
adaptable
to
laboratory
conditions,
the
northern
bobwhite
has
been
used
extensively
under
EPA
and
American
Society
for
Testing
and
Materials
(
ASTM)
test
guidelines
as
a
representative
species
for
toxicological
evaluation
of
pesticides
and
other
environmental
contaminants.
Although
there
are
many
natural
strains
of
bobwhite,
few
domesticated
strains
have
been
intentionally
developed.
Guidelines
specifically
call
for
bobwhite
used
on
test
to
be
phenotypically
indistinguishable
from
the
wild
type
to
retain
capacity
to
represent
wildlife
responses
to
chemicals.
The
life
stages
of
the
bobwhite
are
significantly
longer
than
those
of
the
Japanese
quail,
particularly
the
period
from
growth
to
reproductive
maturity.

4.2.1
Natural
History
The
bobwhite
is
distributed
from
southeastern
Ontario
(
Canada)
to
Guatemala
and
in
Cuba
but
reaches
its
highest
density
in
the
eastern
United
States
and
Mexico.
The
species
has
been
widely
transplanted
from
pen­
raised
and
wild­
trapped
birds
in
the
United
States
and
is
well
established
in
areas
beyond
its
natural
range,
with
disjunct
populations
found
in
Washington,
Oregon,
and
Idaho.
Northern
bobwhite
has
been
introduced
also
to
British
Columbia
(
Canada),
Puerto
Rico,
Hawaii,
and
New
Zealand
(
Hoyo
et
al.
1994).
It
is
a
ground­
dwelling
species
with
forest
and
grassland
affinities.
It
also
has
adapted
to
intensive
agricultural
areas
in
the
United
States
and
to
xeric
ecosystems
of
Mexico
and
Central
America.
Bobwhite
nests
in
the
spring
with
clutch
sizes
of
10
to
15
eggs.
The
incubation
period
is
23
days,
and
nest
success
is
usually
between
20%
to
40%.
After
the
breeding
season,
family
coveys
of
about
12
in
its
southern
range
and
15
in
northern
United
States
are
formed.
Body
size
also
increases
from
south
to
north
in
its
native
range
with
birds
in
Chiapas,
Mexico,
averaging
129
g,
whereas
those
in
the
eastern
United
States
weigh
about
172
g.
The
northern
bobwhite
is
a
sedentary
species
with
a
home
range
of
10
ha
to
31
ha.
It
is
an
opportunistic
feeder,
consuming
mainly
seeds
and
invertebrates.

4.2.2
Availability,
Culture,
Handling
Numerous
commercial
and
game
farm
sources
of
northern
bobwhite
are
available.
Most
sources
supply
phenotypical
wild­
type
birds
for
hunting
and
dog
training.
These
or
similar
wild­
type
strains
are
the
stocks
also
used
or
maintained
by
testing
and
research
laboratories.
Bobwhite
husbandry
is
similar
to
that
of
the
Japanese
quail.
Methods
of
incubation,
brooding,
maintenance
of
juveniles,
and
breeder
support
are
generally
the
same
as
for
the
smaller
Japanese
quail.
However,
space
requirements
are
greater,
400
to
900
cm2
per
adult
bobwhite
(
Olsen
Battelle
Draft
26
April
23,
2003
1993),
and
humidity
requirement
during
incubation
is
higher
(
85%
RH)
for
bobwhite
(
Ottinger
and
Rattner
1999).
Similar
to
Coturnix,
the
northern
bobwhite
has
a
low
photoperiodic
threshold
(
10L:
14D)
for
stimulation
of
the
reproductive
system
(
Kirkpatrick
1955),
and
also
lacks
the
photorefractoriness
of
most
other
birds.

All
life
phases
of
the
bobwhite
are
longer
than
those
of
Japanese
quail.
The
incubation
period
is
23
to
24
days,
development
of
sexually
distinguishable
plumage
occurs
at
about
12
weeks
of
age,
and
sexual
maturity
is
reached
at
24
weeks
of
age
(
Ballard
et
al.
1994).
Peak
production
is
reached
at
about
6
weeks
after
the
onset
of
lay.
The
northern
bobwhite
is
bred
to
retain
its
flightiness,
and
excessive
handling
may
result
in
mortality.
Inbreeding
depression
will
occur
in
bobwhite
stocks
under
200
pairs.
Therefore,
unrelated
breeder
stock
is
introduced
into
the
resource
stocks
of
bobwhite
at
least
every
3
years
to
reduce
inbreeding
(
International
Hatchery
Practice
1989).

4.2.3
Strains
There
are
22
recognized
subspecies
of
bobwhite
(
Hoyo
et
al.
1994).
Five
of
the
subspecies,
C.
v.
marilandicus,
C.
v.
virginianus,
C.
v.
floridanus,
C.
v.
texanus,
and
C.
v.
taylori,
are
native
to
the
United
States
(
Rosene
1969).
The
northern
bobwhite
is
commonly
bred
commercially
or
by
hobbyists
as
wild
stock.
Very
little
deliberate
selective
breeding
of
northern
bobwhite
has
been
practiced,
with
the
exception
of
the
bobwhite
blood­
type
variants
maintained
at
Northern
Illinois
University.
However,
a
few
strains,
such
as
Eastern,
for
example,
are
selected
for
body
weight
for
meat
production.
They
are
phenotypically
similar
to
the
wild
type
except
for
body
weight
and
are
generally
not
used
in
avian
toxicity
tests.
However,
because
most
suppliers
raise
bobwhite
in
game
farm
situations,
there
is
some
use
of
these
meat
strains
by
commercial
testing
laboratories
when
production
of
eggs
and
juveniles
has
decreased
in
winter.
These
larger
strains
are
usually
more
docile
and
lay
fewer
eggs
(
International
Hatchery
Practice
1989).

4.3
Strengths
and
Weaknesses
Table
4­
3
summarizes
major
strengths
and
weaknesses
of
the
two
species
for
two­
generation
tests.
The
major
advantages
of
using
the
Japanese
quail
over
bobwhite
in
a
two­
generation
test
guideline
with
endocrine
endpoints
are
related
to
the
very
rapid
incubation
and
maturation
(
Table
4­
2)
and
high
rate
of
egg
production
of
the
Japanese
quail.
These
traits
allow
for
the
completion
of
a
multigenerational
test
within
a
relatively
short
time,
about
30
weeks,
compared
with
70
weeks
for
a
bobwhite
test.
Further,
they
allow
a
large
number
of
eggs
per
hen
from
which
to
sample
for
egg
quality
and
chemical
residue
analyses.
The
cloacal
gland
of
the
Japanese
quail
provides
an
indirect
measure
of
gonadal
development,
indicating
reproductive
fitness,
and
sexual
maturation
of
the
males.
Because
of
the
extensive
use
of
Coturnix
in
biochemical
and
steroid
research,
endocrine
and
behavioral
patterns
in
this
species
are
well
characterized
(
Hutchinson
et
al.
2000;
Ottinger
and
Brinkley
1978,
1979a,
1979b).
Spermatogenesis
is
most
fully
characterized
in
the
Japanese
quail
(
Lin
and
Jones
1992),
which
provides
the
ability
to
quantify
histopathological
evaluations
of
testicular
function
and
to
determine
length
of
exposure
required
before
spermatogonial
damage
could
be
detected
Battelle
Draft
27
April
23,
2003
extragonadally
(
Section
5.1.1).
No
information
is
available
on
the
seminiferous
cycles
of
bobwhite.

Major
disadvantages
associated
with
the
use
of
the
Japanese
quail
are
related
to
its
extensive
history
of
domestication.
Stock
populations
are
very
sensitive
to
inbreeding
and
loss
of
fertility.
Consequently,
heed
must
be
given
to
appropriate
mating
protocols
in
source
flocks
and
to
the
parental
and
offspring
pairing
in
a
multigenerational
test
protocol.
Also,
many
strains
of
Japanese
quail
are
being
used
around
the
world
for
reproduction
testing.
These
strains
differ
in
body
size,
maturation
rates,
egg
production,
and
lipid
deposition
in
yolk.
The
impact
of
different
strain
selection
on
the
outcome
of
a
two­
generation
study
is
not
known.

In
contrast,
the
northern
bobwhite
is
far
less
domesticated
and
thus
faces
less
inbreeding
pressure
and
presumably
is
more
representative
of
wildlife
responses.
However,
the
comparative
sensitivity
of
the
two
species
to
environmental
chemicals,
particularly
those
with
potential
endocrine
effects,
is
not
known.
A
study
by
Solecki
et
al.
(
1996)
compared
the
reproductive
effects
of
an
OP
pesticide
on
Japanese
quail
with
those
published
by
Bennett
et
al.
(
1990),
but
most
of
the
reproductive
impacts
observed
could
be
attributed
to
parental
toxicity
in
both
studies.
Although
no
sensitivity
comparisons
in
chronic
reproductive
toxicity
tests
have
been
conducted,
comparison
of
the
relative
sensitivity
of
the
Japanese
quail
and
bobwhite
to
acute
dietary
exposure
(
Romijin
et
al.
1995)
and
to
acute
oral
dose
with
pesticides
(
Romijin
et
al.
1995;
Baril
et
al.
1994)
have
been
made
using
data
from
the
open
literature
and
from
studies
in
support
of
registration
of
pesticides.
Both
studies
showed
that
the
northern
bobwhite
tended
to
be
more
sensitive
to
more
compounds
than
the
Japanese
quail,
but
the
difference
was
not
statistically
significant.
In
the
Romijin
et
al.
study,
the
differences
in
sensitivity
between
the
two
quail
species
did
not
exceed
a
factor
of
5.
Sensitivity
to
some
dietary
mycotoxins
was
also
greater
in
bobwhite
chicks
than
in
Japanese
quail
(
Ruff
et
al.
1992).
Aflatoxin,
toxic
metabolites
produced
by
the
mold,
Aspergillus
flavus,
and
T­
2
toxin
resulted
in
40%
and
23%
mortality,
respectively,
in
2­
and
3­
week­
old
bobwhite.
No
mortality
was
observed
in
Japanese
quail
chicks
fed
the
same
contaminated
diets.
Body
weight
loss,
feed
conversion
ratio,
and
mouth
lesions
were
all
less
severe
in
Japanese
quail
(
Ruff
et
al.
1992).
Currently,
a
study
directly
comparing
the
relative
reproductive
sensitivity
of
the
Japanese
quail
and
northern
bobwhite
to
known
EDCs
is
in
progress
at
the
University
of
Maryland
for
the
EPA.
Until
these
results
are
available,
existing
data
indicate
that
the
sensitivity
of
the
two
species
to
environmental
chemicals
is
comparable.
Battelle
Draft
28
April
23,
2003
Table
4­
3.
Major
Strengths
and
Weaknesses
of
Japanese
Quail
and
Northern
Bobwhite
Related
to
Use
in
Avian
Two­
Generation
Reproduction
Toxicity
Tests
Japanese
Quail
Northern
Bobwhite
ADVANTAGES
ADVANTAGES
°
Endocrine,
behavioral
pattern
characterized
°
Small
bird;
occupies
230
cm2
per
bird
°
Reach
sexual
maturity
by
6
weeks
°
Prolific
layer,
close
to
1
egg
per
day
°
Early
maturity
(
36
days
males,
42
days
females)
°
Short
incubation
period
(
16
­
17
days)
°
Males
are
aggressive
breeders
°
Males
maintain
high
fertility
(
90%)
°
Adapts
well
to
breeder
cages
°
Biological,
physiological,
biochemical
data
available
°
Produce
a
large
egg
(
8%
of
body
weight)
°
Naturally
hardy
in
the
laboratory
°
Highly
photosensitive
°
Physiological
aging
is
rapid
and
lifespan
short
°
Dimorphism
of
plumage
color
and
pattern
makes
it
possible
to
identify
sex
at
3
weeks
of
age
°
Cloacal
gland
of
the
male
may
be
used
as
measure
of
maturity
°
History
of
use
in
toxicity
testing
°
Spermatogenesis
is
well
characterized
°
Less
domesticated,
wild
type
°
Small
bird;
occupies
400­
900
cm2
per
bird
°
Prolific
layer
(
somewhat
less
than
Japanese
quail)
°
Males
are
aggressive
breeders
°
Males
maintain
high
fertility
(
95%)
°
Adapts
well
to
laboratory
°
Produces
a
relatively
large
egg
(
8%­
105%
of
body
weight)
°
Hardy
in
the
laboratory
°
Highly
photosensitive
°
Populations
not
prone
to
photoperiodic
drift.
°
Dimorphism
of
plumage
color
°
History
of
use
in
toxicity
testing
°
More
yolk
per
egg
(
39.8%
by
weight)
compared
with
Japanese
quail
(
31.9%)

DISADVANTAGES
DISADVANTAGES
°
Inbreeding
not
tolerated,
(
leads
to
impaired
fertility)
°
Strains
differ
in
body
weight,
maturation
rate,
egg
production,
lipid
deposition
in
body
and
egg.
°
Populations
can
show
marked
photoperiodic
drift
with
large
variability
in
reproductive
response
°
Food
wastage
making
food
consumption
measurement
difficult
°
Most
strains
have
colored
eggshells
that
are
difficult
to
handle.
Mottling
makes
detection
of
cracks
in
shell
difficult
(
some
white
egg
strains
are
available)
°
Less
yolk
per
egg
(
31.9%
by
weight)
compared
with
bobwhite
(
39.8%)
°
Long
incubation
period
°
Long
maturation
period
(
24
months)
°
Food
wastage
making
food
consumption
measurement
difficult
°
Sex
cannot
be
distinguished
by
plumage
until
12
weeks
of
age
°
Lack
cloacal
gland
°
Spermatogenesis
is
not
characterized
Battelle
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29
April
23,
2003
Also
comparable
are
baseline
ranges
of
reproductive
parameters
of
the
two
quail
species.
Tables
4­
4
and
4­
5
show
typical
reproductive
values
for
the
Japanese
quail
and
northern
bobwhite
compiled
in
a
revised
draft
guideline
for
avian
reproduction
toxicity
testing
(
OECD
2001).
For
additional
comparison
are
shown
the
baseline
values
reported
by
Piccirillo
and
Orlando
(
1985)
for
evaluation
of
control
data
for
one­
generation
reproduction
studies
using
the
bobwhite.
Ranges
for
the
Japanese
quail
will
likely
vary,
depending
on
strain
selection.
To
evaluate
which
of
the
quail
species
would
be
the
better
choice
for
increasing
the
power
of
an
avian
reproduction
test
to
detect
effects,
by
displaying
less
background
variation
in
reproductive
endpoints,
Springer
and
Collins
(
1999)
compared
the
results
of
two
simulation
studies.
Using
historical
control
data
of
reproductive
parameters
for
Japanese
quail
(
Baus
et
al.
1999)
and
northern
bobwhite
(
Springer
and
Collins
1999),
the
two
studies
employed
simulation
methods
to
compare
power
of
the
respective
test
to
detect
a
20%
change
in
the
mean
value
of
a
treated
group
relative
to
controls.
Power
estimates
for
both
species
were
high
(>
0.80)
for
ratio­
type
endpoints,
such
as
eggs
hatched
/
fertile
eggs,
but
lower
for
count­
type
endpoints,
such
as
eggs
laid/
hen.
No
similar
power
comparisons
are
available
for
endocrine
endpoints.

Table
4­
4.
Typical
Values
for
Reproductive
Parameters
in
Japanese
Quail
and
Northern
Bobwhite
Parameter
Japanese
Quail
Northern
Bobwhite
OECD
Piccirilo
and
Orlando
1981
No.
eggs
laid/
hen/
day
0.66
to
0.89
0.40
to
0.81
0.6
±
0.004
Percentage
cracked
or
broken
eggs
0%
to
10%
0%
to
6%
8.2%
±
7.2%

Viability
(
percentage
of
fertile
eggs
that
develop
live
embryos
at
2/
3
through
incubation)
85%
to
96%
72%
to
98%
73.6%
±
19.9%

Hatchability
(
percentage
of
viable
eggs
that
hatch)
70%
to
80%
70%
to
98%
82.0%
±
12.8%

Percentage
hatchlings
that
survive
to
14
days
85%
to
97%
69%
to
98%
76.3%
±
5.0%

Mean
number
14­
dayold
survivors/
hen/
day
0.34
to
0.71
0.24
to
0.38
Eggshell
thickness
(
mm)
0.19
to
0.22
0.20
to
0.25
0.31
±
7.2%
Battelle
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23,
2003
(
a)
Data
adapted
from
OECD
(
2000).
Table
4­
5.
Comparison
of
Development
Phases
in
Japanese
Quail
and
the
Northern
Bobwhite(
a)

Species
Candling
for
fertility
and
viability
(
days)
Incubation
(
days)
Hatch
(
days)
Dimorphism
of
Plumage
(
weeks)
Sexual
Maturity
(
weeks)
Peak
Egg
Production
after
Onset
of
Lay
Japanese
quail
8
15
to
16
17
to
18
3
6
3
Northern
bobwhite
11
20
to
21
24
to
25
12
24
6
5.0
EXPERIMENTAL
DESIGN
CONSIDERATIONS
FOR
TWO­
GENERATION
AVIAN
TESTS
5.1
Exposure
Duration
The
exposure
duration
should
be
long
enough
for
the
test
substance
to
reach
equilibrium
in
the
tissues
and
all
possible
effects
on
the
reproductive
processes
to
be
expressed
in
the
selected
endpoints
and
provide
exposure
to
all
life
stages.

5.1.1
Exposure
of
the
Parental
(
P1)
Generation
(
Pre­
or
Post­
Egg­
Laying)

Current
EPA
and
OECD
guidelines
were
originally
designed
to
detect
reproductive
deficits
resulting
from
chronic
exposure
to
bioaccumulating
substances.
Therefore,
birds
are
exposed
under
these
guidelines
well
in
advance
of
egg­
laying
so
that
the
compound
reaches
equilibrium
in
the
tissues
and
presumably
a
maximum
exposure
level
in
eggs.
Newer
pesticides
are
much
less
persistent,
and
significant
reproductive
effects
have
been
detected
with
treatment
periods
of
only
1
to
3
weeks
(
Bennett
and
Bennett
1990;
Bennett
et
al.
1990;
Rattner
et
al.
1982;
Stromborg
1981,
1986).
This
led
several
authors
to
review
the
statistical
and
physiological
advantages
and
disadvantages
of
the
pre­
egg­
laying
and
post­
egg­
laying
exposure
regimens
for
one­
generation
avian
reproductive
toxicity
tests
(
e.
g.,
Collins
1994;
Mineau
et
al.
1994;
Bennett
et
al.
1990;
Bennett
and
Ganio
1991;
David
Farrar,
personal
communication
to
T.
Maciorowski,
D.
Urban,
D.
McLane,
and
D.
Balluff,
Statistical
aspects
of
the
evaluation
of
avian
reproductive
effects:
accomplishments
and
objectives,
1995)
and
for
the
design
of
multiple
generation
tests
that
would
include
endocrine
endpoints
(
Hart
et
al.
1999;
Baus
et
al.
1999;
Springer
and
Collins
1999;
Bennett
et
al.
2001).
These
authors
pointed
out
that
exposure
following
proven
breeding
would
allow
for
the
removal
of
incompatible
or
infertile
birds
prior
to
exposure,
which
would
remove
a
nontreatment­
related
source
of
variation
and
could
increase
the
power
of
the
test.
Pretreatment
measurements
could
also
be
used
as
covariates
in
the
statistical
analysis,
again
potentially
increasing
the
power
of
the
statistical
test.
Battelle
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April
23,
2003
Conversely,
exposure
before
pairs
are
proven
to
be
breeders
risks
a
loss
of
replication
and
corresponding
statistical
power
due
to
infertility
or
incompatibility.
Using
control
data
from
a
variety
of
historical
data
sets,
several
investigators
(
Baus
et
al.
1999;
Springer
and
Collins
1999;
David
Farrar,
personal
communication
to
T.
Maciorowski,
D.
Urban,
D.
McLane,
and
D.
Balluff,
Statistical
aspects
of
the
evaluation
of
avian
reproductive
effects:
accomplishments
and
objectives,
1995)
studied
the
power
of
the
proposed
(
OECD
2000)
design
of
the
avian
reproduction
test
through
modeled
simulations
of
reproductive
effects.
These
investigators
showed
that
use
of
pretreatment
measures
as
covariates
in
simulation
tests
resulted
an
increase
in
the
power
of
the
test
for
number
of
eggs
laid
and
the
number
of
eggs
incubated
that
hatched
for
both
the
Japanese
quail
and
bobwhite
(
Springer
and
Collins
1999).
Although
the
simulation
studies
also
demonstrated
that
significantly
increased
power
can
be
obtained
for
all
count
variables
by
replacing
nonproducing
pairs
of
bobwhite
(
Springer
and
Collins
1999),
replacing
pairs
in
tests
using
Japanese
quail
appeared
to
be
ineffective
(
Baus
et
al.
1999)
or
only
slightly
effective
(
Springer
and
Collins
1999)
in
increasing
the
power
of
the
test.
These
simulation
comparisons,
however,
were
based
on
study
designs
that
included
only
post­
egg­
laying
treatment.

No
simulation
studies
have
been
conducted
that
provide
information
for
selection
of
treatment
period
based
on
power
comparisons
of
pre­
egg­
laying
and
post­
initiation
of
egg­
laying
exposure
scenarios.
However,
Collin
(
1994)
showed
that
the
power
of
the
FIFRA
avian
reproduction
test
(
EPA
1982)
wherein
treatment
is
started
prior
to
egg­
laying
is
low.
He
determined
that
to
have
80%
power
to
detect
a
20%
decline
in
the
number
of
hatchlings
per
eggs
incubated
required
a
sample
size
of
27
pens
for
hatchlings
exposed
to
a
test
substance
for
10
weeks
prior
to
egg­
laying
and
for
8
weeks
after
the
start
of
egg­
laying
.
To
detect
a
reduction
in
the
number
of
eggs
laid
in
the
pre­
egg­
laying
exposure
tests
would
require
74
pens.
In
contrast,
Springer
and
Collins
(
1999)
determined
that
only
16
pens
would
be
required
to
detect
a
20%
reduction
in
these
same
parameters
when
treatment
is
started
after
the
initiation
of
egg­
laying
and
that
only
12
pens
would
be
required
if
the
post­
initiation
of
egg­
laying
test
included
a
covariate.
This
comparison,
however,
is
potentially
confounded
by
differences
in
the
assumptions,
and
the
simulation
and
statistical
methods
between
the
studies.
In
particular,
Collins
(
1994)
used
reproduction
data
from
the
entire
study
period
rather
than
limiting
it
to
the
peak
laying
period
of
5
to10
weeks
used
in
the
simulation
reviewed
by
Springer
and
Collins
(
1999).
Restricting
the
simulations
to
a
period
of
high
egg
production
when
the
number
of
hens
in
production
and
their
rate
of
production
are
most
stable
appears
to
greatly
enhance
the
statistical
power
of
the
test.
Improved
statistical
power
from
this
high­
production
period
is
probably
the
result
of
the
increased
median
value
of
the
endpoints
(
David
Farrar,
personal
communication
to
T.
Maciorowski,
D.
Urban,
D.
McLane,
and
D.
Balluff,
Statistical
aspects
of
the
evaluation
of
avian
reproductive
effects:
accomplishments
and
objectives,
1995).
Even
so,
the
power
values
obtained
in
investigations
that
used
only
data
from
the
peak
production
period
in
bobwhite
varied
greatly
(
Springer
and
Collins
1999).

When
compared
with
the
power
increases
obtained
from
screening
out
nonproductive
pairs
or
using
pretreatment
data
as
covariates,
using
data
from
the
period
of
high
egg
production
appears
to
be
more
influential
in
increasing
the
power
to
detect
effects
in
avian
reproduction
studies.
Battelle
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April
23,
2003
Springer
and
Collins
(
1999)
reported
a
doubling
of
the
power
to
detect
effects
in
data
from
Weeks
3
to
8
after
the
onset
of
laying,
as
compared
with
Weeks
1
to
6
from
onset
of
laying
in
bobwhite.
Smaller
increases
of
24%
to
32%
were
seen
when
nonlayers
were
removed
or
when
a
covariable
was
included
in
the
analysis
from
simulations
based
on
data
from
birds
treated
prior
to
egg­
laying.
Therefore,
it
is
possible
to
increase
the
power
of
a
test
to
detect
effects
in
a
study
where
the
birds
are
exposed
prior
to
maturation
by
analyzing
data
during
peak
egg
production,
that
is,
by
excluding
the
low,
variable
egg
production
interval
from
statistical
analysis.
A
proven­
layer
exposure
also
benefits
from
this
approach,
but
in
addition,
has
the
ability
to
further
improve
the
power
to
detect
effects
through
screening
out
nonlayers
and
using
pretreatment
data
as
covariates.

Only
one
study
was
found
in
the
literature
that
directly
compared
the
results
of
a
pre­
egg
and
a
post­
egg­
laying
exposure
regimen
in
reproductive
toxicity
studies
(
Bennett
et
al.
1990).
Bobwhite
in
the
pre­
egg­
laying
test
received
methyl
parathion,
an
OP
insecticide,
in
their
diet
for
a
total
of
25
weeks.
Birds
receiving
dietary
exposure
of
the
insecticide
after
the
onset
of
egg­
laying
were
treated
for
3
weeks
during
the
peak
egg­
laying
period.
All
dose­
related
reproductive
effects
that
were
found
in
the
pre­
egg­
laying
exposure
study,
including
decreased
food
consumption
and
egg
production,
were
also
seen
in
the
post­
egg­
laying
study.
Unfortunately,
the
advantage
of
using
proven
breeders
to
reduce
variability
in
the
measured
endpoints
was
compromised
by
the
abnormally
large
number
of
injured
and
nonproducing
birds,
a
total
of
40%,
in
the
post­
egg­
laying
study.
The
elevated
number
of
injured
birds
was
attributed
to
the
higher
rate
of
aggression
in
the
post­
egg­
laying
birds
that
was
apparently
a
result
of
changing
abruptly
from
the
short
to
the
long
day­
length
light
cycle.
However,
comparison
of
the
two
exposure
regimens
did
demonstrate
that
initiating
exposure
to
a
test
substance
after
the
onset
of
laying
significantly
improved
the
dose­
response
relationship
of
certain
variables
by
using
pretreatment
means
as
covariates
for
each
hen.
Variables
that
demonstrated
an
improved
dose­
response
were
those
with
lower
within­
pen
variation
than
between
pen
variations
such
as
eggshell
quality
(
Hunt
et
al.
1977;
Thompson
et
al.
1983).

From
the
available
experimental
and
simulation
data,
it
is
clear
that
there
is
insufficient
information
to
determine
whether
a
post­
initiation
of
egg­
laying
exposure
will
provide
significant
improvements
over
pre­
egg­
laying
exposure
in
increasing
the
power
of
statistical
tests
to
detect
reproductive
effects.

Besides
the
potential
statistical
advantages
to
be
gained
from
the
ability
to
remove
nonproductive
birds
from
the
test
before
exposure
begins
and/
or
to
use
pretreatment
data
during
egg­
laying
for
each
bird
as
a
control,
a
proven
breeder
exposure
can
also
provide
information
on
the
rapidity
with
which
many
reproductive
effects
are
manifested
in
exposed
birds.
Information
of
this
type
is
particularly
useful
in
risk
assessments
of
chemicals
that
undergo
rapid
degradation
in
the
environment
or
have
use
patterns
that
preclude
long­
term
exposure
during
the
breeding
season.
Another
advantage
is
the
savings
in
time
and
cost
that
is
attained
with
the
reduction
in
exposure
period
from
the
20
weeks
of
the
prematuration
exposure
regimen
to
less
than
10
weeks
of
the
proven
breeder
regimen.
However,
it
must
be
remembered
that
the
reduced
exposure
period
may
also
not
be
long
enough
for
histological
or
biochemical
lesions
to
develop.
Battelle
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April
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2003
There
are
potential
reproductive
effects,
however,
that
cannot
be
detected
in
the
P1
generation
by
exposing
birds
only
after
reproductive
maturity.
Of
these
effects,
the
age
at
which
sexual
maturation
is
attained
is
of
particular
importance
to
reproductive
success
and
maintenance
in
wild
bird
populations.
Altered
rate
of
maturation
is
emerging
as
one
of
the
more
significant
endpoints
of
endocrine
disruption
in
birds
(
Yoshimura
et
al.
2000).
In
a
study
by
Edens
et
al.
(
1976),
age
at
sexual
maturity
was
among
the
most
sensitive
measures
of
impaired
reproduction
in
the
female
Japanese
quail
exposed
to
dietary
lead
from
hatch
through
12
weeks
of
age.
Delayed
sexual
maturation
was
detected
at
dietary
concentrations
that
caused
no
body
weight
loss
or
overt
signs
of
toxicity.
Onset
of
egg­
laying
was
delayed
by
up
to
2
weeks
in
treated
birds.
In
turn,
peak
egg
production
was
also
delayed
and
in
most
of
the
treatment
groups
was
not
attained
before
termination
of
the
test
at
12
weeks
of
age.
Such
a
delay
in
peak
production
of
treated
birds
could
confound
a
direct
comparison
not
only
of
fecundity
but
also
body
weight,
because
female
quail
typically
increase
in
body
weight
by
20%
to
40%
during
sexual
maturation.
Exposure
duration
may
need
to
be
lengthened
under
these
circumstances
to
characterize
the
delayed
response
in
egg
production.
Conversely,
chronic
treatment
with
tamoxifen,
an
estrogen
antagonist,
resulted
in
precocious
puberty
and
accelerated
spermatogenesis
in
chickens
(
Rozenboim
et
al.
1986),
similarly
offering
the
potential
to
confound
direct
comparison
with
reproductive
parameters
in
control
and
treated
birds.
A
regression
approach
would
capture
the
time­
course
of
such
events.

As
pointed
out
in
Bennett
et
al.
(
2001),
the
effects
of
delayed
or
accelerated
maturation
do
not
determine
the
ability
of
the
F1
generation
to
reproduce
successfully,
because
they
are
not
transgenerational
effects.
Maturation
information
could
be
better
attained
in
separate
tests.
Bennet
et
al.
(
2001)
also
noted
that
it
is
possible
to
obtain
data
to
evaluate
the
effects
of
pre­
egg­
laying
exposure
from
the
F2
chicks,
assuming
the
F1
chicks
are
treated
prior
to
sexual
maturation.
Another
alternative
would
be
to
add
relatively
inexpensive
sexual
maturation
endpoints,
such
as
cloacal
gland
size/
function
and
day
of
first
egg,
to
the
avian
one­
generation
test.
The
additional
observations
would
also
provide
information
on
exposure
duration
to
aid
in
identifying
a
delayed
peak
egg
production
period.
However,
if
Japanese
quail
are
used
in
the
one­
generation
tests,
strain
selection
becomes
an
important
factor
in
assuring
the
pre­
egg­
laying
exposure
is
adequate.
As
noted
earlier,
many
strains
of
Coturnix
exhibit
photoperiod
drift,
resulting
in
the
maturation
of
significant
proportions
of
the
population
under
regimens
of
<
8
h
light.
Yamamoto
et
al.
(
1996)
found
that
the
day
length
had
to
be
reduced
severely
to
prevent
the
birds
from
entering
into
reproduction
prior
to
the
10­
week
prereproduction
period.
The
reduced
day
length
resulted
in
reduced
food
consumption,
and
thus
reduced
uptake
of
the
test
substance
in
the
birds.

Selection
of
an
exposure
regimen
for
the
P1
generation
of
the
avian
two­
generation
test
must
also
take
into
account
the
propensity
of
the
test
substance
to
bioaccumulate.
For
compounds
that
require
a
long
time
to
reach
equilibrium
in
tissues,
an
exposure
period
that
begins
prior
to
egg­
laying
may
be
necessary
in
order
to
reach
a
maximum
exposure
level
in
testicular
tissue
(
Section
5.1.6)
and
in
the
egg.
It
is
not
clear,
however,
to
what
extent
maternal
transfer
of
contaminants
to
the
egg
is
derived
from
stored
lipid.
Although
residues
of
OCs
in
eggs
and
Battelle
Draft
34
April
23,
2003
tissues
of
females
have
been
found
to
be
highly
correlated
(
Mineau
1982;
Custer
et
al.
1990;
Loncore
and
Stendell
1977;
Bogan
and
Newton
1977),
other
studies
indicated
that
dietary,
not
adipose
lipid
may
be
the
primary
source
of
fat­
soluble
contaminants
in
eggs
of
exposed
birds
(
Norstrom
et
al.
1986;
Roudybush
et
al.
1979).
Until
the
source
of
contaminants
in
the
egg
is
resolved
under
the
conditions
of
the
test,
the
potential
for
a
delayed
response
in
the
F1
endpoints
for
bioaccumulating
chemicals
should
be
presumed
and
the
exposure
duration
selected
to
accommodate
the
required
build
up
in
tissues.

5.1.2
Exposure
of
the
Offspring
(
F1)
of
the
Parents
(
No
Exposure
vs.
Exposure
from
Hatch
through
Egg­
Laying)

Bennett
et
al.
(
2001)
reviewed
the
various
potential
exposure
scenarios
of
the
F1
generation.
The
two
major
exposure
regimens
involve
1)
a
worst
case
scenario,
wherein
the
chicks
are
exposed
from
hatch
through
egg­
laying
to
the
same
environmental
concentrations
as
their
parents,
or
2)
a
nonexposure
scenario
wherein
the
chicks
do
not
receive
additional
exposure
other
than
in
ovo.
The
former
scenario
is
similar
to
the
exposure
regimen
of
classical
mammalian
reproductive
toxicity
studies
(
EPA
1982,
1985;
OECD
1981)
allowing
observation
of
reproductive,
developmental,
and
endocrine­
mediated
effects
at
all
susceptible
life
stages,
not
just
those
expressed
in
the
P1
generation
or
resulting
from
in
ovo
exposure.
There
is
a
real
potential,
however,
that
exposure
to
the
test
substance
as
the
chicks
mature
will
result
in
nonendocrine­
related
effects
and
potentially
high
juvenile
mortality.
This
confounding
influence
of
direct
toxicity
could
mask
the
detection
of
endocrine­
mediated
effects.
Limiting
the
F1
exposure
to
in
ovo
exposure
(
e.
g.,
sexual
differentiation)
of
the
developing
embryo
would
eliminate
these
potential
interference
and
interpretative
problems,
and
focus
the
test
on
the
reproductive
success
of
the
F1
chicks.
However,
it
is
likely
that
for
many
chemicals,
chicks
will
be
exposed
in
the
wild.
Elimination
of
exposure
during
vulnerable
growth
and
maturation
stages
may
overlook
impacts
to
reproductive
success.
Currently,
there
is
no
information
available
that
would
help
to
resolve
the
relative
importance
of
treating
the
F1
chicks.
It
is
important
to
the
design
of
a
two­
generation
reproduction
test
to
determine
whether
endocrine
disruption
can
be
detected
amid
the
responses
induced
by
direct
toxicity
of
compounds
such
as
OP
insecticides.

Because
the
potential
exposure
patterns
will
differ
for
different
chemicals,
effects
from
both
the
in
ovo
and
continuous
exposure
regimens
must
be
interpreted
with
caution,
if
the
environmental
exposures
are
likely
to
differ
from
the
exposure
regimen
of
the
test.
Continuous
exposure
is
used
for
chemicals
that
degrade
rapidly
in
the
environment
or
in
cases
in
which
F1
chicks
are
not
exposed
to
chemicals
to
which
birds
in
the
wild
would
be
exposed
throughout
their
life
cycle.

5.1.3
Combined
Exposure
Scenarios
for
P1
and
F1
Generations
The
exposure
scenarios
for
each
generation
should
be
combined
in
such
a
manner
that
the
maximum
number
of
reproductive
processes
and
vulnerable
life
stages
are
exposed
over
the
period
of
the
study.
In
addition,
the
exposures
should
not
mask
endocrine­
related
effects
or
confound
interpretation
of
results.
The
selection
of
exposures
should
also
maximize
time
and
cost­
effectiveness
of
the
test
relative
to
the
information
obtained.
From
the
discussions
above,
a
list
of
desirable
attributes
were
obtained
and
matched
to
the
various
exposure
regimens
(
Table
Battelle
Draft
35
April
23,
2003
5­
1)
regardless
of
the
generation
(
P1,
F1,
or
F2).
In
all
exposure
scenarios,
the
F2
generation
is
not
exposed
to
the
test
substance.
The
combination
of
exposures
that
provides
the
most
comprehensive
number
of
the
attributes
with
minimum
disadvantages
was
determined
(
Table
5­
1):
post­
egg­
laying
production
exposure
of
the
P1
generation;
exposure
of
the
F1
chicks
through
growth
and
egg­
laying;
no
exposure
of
the
F2
chicks.
The
resulting
exposure
combination
provides
a
different
exposure
history
for
each
generation
such
that
the
P1
generation
receives
a
subchronic
exposure
during
the
reproductive
adult
stage;
the
F1
offspring
are
exposed
continuously
from
in
ovo
embryogenesis
through
egg­
laying;
and
the
F2
generation
is
exposed
in
ovo
only.
By
contrasting
the
P1
proven
breeders
and
F1,
the
reproductive
impact
from
full
life
cycle
exposure
from
in
ovo
exposure
by
combined
parental
transfer
and
exposure
of
F1
birds
through
growth
and
breeding
can
be
obtained.
Contrasting
the
F1
with
the
F2
exposures
allows
evaluation
of
the
contribution
of
in
ovo
exposure
alone
to
the
reproductive
and
endocrine­
mediated
effects
of
the
test
substance.
Although
effects
on
sexual
maturation
are
included
in
the
F1
exposure,
it
is
not
separated
from
effects
induced
in
ovo.
If
this
information
were
needed,
a
separate
study
could
be
performed,
or
maturation
endpoints
added
to
existing
one­
generation
tests.
A
combination
that
employs
the
pre­
egg­
laying
exposure
of
the
P1generation
would
provide
similar
information
as
that
previously
described,
but
it
would
add
direct
maturation
information.
However,
acquisition
of
these
data
would
be
at
considerable
time
and
labor
cost
and
with
potential
loss
of
statistical
power
in
ANOVA­
based
comparisons.
Preegg
laying
exposure
could
affect
the
F1
and
consequently
the
F2
generation
by
influencing
the
transport
of
any
of
a
number
of
essential
maternal
substances
that
are
needed
in
the
egg
for
normal
development.
In
addition,
if
egg­
laying
were
delayed
or
accelerated
by
prebreeding
exposure,
comparison
of
the
periods
of
peak
egg
production
between
treated
and
control
birds
would
have
to
be
made
with
caution.

Table
5­
1.
Required
Attributes
of
Parental
and
Offspring
Exposure
Regimens
TREATMENT
Parameter
P1
F1
F2
Pre­
egg
Post­
egg
None
Chicks(
a)
None
Increased
power
of
test
­­­­(
b)
X
­­­­
­­­­
­­­­

Detect
altered
maturation
X
­­­­
­­­­
X
­­­­

Detect
effects
of
in
ovo
exposure
­­­­
­­­­
X
Confound
X
Detect
effects
of
chick
exposure
Partial
­­­­
­­­­
X(
c)
­­­­

Worst­
case
environmental
exposure
Partial
­­­­
­­­­
X
­­­­
Battelle
Draft
36
April
23,
2003
Exposure
with
advantages
that
cannot
be
replaced
by
other
exposure
scenario
without
consideration
of
cost
­­­­­
X
­­­­
X
(
X)(
d)

Relative
time/
cost
savings
­­­­
High
Moderate
­­­­­
­­­­

Exposure
with
advantages
that
cannot
be
replaced
by
other
exposure
scenario
with
consideration
of
cost
­­­­
X
­­­­
X
X
a)
exposed
from
hatch
through
egg­
laying.
b)
­­­­
no
effect.
c)
Possible
confounding
of
endocrine
effects
from
direct
toxicity.
d)
Could
be
replaced
by
F1
no
exposure.

5.1.4
Selection
of
Egg
Cohort
for
F1
Breeding
Pairs
The
egg
cohort
from
which
the
F1
breeding
pairs
will
be
obtained
should
be
selected
at
a
time
during
egg
production
when
the
full
effect
of
the
test
substance
on
the
production
and
quality
of
the
eggs
and
the
viability
of
the
young
have
been
attained.
In
tests
where
an
exposure
initiated
after
egg­
laying
has
begun,
effects
in
eggs
may
not
appear
for
some
time
after
the
initiation
of
the
exposure.
For
example,
yolk
is
recruited
by
ovarian
follicles
up
to
9
days
before
the
egg
is
laid
(
Bacon
et
al.
1973)
possibly
delaying
potential
developmental
effects
of
the
test
substance
on
embryos
after
the
initiation
of
treatment.
If
the
test
substance
has
not
reached
equilibrium
in
the
tissues,
the
period
of
maximum
deposition
of
the
chemical
into
eggs
may
be
further
delayed
and
maximum
effects
observed
even
later.
As
noted
in
Section
5.1.1,
the
source
of
chemical
deposited
in
egg
may
be
from
the
diet
rather
than
fat
tissue,
in
which
case
little
extra
delay
would
be
observed.
Another
source
of
delayed
response
in
birds
exposed
during
breeding
is
the
time
required
(
17
 
21
days)
for
effects
on
early
spermatogenic
processes
to
be
observed
in
eggs
(
see
Section
5.1.6).
Because
of
these
and
other
potential
delays
in
response
after
the
onset
of
exposure,
selection
of
the
egg
cohort
for
the
next
breeding
population
(
F1)
should
be
obtained
as
late
as
possible
in
the
treatment
period.
Therefore,
the
last
weekly
egg
batch
at
the
end
of
the
parental
treatment
period
should
be
used
for
the
F1
breeding
pairs.

If
treatment
of
the
P1
birds
results
in
a
decrease
in
the
number
of
eggs
or
in
reduced
hatchability,
then
additional
eggs
should
be
acquired
by
accumulating
eggs
from
the
last
two
batches
of
eggs.
Eggs
can
be
maintained
for
up
to
14
days
in
cool
storage.
Care
should
be
taken
to
reduce
evaporation
in
the
eggs
during
storage
to
extend
their
shelf
life.
Egg
batches
from
which
the
F1
pairs
will
be
obtained
should
be
retained
and
composited
before
incubation
and
not
incubated
separately,
such
that
two
batches
of
chicks
of
different
ages
are
produced.
Maintaining
pairs
of
Battelle
Draft
37
April
23,
2003
different
ages
would
be
difficult
in
practice
and
greatly
add
to
the
complexity
of
the
experimental
design.

5.1.5
Selection
of
P1
and
F1
Birds
for
Pairing
and
Breeding
For
the
formation
of
the
P1
breeding
population,
birds
should
be
randomly
paired
and
allocated
to
a
treatment.
More
pairs
than
are
required
on
test
should
be
selected
to
ensure
that
there
is
the
required
number
of
breeding
pairs
in
the
control
group
at
the
end
of
the
treatment
period.
This
number
can
be
estimated
from
the
fertility
rate
of
the
source
flock
and
the
loss
expected
from
aggression.
If
a
proven
breeder
exposure
regimen
is
used,
pairs
selected
to
be
on
test
should
have
laid
at
least
one
fertilized
egg.

Protocols
for
selecting
chicks
for
the
F1
breeding
population
must
ensure
that
inbreeding
is
minimized
and
that
the
population
is
representative
of
the
parental
population.
To
minimize
inbreeding,
chicks
are
marked
at
hatch
so
that
they
can
be
traced
to
parental
origin.
When
the
gender
of
the
chicks
can
be
determined
by
plumage,
the
chicks
should
be
randomly
chosen
and
paired
from
the
pool
of
available
chicks
for
each
P1
pair.
The
random
pairing
protocol
will
provide
a
mechanism
for
avoiding
brother­
sister
pairing.
If
possible,
the
same
number
of
chicks
from
each
P1
pair
both
within
treatments
and
between
treatments
will
be
represented
in
the
F1
breeding
population.

If
exposure
to
the
test
substance
has
resulted
in
reduced
chick
production
or
an
altered
ratio
of
males
and
females,
Bennett
et
al.
(
2001)
suggested
that
it
is
permissible,
within
reason,
to
randomly
select
chicks
from
other
P1
pairs
within
the
same
treatment
to
fill
out
the
required
number
of
pairs
per
group
for
the
F1
breeding
population.
This
unequal
selection
of
the
F1
chick
population
must
be
done
with
caution,
because
it
could
result
in
a
cohort
of
birds
less
sensitive
to
the
chemical
than
the
average
of
the
P1
generation
such
that
subsequent
generations
show
a
reduced
response.
It
may
be
possible
to
avoid
back­
filling
the
groups
by
accumulating
eggs
from
the
last
14
days
of
the
treatment
period
rather
than
the
last
7
days.

5.1.6
Comparison
of
One­
Generation
and
Two­
Generation
Exposure
Regimens
Originally
designed
to
detect
reproductive
failure
in
birds
exposed
to
bioaccumulating
chemicals,
the
one­
generation
avian
reproduction
toxicity
studies
have
extensive
exposure
periods.
In
the
one­
generation
tests
in
current
use
(
ASTM
Method
E1062;
OECD
Guideline
206;
OPPT
Guideline
850.2300),
the
parental
birds
are
exposed
to
the
test
substance
for
8
to
12
weeks
prior
to
egg­
laying.
After
egg­
laying
begins,
the
birds
receive
an
additional
8
to
10
weeks
of
exposure.
In
the
ASTM
procedure,
the
duration
of
exposure
after
egg­
laying
has
begun
can
be
reduced
to
the
equivalent
of
two
clutches
of
eggs.
Thus,
the
treatment
period
continues
until
the
controls
produce
25
eggs.

OECD
(
2001)
proposed
a
two­
generation
avian
reproduction
toxicity
test
guideline;
however,
consensus
on
the
appropriate
exposure
regimen
has
not
been
reached.
Several
combinations
of
exposure
scenarios
have
been
explored
by
the
OECD
Expert
Group
on
Assessment
of
Endocrine
Battelle
Draft
38
April
23,
2003
Disrupting
Effects
in
Birds
for
the
Endocrine
Disruptor
Testing
and
Assessment
Task
Force.
In
one
scenario,
the
parental
birds
are
exposed
prior
to
sexual
maturation
and
for
8
weeks
after
the
beginning
of
egg­
laying,
much
like
in
the
exposure
regimen
for
the
one­
generation
studies.
The
F1
generation
is
either
not
exposed
or
exposed
during
all
or
certain
stages
of
the
birds'
life
cycle.
The
second
P1
exposure
scenario
was
proposed
to
address
the
more
contemporary,
short­
lived
compounds
and
to
take
advantage
of
enhanced
statistical
power
from
using
proven
breeders
(
Section
5.1.1).
In
this
exposure
regimen,
the
test
substance
is
not
administered
until
the
females
are
proven
egg­
layers.
Specifically,
to
be
allocated
to
the
test,
a
pair
must
have
produced
at
least
one
egg
during
the
last
week
prior
to
the
start
of
a
pretreatment
period.
Treatment
begins
2
weeks
after
the
beginning
of
the
pretreatment
period
to
coincide
with
the
peak
period
of
egg
production
in
the
Japanese
quail.
Birds
are
exposed
to
the
test
substance
for
6
weeks.
The
various
options
for
the
F1
exposures
are
the
same
as
for
those
that
could
be
combined
with
the
first
P1
exposure
regimen.

Because
the
exposure
is
relatively
short,
it
is
critical
that
the
6­
week
exposure
will
allow
the
detection
of
effects
on
germ
cells
to
be
observed.
For
female
Japanese
quail,
depending
on
strain
and
age
of
the
birds,
4
to
9
days
are
required
for
the
follicles
to
recruit
yolk
(
Bacon
and
Koontz
1971;
Bacon
et
al.
1973;
number
of
days
may
differ
for
other
birds
 
see
p.
54
this
report
and
Bacon
et
al.
1973),
and
24
h
are
needed
for
fertilization,
formation
of
the
shelled
egg
and
oviposition
(
Johnson
2000).
Assuming
the
test
substance
rapidly
reaches
steady
state
in
adipose
tissue
and
that
the
source
of
chemical
is
the
adipose
tissue
rather
than
the
diet
(
Section
5.1.1),
the
potential
delay
in
effects
would
be
at
least
1
week.
In
contrast,
some
effects
could
not
be
observed
for
3
weeks
(
half
the
exposure
period)
in
male
Coturnix.

To
be
able
to
detect
any
adverse
effect
on
the
earliest
stages
of
sperm
development,
spermatogonial
stem
cells,
would
require
a
minimum
exposure
period
of
17
to
21
days.
Spermatogenesis,
the
time
between
the
first
division
of
the
spermatogonia
and
the
freeing
of
sperm
by
the
Sertoli
cell
into
the
lumen
of
the
seminiferous
tubule
(
spermiation),
occurs
in
12.8
days
in
Coturnix
(
Lin
and
Jones
1992).
During
this
time,
germ
cells
within
the
seminiferous
epithelium
become
committed
to
divide
synchronously
in
stages
of
development
and
occupy
a
defined
area
within
the
tubule
(
Kirby
and
Froman
2000).
For
Japanese
quail,
the
duration
of
one
complete
cycle
of
these
stages
is
2.69
days.
Thus,
sperm
cells
released
from
the
germinal
epithelium
arise
from
spermatagonia
that
began
differentiating
4.75
cycles
earlier.
Assuming
that
the
test
substance
comes
to
equilibrium
in
the
tissue
within
one
cycle
of
the
seminiferous
epithelium,
a
minimum
of
about
6
cycles
of
the
germinal
epithelium,
equal
to
16
days,
would
be
required
to
ensure
that
impact
on
early
spermatogenic
processes
could
be
detected
in
lumenal
sperm.
An
additional
24
h
are
needed
for
the
sperm
to
move
through
the
excurrent
ducts
of
Japanese
quail
(
Clulow
and
Jones
1988)
to
be
collected
in
extragonadal
(
deferent
duct
or
ejaculate)
sperm
samples.
Longer
exposure
periods
will
be
needed,
if
the
test
substance
does
not
reach
equilibrium
in
the
tissue
within
the
short
2.69­
day
cycle
of
the
seminiferous
epithelium.
After
copulation,
the
sperm
are
taken
up
by
sperm
storage
tubules
in
the
oviduct
of
the
female,
where
they
can
be
stored
for
7
to
9
days
(
Schom
and
Abbot
1974).
This
could
mean
that
an
additional
7
to
9
days
would
be
required
before
the
first
sperm
exposed
as
spermatagonia
would
be
available
to
fertilize
eggs.
However,
last­
male
precedence
phenomenon,
by
which
the
sperm
Battelle
Draft
39
April
23,
2003
from
the
last
male
or
last
copulation
will
fertilize
a
disproportionate
number
of
eggs
as
time
passes,
exists
in
birds
and
probably
reduces
the
delay
in
sperm
availability
in
the
storage
tubules
to
fewer
than
4
days
(
Birkhead
and
Moller
1992).
Therefore,
in
a
6­
week
exposure
period,
only
during
the
final
3
weeks
of
this
period
will
the
full
effects
of
the
test
substance
on
fertility,
embryo
viability,
hatching
success,
and
sperm
quantity
and
quality
be
observed.
No
effective
statistical
method
has
been
identified
for
analyzing
such
a
delayed
treatment
effect.

Springer
and
Collins
(
1999)
conducted
simulation
tests
using
an
adaptation
of
the
Roth
step­
down
trend
test
incorporating
covariates
in
the
last
3
weeks,
Weeks
8
through
10,
of
a
standard
bobwhite
quail
reproduction
toxicity
test.
The
use
of
these
selected
data
resulted
in
a
decline
in
the
power
of
the
test
to
detect
two
important
variables,
the
number
of
chicks
that
hatched
per
number
of
eggs
incubated
and
the
number
of
hatchlings
that
survived
14
days.
Little
difference
in
power
was
observed
for
the
other
variables.
Because
Coturnix
sustain
peak
production
much
longer
than
bobwhite,
it
may
be
that
at
the
variability
in
production
variables
would
be
less
in
Japanese
quail
at
the
equivalent
treatment
period.
If
test
substances
required
additional
time
to
reach
equilibrium
than
accounted
for
in
the
first
cycle
of
the
germinal
epithelium,
<
3
days,
then
the
6­
week
exposure
should
be
extended.
Extending
the
exposure
period
from
6
to
8
weeks
may
be
prudent
both
to
better
detect
effects
on
early
spermatogenesis
and
to
avoid
increased
variability
in
preproduction
in
aging
birds.
The
time
course
for
spermatogenesis
has
not
been
characterized
for
bobwhite,
and
therefore,
the
necessary
treatment
period
to
detect
effects
in
early
spermatogenic
processes
is
unknown.

In
F1
exposure
scenarios
that
extend
through
egg­
laying,
the
statistical
benefits
from
using
pretreatment
data
as
covariates
and
eliminating
nonproductive
pairs
from
test
before
treatment
begins
are
not
available.
Therefore
it
is
important
to
reduce
variation
within
the
test
by
comparing
egg
production
and
associated
parameters
during
the
more
stable
peak
egg­
laying
period.

5.2
Route
of
Administration
Peroral
routes
of
administration
are
the
most
common
procedures
used
to
introduce
a
chemical
into
test
animals
to
assess
its
toxic
properties
(
Tyler
1999).
These
routes
are
commonly
used,
because
they
can
provide
realistic
uptake
scenarios
and
relative
ease
in
quantifying
dose.
Choosing
the
route
of
administration
should
be
based
on
the
type
of
exposure
encountered
by
birds
in
the
environment.
Exposure
via
food
or
water
is
the
most
realistic
of
the
routes,
although
continuous
exposure
at
constant
concentrations
is
unlikely,
given
the
foraging
ranges
of
most
bird
species.
Compounds
that
are
insoluble
in
water,
or
that
are
volatile
or
reactive
could
need
to
be
administered
orally
by
bolus.
The
strengths
and
weaknesses
of
these
three
peroral
routes
as
they
pertain
to
avian
reproductive
tests
are
discussed
below.
Battelle
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40
April
23,
2003
5.2.1
Food
Oral
exposure
through
contaminated
food
is
the
most
common
route
used
for
chronically
exposing
birds
to
environmental
toxicants.
This
route
is
used
because
administration
of
the
test
substance
by
diet
with
ad
libitum
access
allows
for
a
more
natural
exposure
throughout
the
duration
of
the
study
and
avoids
the
intermittent
high
body
loading
of
bolus
(
also
called
gavage,
or
forced­
feed)
dosing.
Although
birds
consume
food
throughout
the
day,
a
bolus­
like
exposure
does
occur
with
the
typical
gorge
feeding
at
dawn
and
dusk
(
just
after
lights
on
and
just
before
lights
off
in
the
laboratory
setting)
observed
in
many
bird
species;
however,
it
is
unlikely
that
the
dietary
bolus
effect
is
as
pronounced
as
that
from
gavaged
doses.

A
major
disadvantage
of
dietary
exposure
compared
with
gavage
dosing
is
that
dose
estimation
(
milligram
per
kilogram
per
day)
is
much
less
precise,
because
the
test
substance
is
not
delivered
directly
into
the
digestive
tract
of
the
bird.
The
feeding
habit
of
caged
quail
results
in
wastage
of
a
great
amount
of
food,
which
can
increase
feed
requirements
from
14­
18
g/
day
to
30­
35
g/
day
(
NRC
1969),
making
accurate
estimates
of
food
consumption,
and
accordingly,
the
ingested
dose
difficult.
Use
of
wire
mesh
over
feed
dispensers
and
other
restrictions
reduce
billing
out
of
feed,
but
any
such
control
measures
must
not
restrict
ad
libitum
access
to
food.
Also,
some
test
substances
can
be
unpalatable
to
the
birds
or
cause
anorexia
(
e.
g.,
Stromborg
1986a,
1986b;
Bennett
and
Bennett
1990)
and
consequently
result
in
diet
avoidance.
In
mammalian
studies,
bolus
dosing
would
be
employed
to
determine
the
toxicity
and
endocrine
system
impacts
of
such
chemicals
(
Wilson
and
Hayes
1994).
However,
food
avoidance
or
anorexia
in
birds
is
considered
an
important
consequence
of
dietary
exposure
to
chemicals
in
the
environment
(
Bennett
and
Ganio
1991;
Bennett
et
al.
2001)
and
bolus
dosing
is
not
often
employed
to
otherwise
determine
impacts
in
avian
reproductive
tests.
Therefore,
measures
of
food
consumption
are
essential
to
the
interpretation
of
results
from
feeding
studies.
Typically,
dietary
exposures
are
administered
continuously
at
constant
concentrations
throughout
an
exposure
period
of
several
weeks.
Continuous
exposure
to
consistent
concentrations
for
several
weeks
will
almost
always
expose
birds
to
a
great
deal
more
test
substance
than
they
would
encounter
in
the
wild
environment,
because
they
do
not
feed
on
100%
contaminated
diet
in
the
latter
setting
due
to
the
large
foraging
ranges
of
most
bird
species
and
the
relatively
short
environmental
half­
life
of
contemporary
agrichemicals..
More
realistic
exposures
could
be
achieved
by
reducing
the
concentration
of
chemicals
in
the
diet
based
on
natural
degradation
rates
and
the
frequency
of
field
applications.
However,
such
dietary
adjustments
add
complexity
and
cost
to
the
exposure
regimen.

Stability
of
the
chemical
in
the
feed
must
be
verified
to
determine
the
frequency
of
diet
preparation
that
will
ensure
that
the
birds
receive
a
continuous
exposure
to
the
target
concentration
or
one
that
is
typical
of
attenuation
rates
in
the
environment.
Most
test
guidelines
require
that
the
dietary
concentrations
not
fall
below
80%
of
the
initial
concentration
(
OPPT
Guideline
850.2300;
OECD
Guideline
206).
It
is
also
essential
to
ensure
that
the
test
material
is
homogeneously
distributed
in
the
diet.
Because
some
chemicals
may
bind
to
the
feed,
thus
greatly
reducing
bioavailability
of
the
substances,
extraction
tests
should
also
be
conducted
to
assure
full
exposure
to
the
chemical.
The
chemical
analyses
required
for
these
verification
tests,
Battelle
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41
April
23,
2003
such
as
stability,
homogeneous
distribution,
and
bioavailability,
typically
exceed
the
analytical
costs
incurred
in
bolus
treatments.
However,
the
savings
in
labor
from
administering
the
chemical
in
the
diet
compared
with
the
intensive
labor
required
for
daily
bolus
dosing
(
Section
5.1.3)
usually
mitigates
the
analytical
costs
of
dietary
treatment.

Incorporation
of
the
test
substance
into
commercially
available
game
bird
chow
at
different
concentrations
is
relatively
simple
and
should
be
accomplished
whenever
possible
without
the
use
of
a
carrier.
Evaporative
solvents
or
low­
volatility
solvents
common
to
the
food
industry
are
used
when
needed
to
minimize
confounding
toxicity
from
carrier
substances.
However,
plant­
based
carriers
such
as
corn
oil
can
contain
endocrine­
active
compounds
and
should
be
tested
for
phytoestrogen
content
and
either
stripped
of
these
materials,
or
an
alternative
noncontaminated
carrier
should
be
used.
The
stripping
process
should
not
add
additional
contaminants
or
alter
the
palatability
of
the
feed.
Endocrine­
active
compounds
in
the
diet,
whether
feed
is
used
as
an
exposure
route
or
not,
are
also
of
concern.
For
example,
soy
and
corn,
which
contain
variable
amounts
of
phytoestrogens,
are
the
leading
constituents
of
most
commercial
game
bird
and
poultry
diets.
The
effect
on
test
results
of
diets
containing
natural
estrogenic
compounds
is
unknown,
as
is
the
effect
of
removing
phytoestrogens
from
the
diet.
Diets
amended
with
clover
extract
containing
elevated
levels
of
phytoestrogens
delayed
the
onset
of
egg
laying
and
reduced
egg
production
in
captive
California
quail
(
Leopold
et
al.
1975).
In
a
comparative
study
of
the
relative
potency
of
phytoestrogen
and
several
synthetic
estrogens,
the
synthetic
estrogens
 
estriol,
ß­
estradiol­
3­
benzoate
and
diethylstilbestrol
affected
bobwhite
reproduction
at
dietary
concentrations
of
100
:
g/
bird/
day.
The
phytoestrogen
biochenin
A
had
no
effect
on
reproduction
at
concentrations
up
to
1000
:
g/
bird/
day
(
Lien
et
al.
1985).

Overall,
administering
the
test
substance
in
feed
provides
for
a
more
environmentally
realistic
 
if
expected
environmental
concentration
and
chemical
decomposition
are
taken
into
account
 
and
cost­
effective
exposure
than
the
bolus
route
(
discussed
in
Section
5.2.3)
and
for
a
greater
range
of
compounds
than
can
be
delivered
in
water
(
see
Section
5.1.2).

5.2.2
Water
Water,
like
dietary
treatment,
is
an
ecologically
relevant
route
of
exposure
for
water­
soluble
chemicals,
as
long
as
adequate
dose
can
be
achieved.
Because
of
the
difficulty
in
maintaining
uniform
suspensions
or
emulsions
of
nonsoluble
substances
in
water,
this
route
is
seldom
used
as
the
route
of
exposure
for
compounds
with
low
water
solubility.
Concurrent
contamination
of
the
test
substance
with
endocrine­
active
compounds
or
other
contaminants
in
water
is
not
as
great
a
concern
as
it
is
in
dietary
exposures,
because
it
is
relatively
easily
purified
prior
to
mixing.
Dispersion
is
also
more
easily
achieved
for
water­
soluble
test
substances
administered
in
drinking
water.
However,
evaporation
may
result
in
concentration
of
the
test
substance
and
water
dispensers
that
minimize
evaporation
should
be
used
in
place
of
trough
feeders.
The
water
dispenser
should
also
provide
a
means
of
determining
the
amount
of
liquid
consumed
so
that
an
accurate
dose
can
be
calculated.
Water
spillage
is
more
serious
than
is
feed
spillage
in
determining
dose,
because
recovery
of
spilled
water
is
seldom
feasible;
therefore,
careful
monitoring
of
the
water
dispensers
must
be
maintained
throughout
the
dosing
phase
of
a
study.
Battelle
Draft
42
April
23,
2003
The
same
precautions
related
to
maintaining
test
concentrations
of
volatile
chemicals
in
feed
also
pertain
to
those
dispensed
in
water.

5.2.3
Bolus
Bolus
is
a
peroral
procedure,
in
which
the
test
substance
is
fed
to
the
bird
through
an
intubation
tube
directly
into
the
crop
or
proventriculus.
Although
usually
delivered
in
liquid
form
by
this
method,
material
can
also
be
delivered
in
gelatin
capsules.
It
is
used
to
simulate
oral
uptake
when
chemicals
have
physical
and/
or
chemical
properties
that
are
not
suitable
for
formulation
in
water
or
feed
due
to
problems
of
stability
or
volatility,
for
example,
or
when
the
chemical
is
unpalatable.
However,
palatability
and
avoidance
are
important
factors
influencing
reproduction
in
birds
through
decreased
caloric
intake
as
observed
for
some
OPs
(
e.
g.
Bennett
et
al.
1990;
Stromborg
1986a,
1986b;
Bennett
and
Ganio
1991)
and
provides
information
that
would
be
overlooked
with
bolus
dosing.
Although
diet
avoidance
data
are
important,
it
must
be
remembered
that
contaminated
feed
can
be
replaced
by
other
choices
in
food
types
and
location
in
the
field.

The
greatest
advantage
of
administering
the
test
substance
by
bolus
is
that
it
can
provide
the
most
accurate
dose
estimate.
The
researcher
directly
administers
a
known
amount
of
test
substance
to
each
bird
based
on
the
bird's
body
weight
and
does
not
have
to
account
for
spillage,
evaporation,
or
consumption
rates
to
calculate
the
dose.
But
because
each
bird
must
be
weighed
prior
to
each
dosing
event
to
calculate
the
dosing
volume
to
be
delivered,
and
each
bird
must
be
dosed
by
hand
each
day,
this
method
is
very
labor­
intensive
over
the
course
of
a
study.
Assuming
three
treatment
groups
and
a
control
group
with
16
pairs
of
birds
in
each
and
a
capture,
weighing,
dose
calculation,
and
dose
delivery
time
of
only
5
min/
bird,
labor
for
dosing
would
exceed
10
h/
day.
Dosing
a
large
number
of
animals
rapidly
also
greatly
increases
the
variability
in
bolus
accuracy
among
technicians,
and
deaths
from
delivery
of
the
dose
to
the
trachea
and
lungs
are
not
uncommon
(
Robens
et
al.
1994).
Handling
stress
can
also
have
impact
to
the
health
of
the
birds
and
has
been
shown
to
affect
plasma
levels
of
steroid
and
thyroid
hormones
(
Williamson
and
Davison
1985a;
Davison
et
al.
1985).

In
contrast
to
the
more
gradual
dosing
by
ingestion
of
a
test
compound
in
feed
or
water,
administration
of
a
daily
dose
of
chemical
in
a
bolus
can
alter
absorption
rates
and
saturate
hepatic
metabolic
enzyme
systems
(
Tyler
1999).
Greater
absorption
rates
result
in
higher
peak
plasma
concentrations,
and
thus,
lower
test
concentrations
can
be
tolerated
in
bolus­
dosed
animals
than
in
animals
receiving
feed
or
water
treatment.
Bolus
dosing
therefore
can
limit
the
achievable
dose
range.
Also,
saturation
of
hepatic
enzymes
from
bolus
doses
can
result
in
breakthrough
of
the
parent
chemical
and
higher
concentrations
(
or
lower
concentrations
of
activated
metabolites)
in
the
systemic
circulatory
system
than
occur
from
the
more
natural
dietary
exposure,
potentially
affecting
toxic
and
endocrine
responses.

The
timing
and
volume
of
bolus
dosing
are
also
important
to
the
outcome
of
the
test.
Because
gastric
emptying
time
can
affect
the
absorption
and
bioavailability
of
test
substances,
the
dose
is
generally
administered
when
little
feed
is
present
to
interfere
with
absorption
and
that
the
various
concentrations
(
milligram/
milliliter)
be
delivered
in
a
constant
volume
Battelle
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April
23,
2003
(
milliliter/
kilogram
body
weight).
The
former
requires
awareness
of
the
feeding
pattern
of
the
birds.
Also,
dosing
on
an
empty
gastrointestial
tract
is
not
similar
to
natural
exposure,
whereby
food
material
already
could
be
present
to
influence
the
availability
and
uptake
of
the
test
substance
in
the
gut.
In
addition,
regurgitation
can
result
in
significant
(
37%
to
72%)
loss
of
gavaged
dose
(
Hart
and
Thompson
1995).
Neither
volume
of
administered
dose
up
to
0.2%
of
body
weight
nor
prior
fasting
affected
the
extent
of
regurgitation
observed
in
starlings
(
Sturnus
vulgarus)
gavaged
with
chlorfenvinphos.
Use
of
gelatin
capsules
did
not
reduce
the
regurgitation
of
the
emetic
pesticide
(
Hart
1993).
However,
gelatin
capsules
may
allow
administration
of
larger
volumes
of
compounds
without
emetic
properties.
Splitting
the
dose
volume
between
morning
and
evening
periods
may
also
aid
in
dose
administration
by
capsule
or
liquid
gavage.
(.
It
should
be
noted
that
although
vomiting
has
been
reported
for
a
wide
range
of
avian
species
(
Prys­
Jones
et
al.
1973,
Tomacc
1975,
Zack
and
Falls
1976,
Hart
and
Thompson
1995),
the
extent
of
regurgitation
varies
between
the
species
and
between
chemicals
tested.
When
the
test
substance
is
not
water­
soluble,
a
corn
oil
vehicle
is
commonly
used
to
dilute
the
test
substance
and
administer
the
dose
to
the
animal.
However,
it
is
increasingly
recognized
that
use
of
this
vehicle
and
other
vegetable
oils,
mineral
oil,
and
some
emulsifying
agents
can
have
a
profound
effect
on
outcome
of
the
test.
In
general,
the
uptake,
peak
plasma
level,
and
toxicity
of
test
compounds
appear
to
be
exacerbated
by
corn
oil
(
e.
g.,
Farooqui
et
al.
1995;
Bull
et
al.
1986;
Chieco
et
al.
1981).
Use
of
corn
oil
increases
lymph
flow
and
the
uptake
of
lipophilic
compounds
such
as
p,
p'­
DDT
through
the
lymphatic
system
(
Sieber
1976),
thus
potentially
increasing
the
transfer
of
these
compounds
to
the
yolk
and
reducing
the
effect
of
hepatic
portal
first­
pass
circulation.
In
addition,
corn
oil
contains
endocrine­
active
substances
that
could
confound
reproductive
and
endocrine­
related
test
results.
Therefore,
aqueous
suspensions
of
less
readily
soluble
test
materials
are
often
preferred
over
food
oil
vehicles.

There
are
also
potential
problems
with
intubation
trauma
of
the
tissues
of
the
upper
gastrointestinal
tract
that
come
in
direct
contact
with
the
delivered
bolus
of
chemical
and
of
handling
stress,
particularly
of
laying
hens,
during
the
daily
dosing
regimen.
Handling
of
laying
hens
can
seriously
reduce
egg
production.

Overall,
the
disadvantages
of
this
dosing
procedure
 
elevated
cost,
excessive
handling,
regurgitation
of
dose,
potential
for
direct
tissue
injury,
technician
variability,
loss
of
test
subjects
due
to
misplacement
of
dose,
the
influence
of
oil
vehicles
on
response
parameters,
and
limitations
on
dosage
range
 
outweigh
the
advantage
of
accurate
dose
estimates.
It
should
only
be
used
when
the
stability
or
volatility
of
the
test
substance
precludes
using
feed
or
water
routes
of
exposure
or
when
the
test
substance
is
not
emetic,
is
administered
in
capsules,
and
sufficient
experienced
labor
or
reduced
number
of
birds
can
be
employed
to
minimize
time
and
stress
of
dosing.

5.3
Dose
Selection
Dose
refers
to
the
amount
in
milligrams
of
test
substance
administered
to
the
treated
animal.
Dose
can
be
administered
as
a
constant
daily
dosage,
amount
per
unit
body
weight,
or
as
a
constant
dietary
concentration.
Constant
dosage
adjustments
to
account
for
different
and
changing
food
consumption
rates
in
growing
birds
provide
a
means
of
comparing
effects
Battelle
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April
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2003
between
life
stages
and
species
of
different
body
size.
Dose
adjustments
are
made,
however,
at
the
expense
of
worst­
case
exposures
in
young
birds
and
the
ability
to
compare
results
with
environmental
concentrations.
Because
both
exposure
methods
are
highly
artificial
compared
with
the
dynamic
changes
in
diet
composition,
contaminant
loading,
and
consumption
rates
of
wild
birds
during
the
reproductive
season,
the
selection
of
exposure
method
should
be
determined
by
data
needs
of
the
risk
assessment
process
that
will
be
employed.

5.3.1
Dose
Adjustment
for
Size
Selection
of
test
concentrations
for
reproductive
studies
where
the
test
substance
is
administered
in
feed
is
often
based
on
providing
a
constant
daily
dose
to
the
animal.
An
expected
outcome
of
these
tests
is
the
determination
of
an
NOAEL
and/
or
a
lowest
observable
adverse
effect
level
(
LOAEL),
both
reported
in
milligrams
of
test
substance
ingested
per
kilogram
of
body
weight
per
day,
because
feed
is
not
measured
for
individual
birds,
and
body
weight
can
change
throughout
the
test;
further,
no
reproduction
study
requires
daily
weighing
of
birds.
These
and
other
dosage
values,
such
as
toxicity
values
expressed
as
the
amount
of
test
substance
administered
per
unit
body
weight
to
the
animal,
for
example,
provide
a
basis
for
comparing
effects
among
individuals
and
species
of
widely
varying
body
size.
Also,
during
the
rapid
growth
phase,
young
animals
consume
more
food
per
body
weight
than
adults,
and
thus
consume
more
test
substance
per
body
weight
per
day
than
adults.
Weight
changes
and
energy
requirements
of
reproductive
adults
also
differ
greatly
from
nonreproductive
adult
birds.
Therefore,
dietary
concentrations
of
the
test
substance
are
adjusted
to
attain
constant
dosage
among
all
animals
within
a
treatment,
regardless
of
size.

Maintenance
of
a
constant
daily
dose
ensures
that
the
young
are
not
exposed
to
concentrations
above
the
maximum
tolerated
dose
(
MTD)
and
that
the
relative
sensitivity
of
the
life
stages
to
the
test
substance
can
be
evaluated.
However,
frequent
diet
adjustments
would
have
to
be
made
during
the
rapid
growth
phase
to
maintain
a
constant
dosage
throughout
the
treatment
period.
The
frequency
of
dietary
treatment
changes
should
minimize
the
dosage
differences
between
the
beginning
of
an
adjusted
dose
period
and
its
termination.
A
weekly
interval
is
commonly
used
in
mammalian
studies.
However,
for
some
portions
of
the
growth
curve,
more
frequent
dose
adjustments
would
be
needed
for
the
Japanese
quail,.
For
example,
a
weekly
dosing
interval
could
result
in
the
chicks'
consuming
a
dose
at
the
beginning
of
the
period
that
is
43%
greater
than
that
at
the
end
of
the
same
dosage
interval
(
Figure
5­
1).
Also,
individual
birds
vary
in
their
rate
of
food
consumption
and
body
weight
at
any
given
age,
resulting
in
variable
doses
within
treatments,
even
when
the
diet
is
adjusted.
Adjusting
dietary
concentrations
to
compensate
for
changing
food
consumption
rates
requires
weighing
the
birds
to
obtain
weight­
gain
data
on
which
to
base
the
diet
adjustments,
and
thereby
introduces
a
great
deal
of
handling
stress
into
the
study,
as
well.
Labor
and
analytical
costs
to
verify
treatment
concentrations
are
also
increased
by
frequent
diet
concentration
adjustments.
Battelle
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April
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5.3.2
Dose
Adjustment
by
Life
Stage
(
Young
vs.
Adult)

The
goal
of
avian
reproductive
studies
has
been
to
estimate
risk
to
wildlife
from
exposure
to
expected
field
levels
of
a
contaminant.
Because
chicks
that
will
become
the
reproductive
adults
of
the
F1
generation
would
be
exposed
under
worst­
case
scenarios
to
the
same
field
levels
of
a
contaminant
as
they
would
as
adults,
the
effects
of
exposure
during
this
life
stage
are
important
to
the
outcome
of
the
test.
That
is,
the
results
from
tests
designed
around
a
constant
dietary
exposure
are
more
directly
comparable
to
environmental
concentrations
than
those
that
adjust
exposure
by
life
stage.

There
are
disadvantages
to
this
approach.
The
range
of
doses
in
reproduction
studies
are
based
on
adult
exposure
to
ensure
maximum
challenge
to
the
reproductive
parent;
therefore,
young
birds
consuming
a
proportionately
greater
amount
of
contaminated
feed
during
their
growth
phase
acquire
a
daily
dose
through
their
diet
that
will
be
considerably
higher
than
that
which
they
will
ingest
as
adults.
This
can
result
in
dose
overlap,
whereby
the
young
are
virtually
in
a
different
dose
group
than
their
parents
for
a
period
of
time
(
Wilson
and
Hayes
1994).
In
Figure
5­
1,
growth
and
food
consumption
data
from
Marks
(
1991)
were
used
to
calculate
the
ingested
dose
at
weekly
intervals
of
birds
that
were
consuming
a
diet
containing
2­
week­
old
Japanese
quail
and
the
7­
week
old
quail
fed
the
same
100
ppm
diet
are
significantly
different
(
15.8
mg/
kg
and
9
mg/
kg,
respectively).
If
the
dietary
treatments
were
separated
by
a
geometric
factor
of
0.6,
the
next
highest
treatment
would
be
about
167
ppm.
The
ingested
dose
in
a
7­
week­
old
would
be
about
15
g
or
less
than
the
amount
ingested
by
the
2­
week­
old
chick
at
the
lower
treatment
level.
Dose
concentrations
are
usually
based
on
either
MTD
of
the
reproductive
adult
or
a
multiple,
such
as
five
times,
for
example,
the
expected
field
concentration,
to
provide
sufficient
challenge
to
observe
effects
in
reproduction
studies.
Therefore,
by
not
adjusting
down
the
dietary
concentration
to
account
for
the
higher
rate
of
intake,
the
F1
chicks
will
receive
higher
dosages
of
chemical
than
reflected
in
the
adult
weight­
based
test
concentrations.
The
greater
ingested
doses
can
be
lethal
to
the
F1
chicks
and
hinder
evaluation
of
the
effects
of
the
test
substance
on
the
second
generation.
However,
mortality
of
chicks
due
to
direct
acute
toxicity
may
occur
in
the
environment
and
could
cause
a
valid
reduction
in
recruitment
for
exposed
populations.
Battelle
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April
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2003
0
5
10
15
20
25
30
1
2
3
4
5
6
7
8
Age
(
weeks)
Dosage
(
mg/
Kg)
at
Constant
Exposure
100
ppm
167
ppm
Figure
5­
1.
Calculated
Dose
at
Weekly
Intervals
of
Growing
Japanese
Quail
Consuming
a
Diet
Amended
with
100
or
167
ppm
of
Test
Substance
(
body
weight
and
food
consumption
data
from
Marks
[
1991])

Comparison
of
chemical
sensitivity
among
life
stages
is
also
complicated
by
the
unequal
daily
doses
among
individuals
and
at
different
life
stages.
However,
the
logistics
of
periodically
adjusting
the
dietary
concentrations
is
eliminated
by
the
consistent­
exposure
design,
reducing
both
labor
and
cost
of
analytical
verification
of
the
different
test
concentrations.
This
is
particularly
important
when
using
the
Japanese
quail
as
the
test
species,
because
numerous
adjustments
of
test
concentrations
would
be
required
to
compensate
for
their
rapid
changes
in
body
weight
and
to
provide
a
reasonably
constant
daily
dose
at
both
the
beginning
of
an
adjusted
dose
period
and
its
termination.
If
an
NOAEL
or
LOAEL
is
reported
from
data
obtained
under
a
constant
exposure
in
feed,
care
should
be
taken
that
the
value
is
represented
as
a
dietary
concentration
in
parts
per
million.
Reporting
values
based
on
adult
daily
dosage
(
milligram/
kilogram/
day)
is
not
representative
of
the
doses
experienced
by
the
birds
over
the
entire
exposure
period.

5.4
Statistical
Considerations
Statistical
approaches
for
interpreting
avian
reproductive
toxicity
tests
have
been
reviewed
by
a
number
of
authors.
Bennett
et
al.
(
1990,
2001),
Mineau
et
al.
(
1994),
and
Schlatterer
et
al.
(
1993)
discussed
the
effects
of
mortalities
and
incompatibility
of
birds
on
sample
size
associated
1Benchmark
dose
(
BMD)
is
defined
as
"
a
statistical
lower
confidence
limit
on
the
dose
producing
a
predetermined
level
of
change
in
an
adverse
response
compared
to
[
sic]
a
response
in
untreated
animals"
(
EPA
1995).
For
example,
a
BMD10
would
be
the
95%
lower
confidence
limit
on
a
dose
that
produces
a
10%
increase
in
an
adverse
effect,
such
as
the
number
of
chicks
with
ovatestes.

Battelle
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47
April
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2003
with
the
timing
and
duration
of
treatment
in
reproductive
studies
using
bobwhite
and
Japanese
quail.
A
reduction
in
the
sample
size
directly
affects
the
power
of
hypothesis
testing
and
the
estimation
of
the
NOAEL.
Bennett
and
Ganio
(
EPA
1991)
and
MacLeod
(
1994)
provided
recommendations
on
determining
which
multiple
comparison
procedure
should
be
used
and
how
the
testing
should
be
carried
out
to
estimate
the
NOAEL.
Farrar
(
David
Farrar,
personal
communication
to
T.
Maciorowski,
D.
Urban,
D.
McLane,
and
D.
Balluff,
Statistical
aspects
of
the
evaluation
of
avian
reproductive
effects:
accomplishments
and
objectives,
1995)
and
Collins
(
1994)
compared
the
power
of
hypothesis­
testing
using
specific
endpoints
measured
in
mallard
ducks
and
bobwhite
quail,
and
Springer
and
Collins
(
1999)
evaluated
the
power
of
hypothesis­
testing
between
bobwhite
and
Japanese
quail.
Chapman
et
al.
(
1996),
Hart
et
al.
(
1999),
and
Crane
and
Newman
(
2000)
suggested
that
the
use
of
regression
methods
is
a
better
approach
to
evaluating
toxicity
in
avian
reproduction
tests,
because
they
are
less
affected
by
the
loss
of
replicates
than
are
ANOVA
methods
and
they
estimate
a
dose­
response
curve
that
can
be
used
to
compare
sensitivity
both
between
endpoints
and
species.
Baril
et
al.
(
1994)
and
Hart
et
al.
(
1999)
discussed
risk
assessment
using
the
estimated
median
lethal
concentration
(
LC50)
from
the
regression
approach
and
alternative
design
strategies
to
reduce
the
number
of
birds
on
test.
Recently,
EPA
evaluated
and
adopted
a
method
based
on
dose­
response
curves
as
an
alternative
to
the
NOAEL
approach
to
human
health
risk
assessment
(
EPA
1995,
1999).
This
benchmark
dose
method1
can
evaluate
both
quantal
and
continuous
data,
and
provide
a
risk
reference
dose
that
can
be
used
like
a
NOAEL
or
LOAEL.
Because
this
approach
is
based
on
regression
methods,
it
incorporates
information
on
the
shape
of
the
dose­
response
curve
and
can
be
used
for
both
threshold
and
nonthreshold
effects.

The
objective
of
an
avian
two­
generation
reproductive
and
developmental
toxicity
test
is
to
provide
the
most
precise
and
accurate
estimate
of
toxicity
associated
with
endocrine
disruption
and
reproductive
fitness
for
an
identified
potential
EDC.
The
results
of
the
Tier
2
testing
should
be
conclusive
in
documenting
a
discernible
cause­
and­
effect
relationship
of
chemical
exposure
to
measurable
manifestation
in
the
test
organisms.
The
test
protocol
will
be
designed
to
be
capable
of
the
following:
°
to
determine
whether
effects
are
a
primary
or
secondary
disturbance
of
endocrine
function
°
to
establish
exposure/
concentrations/
timing
and
effects
relationships
°
to
be
sensitive
and
specific
°
to
assess
relevant
endpoints
°
to
include
a
dose
range
for
full
characterization
of
effects
(
EDSTAC
1998).

Thus,
the
assay
must
be
biologically
sensitive,
have
minimal
variability
associated
with
dose
exposure
throughout
the
test
duration,
and
have
a
statistically
powerful
inference.
Biological
sensitivity
is
a
function
of
the
choice
of
species
tested,
the
relevance
of
the
endpoints
measured
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to
species
productivity
and
survival,
and
the
route,
duration,
and
level
of
the
chemical
exposure.
Design­
associated
variability
in
dose
exposure
is
a
function
of
exposure
route
and
duration,
chemical
stability
and
purity
within
the
testing
environment,
and
the
testing
protocol.
The
power
of
a
statistical
inference
is
a
function
of
the
inherent
variability
in
response;
design­
associated
variability;
the
degrees
of
freedom
and
the
source
of
variability
for
testing;
and
the
estimation
process
and
decision
criteria.

Ideally,
an
experimental
design
incorporates
randomness,
independence,
and
replication
(
Cochran
and
Cox
1957).
Randomness
is
used
to
remove
noise,
independence
is
used
to
extend
the
inferences
made,
and
replication
provides
a
measure
of
variability
for
testing
(
Chapman
et
al.
1996).
Randomization
of
1)
experimental
containers
within
a
testing
environment,
2)
treatment
application
to
experimental
containers,
and
3)
assignment
of
organisms
to
experimental
containers
allows
one
to
incorporate
the
variability
associated
with
the
environmental
conditions,
the
containers,
and
the
organism
equally
across
all
treatments.
Thus,
when
one
evaluates
the
difference
between
treatment
means,
the
variability
associated
with
experimental
environment,
experimental
containers,
and
organisms
being
treated
is
removed
and
only
the
effect
of
the
treatment
remains.

Independence
of
treatment
application
and
the
creation
of
the
treatment,
and
thus,
the
inference
associated
with
the
treatments
under
test,
incorporates
the
variability
associated
with
more
than
one
individual,
in
more
than
one
location,
making
and
applying
the
same
treatment.
The
random
sample
of
organisms
from
a
given
population
actually
limits
the
inference
to
that
population.
However,
one
can
evaluate
the
stability
of
the
inherent
variability
of
the
population
over
time.
An
experimental
unit
is
defined
as
the
group
of
material
or
individuals
to
which
a
treatment
is
applied
independently
in
a
single
trial
of
the
experiment
(
Cochran
and
Cox
1957).
Replication
of
experimental
units
for
each
treatment
provides
a
measure
of
all
the
necessary
sources
of
variability
needed
to
extend
the
inference
across
time
and
space.
A
reduction
in
the
sources
of
variability
that
are
truly
independent
constrains
the
inference
(
Hurlbert
1984).
Thus,
if
only
one
mix
of
each
treatment
is
made
and
then
divided
among
replicates,
the
source
of
variation
associated
with
making
the
treatment
is
not
included
in
the
variability
for
testing,
and
the
inference
is
limited.
Some
would
say
that
this
variability
is
nuisance
noise,
too
small
to
be
of
concern.
Therefore,
if
this
source
of
variability
is
not
included,
it
should
at
least
be
acknowledged.
The
variability
among
replicate
experimental
units
could
also
include
noise
that
was
not
randomized
out
due
to
a
poor
randomization
or
variable
measurement
error.
These
sources
of
variability
can
be
reduced
without
loss
to
inference.

Statistical
power
is
the
probability
of
rejecting
the
null
hypothesis
of
equal
means
when
the
alternative
is
true
 
that
is,
detecting
a
difference
when
there
is
a
difference.
Statistical
power
is
a
function
of
the
variability
among
replicate
experimental
units
within
a
treatment,
the
number
of
replicate
experimental
units,
the
size
of
the
Type
I
error,
and
the
percentage
difference
one
wishes
to
detect.
One
can
control
the
latter
three
components;
however,
the
variability
in
response
is
inherent
in
the
test
organism.
Thus,
the
choice
of
species
to
test
and
the
relevant
endpoints
to
measure
should
include
a
comparison
of
inherent
variability
or
coefficients
of
variation
(
CVs),
defined
as
standard
deviation/
mean
x
100%.
High
CVs
have
low
power
for
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April
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detecting
small­
scale
differences.
For
example,
with
16
replicates
per
treatment,
a
CV
of
50%
would
rarely
detect
differences
less
than
30%
between
the
test
and
reference
treatment
response
at
a
Type
I
error
rate
of
"
=
0.05
(
Figure
5­
2).
For
a
given
CV,
one
can
increase
power
by
increasing
the
number
of
replicates.
Test
species
and
endpoints
with
the
least
inherent
variability,
by
default,
require
the
least
replication
for
a
given
level
of
power
and
thus
are
more
cost­
effective.

Life­
cycle
studies
provide
a
number
of
continuous
and
discrete
random
variables
over
the
course
of
the
exposure.
Examples
of
continuous
data
include
growth
measured
as
size
and
weight,
the
gonadal
somatic
index,
biochemical
markers,
and
time
to
first
laying.
These
data
are
analyzed
using
ANOVA
and
pair­
wise
comparison
techniques
to
determine
difference
between
treatments
and
controls,
and
regression
or
maximum
likelihood
techniques
to
estimate
effective
concentrations.
Life­
cycle
studies
also
produce
a
large
number
of
discrete
data
points,
such
as
sex
ratios,
histopathology
records,
secondary
sex
characteristics,
behavioral
observations,
survival,
and
fertilization
or
hatch
success.
These
data
can
be
analyzed
by
ANOVA
if
arcsine
square
root
transformed,
and
by
pair­
wise
comparison
techniques,
contingency
table
techniques
to
assess
association,
and
regression
or
maximum
likelihood
techniques
to
estimate
effective
concentrations.
Fisher's
Exact
Test
is
an
example
of
a
technique
for
comparing
two
sets
of
discrete
quantal
data.

Data
collected
by
Schlatterer
et
al.
(
1993)
for
an
interlaboratory
comparison
study
on
Japanese
quail
can
be
used
to
compare
the
CVs
for
a
range
of
endpoints
(
Table
5­
2).
Five
laboratories
obtained
8­
to
12­
week­
old
birds
from
the
same
breeder
and
randomly
assigned
12
pairs
to
the
control
diet.
Fitness
measures
were
then
taken
weekly
for
6
weeks
or
from
eggs
laid
during
Weeks
5
and
6.
The
body
weight
of
hatchlings,
percentage
of
fertile
eggs,
and
percentage
hatch
of
fertile
eggs
had
the
smallest
CVs
for
measurements
with
10
observed
means.
Endpoints
measured
on
eggs
were
not
significantly
different
between
Weeks
5
and
6.
The
CVs
generally
increased
with
dose;
thus,
these
estimates
are
potentially
quite
low.
In
general,
there
are
few
data
for
which
a
power
analysis
can
be
conducted
with
great
confidence.
An
increase
in
CV
as
a
function
of
dose
is
dependent
on
the
organism
under
test,
the
exposure
chemical,
and
its
toxicity
to
the
endocrine
system.
A
simulation
study
by
Collins
(
1994)
using
control
data
from
mallard
ducks
and
bobwhite
quail
concluded
that
for
birds
caged
1:
1,
exposed
over
an
8­
week
period,
40
and
35
pens
per
treatment,
respectively,
were
required
to
provide
80%
power
for
detecting
a
20%
change
in
the
affected
variable.
High
CVs
for
these
birds
were
confirmed
by
Farrar
(
David
Farrar,
personal
communication
to
T.
Maciorowski,
D.
Urban,
D.
McLane,
and
D.
Balluff,
Statistical
aspects
of
the
evaluation
of
avian
reproductive
effects:
accomplishments
and
objectives,
1995).
Battelle
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0%
20%
40%
60%
80%
100%

0%
10%
2
0%
30%
40%
50%
60%
70%
80%

%
Reduction
of
Control
Mean
Power
of
Test
CV
=
5%

CV
=
10%

CV
=
15%

CV
=
20%

CV
=
30%

CV
=
40%

CV
=
50%

CV
=
60%

CV
=
70%

Figure
5­
2.
Power
of
a
One­
Sided
Independent­
Samples
T­
Test
as
a
Function
of
the
Percentage
Reduction
Detected
Between
the
Test
and
Reference
Means,
with
16
Replicates
per
Treatment
("
=
0.05)
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Table
5­
2.
Control
Data
Coefficient
of
Variation
(
CV%)
for
the
Raw
and
Transformed
Endpoints
Measured
from
an
Interlaboratory
Comparison
Study
on
Japanese
Quail
Exposed
to
Bis(
tri­
 
butyltin)
oxide(
a)

Variable
N
Raw
Data
(%)
Transform
ed
Data
(%)
Dose
Effect
Detecte
d
Number
eggs
laid
per
week
(
intercept)
5
14
NA(
b)
NS(
c)

Mean
egg
weight
per
week
(
intercept)
5
3
NA
NS
Mean
egg
weight
per
week
2
6
NA
NS
Percentage
cracked
eggs
3
79
54
*(
d)

Percentage
fertile
eggs
10
8
10
**(
e)

Percentage
hatch
of
fertile
eggs
10
8
8
**

Percentage
chicks
dead
in
shell
4
22
12
**

Number
hatched
chicks
10
8
8
**

Body
weight
of
hatchlings
10
6
3
NS
Number
14­
day­
olds
10
17
9
**

Weight
of
14­
day­
olds
10
21
6
NS
Percentage
surviving
chicks
hatched
in
Weeks
5
and
6
10
14
15
**

a)
Table
adapted
from
Schlatterer
et
al.
(
1993).
b)
NA
Not
applicable.
c)
NS
Not
specified.
d)
*
P<
0.05.
e)
**
P
<
0.01.

Springer
and
Collins
(
1999)
stated
that
it
is
very
difficult
to
make
comparisons
of
power
for
hypothesis
testing
between
bobwhite
and
Japanese
quail,
because
the
design
of
the
studies
have
different
starting
times
of
exposure
within
the
life
cycle
of
the
birds:
that
is,
before
maturation
and
after
egg­
laying,
respectively.
The
effect
of
the
different
starting
times
of
exposure
is
further
confounded
with
the
time
required
to
reach
chemical
equilibrium
in
the
bird's
tissues.
Thus,
effects
observed
within
Weeks
5
to
10
in
bobwhite
quail
dosed
at
3weeks
may
not
be
observed
in
Japanese
quail
dosed
post
egg­
laying,
until
Week
12.
Additionally,
one
must
consider
the
decrease
in
variability,
with
increased
power
for
testing,
in
the
number
of
eggs
laid
as
birds
approach
peak
egg
production
(
Springer
and
Collins
1999).
Peak
egg
production
generally
occurs
at
Weeks
5
to
6
and
Weeks
10
to
11
for
Japanese
quail
and
bobwhite,
respectively.
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5.4.1
Sample
Size
5.4.1.1
Ensuring
Adequate
Number
of
Fertile
Pairs.
In
the
OECD
draft
guideline
(
OECD
1999),
the
initial
test
groups
would
consist
of
20
replicate
pens
to
increase
the
likelihood
that
at
least
16
of
them
remain
in
each
group
at
test
termination.
The
highest
concentration
tested
will
be
below
levels
shown
to
cause
mortality
or
severe
signs
of
parental
toxicity
from
range­
finding
screening
tests
or
from
existing
toxicological
data,
but
will
be
of
a
level
that
is
expected
to
reveal
significant
effects
on
reproductive
and
endocrine
endpoints.
Dosing
of
the
P1
will
commence
at
4
weeks
of
age
under
one
scenario,
or
after
2
weeks
of
egg­
laying
by
proven
breeders
under
a
second
scenario,
and
continue
until
test
termination,
depending
on
the
resulting
inherent
variability
observed
in
the
preliminary
tests.
Both
exposure
scenarios
have
benefits,
which
are
discussed
in
Bennett
et
al.
(
2001).
Exposure
before
sexual
maturation
allows
quantification
of
a
delay
in
the
onset
of
laying
and
gonadal
development.
Further,
if
the
exposure
chemical
requires
a
period
of
time
to
build
up
in
the
tissues
before
the
maximum
exposure
level
in
the
eggs
is
reached,
a
delayed
response
in
the
F1
endpoints
may
be
observed.
In
contrast,
exposure
before
pairs
are
proven
breeders
risks
a
loss
of
replication
 
and
statistical
power
 
due
to
incompatibility
or
infertility.
Indeed,
Bennett
et
al.
(
1990)
reported
that
24
out
of
60
pairs
(
40%)
were
removed
from
the
test
during
the
pre­
exposure
period
due
to
mortality,
injury,
or
failure
to
produce
adequate
numbers
of
eggs.
Mineau
et
al.
(
1994),
who
reviewed
134
avian
exposure
studies,
confirmed
this
high
rate
of
infertility
and
mortality.
For
either
exposure
scenario,
it
is
recommended
that
a
population
of
birds
not
be
used
if
more
than
5%
of
either
sex
becomes
debilitated
in
the
7­
day
period
immediately
prior
to
test
initiation.

Hart
et
al.
(
1999)
and
Bennett
et
al.
(
2001)
both
reported
that
exposure
following
proven
breeding
allows
the
pretreatment
measurements
of
fitness
to
be
used
as
covariates
in
the
statistical
analysis.
In
some
cases,
the
covariate
data
can
increase
the
power
of
the
statistical
test.
Removal
of
incompatible
or
infertile
birds
prior
to
exposure
also
removes
nontreatment­
related
sources
of
variation,
which
again
can
increase
the
power
of
the
test.

5.4.1.2
Ensuring
Adequate
Number
of
Offspring
For
Testing
of
Successive
Generations.
The
variability
of
an
endpoint
increases
with
the
number
of
phases
of
the
reproductive
process
being
tested.
Thus,
there
is
less
power
in
detecting
differences
in
14­
day­
old
survivors
than
for
detecting
differences
in
the
number
of
eggs
laid
(
Hart
et
al.
1999).
Additionally,
oral
exposure
could
reduce
food
intake,
thus
affecting
the
exposure
dose
and
resulting
egg
weights
for
several
days
of
testing.
Schlatterer
et
al.
(
1993)
and
Bennett
et
al.
(
1990)
showed
that
a
reduced
food
intake
at
higher
doses
was
correlated
also
with
greater
impairment
of
fertility
rate
or
a
cessation
of
hatching
for
Japanese
quail
and
bobwhite,
respectively.
For
exposure
scenarios
in
which
treatment
is
initiated
after
a
period
of
proven
egg
production,
chicks
raised
for
the
F1
breeding
population
should
be
taken
from
egg
batches
produced
at
the
end
of
the
parental
treatment
period,
thus
allowing
time
for
the
full
effect
of
the
treatment
to
be
established
on
the
egg
production
and
viability
of
young.

The
F1
populations
will
be
divided
into
groups
of
20
replicate
pens
according
to
their
parental
test
diet
and
will
either
receive
no
treatment
or
the
same
treatment
as
the
parental
diet.
If
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treatment
groups
experience
a
significant
reduction
in
the
number
of
F1
chicks
produced,
eggs
produced
over
the
last
10
to
14
days
can
be
accumulated.
Thus,
all
F1
birds
will
be
the
same
age.
For
treatments
with
insufficient
chicks
for
pairing,
either
back­
filling,
the
effects
of
which
are
discussed
in
Section
5.1.5,
would
be
employed,
or
the
test
continued
with
an
unbalanced
number
of
replicates,
or
the
treatment
discontinued.
For
any
of
these
strategies,
one
must
account
for
the
objective
of
this
experiment,
which
is
to
produce
the
worst­
case
scenario;
however,
the
natural
variation
in
the
species
to
respond
to
the
contaminant
will
by
default
produce
more
successful
F1
pairs
that
are
not
as
susceptible
to
the
contaminant.

5.4.2
Hypothesis­
Testing
or
Regression
Analysis
There
has
been
much
debate
over
the
use
of
the
NOAEL
in
toxicity
assessment
and
the
associated
risk
analysis
(
Bennett
et
al.
2001;
Crane
and
Newman
2000;
Chapman
et
al.
1996).
The
debate
stems
from
the
perceived
goal
of
the
avian
reproductive
test:
to
detect
effects
on
the
reproduction
of
the
test
population
at
the
lowest
dietary
concentrations
that
produce
biologically
significant
effects
(
EPA
1991).
The
desire
to
detect
effects
implies
a
comparison
of
means.
ANOVA
methods
are
appropriate
for
comparing
means,
asking
the
question
of
whether
the
treatment
means
are
statistically
different
from
the
control,
such
as
in
a
screening
test
or
a
validation
test.
However,
ANOVA
methods
are
not
appropriate
when
a
precise
and
accurate
estimate
of
toxicity
and
the
pattern
of
response
are
required.
There
is
also
a
false
positive
error
rate
in
ANOVA,
because
the
many
parameters
assessed
are
not
all
independent.
Regression
techniques
provide
an
estimate
of
the
level
of
effect
as
a
function
of
exposure
(
nominal
or
actual
concentration)
and
the
functional
relationship
between
dose
and
response.
Further,
by
analyzing
the
different
dose­
response
relationships,
one
can
compare
the
sensitivity
and
potential
thresholds
of
effect
for
different
endpoints.

Although
the
NOAEL
is
used
widely,
it
should
not
be
relied
on
as
the
sole
indicator
of
low
toxicity
(
Crane
and
Newman
2000;
Chapman
et
al.
1996;
EPA
1991).
The
largest
dose
for
which
statistical
differences
have
failed
to
be
detected
is
a
direct
function
of
the
power
of
the
test:
failure
to
reject
the
null
hypothesis
of
no
difference
does
not
mean
that
there
was
no
effect.
For
example,
for
certain
endpoints
with
CVs
greater
than
or
equal
to
40%,
it
is
unlikely
that
reductions
less
than
30%
will
be
detected
with
16
replicates
per
treatment
(
Figure
5­
2).
It
is
also
conceivable
that
short­
term
range­
finding
experiments
will
have
difficulty
in
predicting
the
location
of
a
NOAEL
in
a
multigenerational
test.
It
may
also
prove
difficult
to
achieve
effects
bracketing
the
50%
response
in
the
F1
population.
However,
effect
concentration
calculations
are
an
appropriate
alternative
for
estimating
doses
associated
with
low
to
medium
toxicity.
Care
must
be
taken
not
to
estimate
an
effects
concentration
that
is
more
sensitive
than
the
data
and
the
experimental
design
will
allow.
Precision
and
accuracy
of
the
effects
concentration
is
a
function
of
the
spread
between
treatment
concentrations
and
the
number
of
concentrations
tested
(
Chapman
et
al.
1996).

The
design
and
analysis
requirements
for
estimating
the
NOAEL
differ
from
those
for
fitting
a
dose­
response
model
(
Chapman
et
al.
1996;
Stephan
and
Rogers
1985).
ANOVA
methods
require
experimental
unit
replication
and
achieve
greater
power
in
testing
as
a
function
of
the
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number
of
replicates.
As
shown
in
Figure
5­
2
and
Table
5­
2,
the
different
endpoints
would
require
different
amounts
of
replication
to
achieve
the
same
level
of
power.
However,
16
replicates
provide
greater
than
80%
power
for
detecting
less
than
a
20%
change
at
"
=
0.05
from
the
control
for
most
of
the
endpoints
in
Table
5­
2,
assuming
the
CVs
do
not
increase
with
dose
beyond
20%.
Transformation
of
the
data
to
satisfy
homogeneity
of
variance
is
required
for
the
parametric
test
and
the
regression
approach.
Estimation
of
the
NOAEL
does
not
require
the
assumption
of
a
specific
model,
such
as
log­
normal,
and
ANOVA
methods,
such
as
the
t­
test
and
Dunnett's
test,
are
robust
to
non­
normal
errors
(
Scheffé
1959).

The
design
of
a
study
intended
for
dose­
response
modeling
does
not
require
replication
of
the
treatments
(
Snedicor
and
Cockran
1980).
Each
individual
responses
is
assumed
to
be
a
random
response
from
a
normal
population
of
responses
for
a
given
dose.
The
variance
is
assumed
to
be
equal
for
each
population.
Replication
of
doses
provides
a
test
of
equal
variance
and
lack­
of­
fit
(
Draper
and
Smith
1981).
Further,
because
of
the
unpredictable
nature
of
survival,
fertility,
and
compatibility
of
birds
in
the
two­
generation
test
and
the
large
variability
in
specific
endpoints,
it
is
desirable
to
have
some
level
of
treatment
replication
to
provide
a
more
accurate
estimate
of
the
mean
population
response
for
a
given
dose.
The
number
of
replicates
would
depend
on
the
maximum
expected
variability
in
response
for
each
dose.
The
variability
in
response
may
be
a
function
of
the
dose.
In
this
case,
either
a
weighted
analysis
should
be
conducted
or
a
data
transformation
applied
that
satisfies
the
assumption
of
homogeneity
of
variance.

Benefits
of
the
regression
approach
include
1)
estimation
of
the
pattern,
or
slope,
of
toxicity
as
a
function
of
dose;
2)
estimation
of
the
distance
between
effect
concentrations
and
environmental
concentrations;
3)
estimation
of
effective
doses
(
EDx)
and
their
associated
confidence
intervals
for
x
equal
to
a
low
to
medium
effect;
4)
estimation
of
EDx
not
limited
to
doses
on
test;
5)
use
of
both
measured
and
nominal
concentrations;
and
6)
ability
to
compare
dose­
response
curves
across
endpoints
(
Chapman
et
al.
1996;
EPA
1991).
The
size
of
the
resulting
confidence
intervals,
indicating
the
precision
of
the
estimated
EDx,
is
a
function
of
the
inherent
variability
in
the
response,
and
the
number
and
spacing
of
the
concentrations
tested.
Guidelines
often
require
five
concentrations
that
are
geometrically
spaced
and
sublethal,
plus
a
no­
dose
control.
Thus,
a
range­
finding
test
would
be
required
to
determine
appropriate
dietary
concentrations.

Regression
modeling
is
sufficiently
flexible
to
handle
a
wide
range
of
dose­
response
patterns,
including
nonmonotonic.
If
only
one
or
two
responses
are
not
either
0%
or
100%
affected
and
at
least
one
is
greater
than
50%
affected,
the
Spearman­
Karber
nonparametric
method
can
be
used
to
estimate
a
median
effective
concentration
(
EC50).
Finally,
the
regression
approach
can
handle
a
wide
range
of
responses,
including
continuous
responses,
counts,
and
quantal
data,
by
re­
expressing
or
transforming
the
data
(
e.
g.,
log[
y+
c],
[
y+
c]
1/
2),
and
probit,
respectively).

Regression
analysis
on
the
weekly
post­
treatment
responses
also
allows
an
evaluation
of
the
resulting
time­
series
and
a
potential
redefinition
of
the
effect
of
treatment
(
Hart
et
al.
1999).
For
example,
a
regression
of
the
rate
of
egg
production
against
time
elapsed
can
be
used
to
assess
the
shape
of
the
response,
the
daily
within­
class
variation
in
response,
and
the
potential
time­
lag
between
exposure
and
response.
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An
alternative
design
that
has
been
suggested
for
estimating
a
median
lethal
dose
(
LD50)
and
its
associated
confidence
interval
uses
an
up­
and­
down
procedure.
The
intent
of
the
design
is
to
minimize
the
number
of
animals
tested.
A
simulation
experiment
(
Hart
et
al.
2001)
concluded
that
the
precision
of
the
up­
down
estimate
was
not
as
good
as
that
achieved
from
the
regression
analysis.
However,
the
achieved
precision
from
the
up­
down
procedure
may
be
acceptable.
For
steep
dose­
response
relationships,
the
starting
doses
and
step
lengths
did
not
seem
to
affect
either
the
estimate
or
the
precision.
Often,
the
number
of
birds
required
was
too
low
for
an
estimate
of
the
slope
of
the
response.
For
shallow
slopes,
the
precision
was
much
less
than
that
achieved
by
regression,
and
the
step
length
seemed
to
affect
the
outcome.
It
is
not
clear
whether
this
type
of
design
would
be
applicable
to
two­
generation
testing.

6.0
ASSAY
ENDPOINTS:
FITNESS
END
POINTS
Fitness
endpoints
are
those
that
contribute
to
the
normal
functions
of
the
animal,
and
thus
relate
to
survival,
growth,
reproduction,
and
behavior
of
a
test
subject.
Endpoints
useful
to
assessing
the
reproductive
and
developmental
toxicity
of
chemicals
over
two
generations
include
those
that
provide
information
on
general
toxicity
of
the
compound
related
to
reproductive
success,
and
those
that
measure
the
disruption
of
endocrine­
mediated
processes
that
can
affect
production.
There
are
four
life
stages
in
precocial
birds,
such
as
the
Japanese
quail
and
northern
bobwhite,
during
which
critical
endocrine­
mediated
processes
occur.
These
potentially
endocrine
disruption­
sensitive
life
stages
are
1)
the
sexual
maturation
period
of
the
P1
and
F1
generations;
2)
egg­
laying
of
the
P1
and
F1
generations;
3)
embryonic
development
(
in
ovo);
and
4)
early
chick
growth
(
F1
and
F2).
Table
6­
1
lists
the
potential
fitness
endpoints
that
could
be
used
in
a
two­
generation
reproduction
test.
In
the
table
and
in
the
discussion
below,
the
endpoints
are
related
to
the
life
stage
in
which
the
endpoint
could
be
measured.
This
table
is
adapted
from
a
discussion
paper
on
prevalidation
of
an
avian
two­
generation
toxicity
test
by
Bennett
et
al.
(
2001)
for
the
OECD
Expert
Group
on
Assessment
of
Endocrine
Disrupting
Effects
in
Birds.
The
table
is
modified
to
reflect
those
endpoint
measures
that
tend
to
cluster
or
segregate
into
groups
of
highly
related
variables
in
avian
reproduction
studies
(
Mineau
et
al.
1994)
and
to
include
those
recommended
by
EDSTAC
(
EPA
1998).
These
clustered
variables
reflect
effects
of
the
test
substance
on
eggshell
quality,
developmental
effects,
and
parental
toxicity,
and
are
so
indicated
on
the
table.
Because
an
alternative
to
proscribing
a
fixed
set
of
endpoints
for
all
compounds
tested
in
a
test
guideline,
is
to
design
a
test
with
a
core
set
of
endpoints
to
which
can
be
additional
endpoints
based
on
anticipated
mechanism
of
action,
the
potential
underlying
estrogenic,
androgenic
and
thyrogenic
endocrine
mechanisms
are
also
provided
in
the
table
(
after
Bennett
et
al.
2001).
Battelle
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2003
Table
6­
1.
Fitness
Endpoints
Specific
to
Endocrine
Active
Substances(
a)

Fitness
Endpoints
Critical
Life
Stages
Type
of
Endocrine
Activity
Embryo
genesis
Early
development
Sexual
maturation
Egg­
laying
Estrogenic
Androgenic
Thyroidogenic
For
F1
and
F2
chicks
number
eggs
laid
per
pair
yes
+(
b)

number
fertile
eggs
per
eggs
laid
PTOX(
d)
yes
+/
 
(
c)
+/­­

number
cracked
eggs
at
set
and
at
2
weeks
ETOX(
e)
yes
number
eggs
hatched
per
eggs
set
DTOX(
f)
yes
yes
+/­­
+/­­

number
chicks
surviving
to
7
and
14
days
per
eggs
set
and
per
eggs
hatched
DTOX
yes
growth
rate
of
chicks
(
weight
at
Days
1,
7,
14)
yes
+/­­
+/­­
+/­­

eggshell
strength
or
thickness
ETOX
yes
early
and
late
viability
per
eggs
set
DTOX
yes
+/­­
+/­­

sex
ratio
of
chicks
yes
yes
+/­­
+/­­

behavior
at
14
days
of
age
Visual
cliff
test
Cold
stress
test
Nest
attentiveness
yes
yes
yes
 
 
 
+

++

For
Parents
body
weight
at
start
and
end
of
treatment
PTOX
yes
yes
+/­­
+/­­

food
consumption
weekly
during
treatment
PTOX
yes
yes
+/­­
+/­­

male
copulatory
behavior
yes
yes
signs
of
toxicity
yes
yes
 
+

survival
yes
yes
Battelle
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Fitness
Endpoints
Critical
Life
Stages
Type
of
Endocrine
Activity
Embryo
genesis
Early
development
Sexual
maturation
Egg­
laying
Estrogenic
Androgenic
Thyroidogenic
For
F1
Juveniles
to
Adults
age
at
onset
of
egg­
laying
yes
+/­­

food
consumption
weekly
during
treatment
yes
yes
yes
+/­­
+/­­

body
weight
weekly
yes
yes
yes
+/­­
+/­­

a)
Adapted
from
Bennett
et
al.
(
2001).
b)
+
indicates
positive
activity
(
e.
g.,
estrogenic,
androgenic,
thyroidogenic).
c)
 
indicates
negative
activity
(
e.
g.,
antiestrogenic,
antiandrogenic,
antithyroidogenic).
a)
PTOX
indicates
endpoints
that
segregate
into
a
group
that
reflect
effects
on
parental
health
and
reproduction.
e)
DTOX
indicates
endpoints
that
segregate
into
a
group
that
reflect
effects
on
development.
f)
ETOX
indicates
endpoints
that
segregate
into
a
group
that
reflect
effects
on
egg
shell
quality.

6.1
Growth
Rate,
Food
Consumption
Food
consumption,
maintenance
of
adult
body
weight,
and
growth
of
chicks
are
important
indices
of
health
status
and
reproductive
fitness
of
each
generation.
Body
weight
is
often
the
most
sensitive
measure
of
effect
in
animals
exposed
to
xenobiotic
chemicals
and
is
analyzed
either
as
change
in
body
weight
or
as
absolute
body
weight.
Using
rates
of
body
weight
change
has
the
advantage
of
increasing
the
initial
sensitivity
of
statistical
analysis.
The
increase
in
sensitivity
is
a
result
of
setting
the
initial
weights
as
a
zero
value,
thus
reducing
the
amount
of
initial
variability
(
Gad
2001).
Body
weight
is
also
used
to
ensure
that
treatment
and
control
groups
contain
animals
of
equivalent
health
status
and
body
condition
at
the
beginning
of
toxicity
tests.
Proper
randomization
of
the
animals
accomplished
when
no
group
differs
significantly
in
body
weight
from
the
mean
body
weight
of
the
other
groups,
and
all
the
animals
on
test
are
within
two
standard
deviations
of
the
overall
mean
body
weight
(
Wilson
and
Hayes
1994).

Food
consumption,
by
affecting
body
condition,
can
influence
sexual
maturation
and
the
quality
and
number
of
eggs
and
subsequent
hatchlings
produced.
In
a
statistical
evaluation
of
134
avian
reproductive
toxicity
studies,
Mineau
et
al.
(
1994)
showed
that
the
average
adult
body
weight
and
food
consumption
during
egg­
laying
correlated
highly
with
the
number
of
eggs
laid.
To
produce
eggs,
quail
must
nearly
double
their
daily
food
consumption
(
Case
1972).
Thus,
reduced
caloric
intake,
resulting
in
poor
body
condition,
as
well
as
direct
effects
on
reproductive
processes,
can
result
in
reduced
fecundity
in
birds
(
Mobarak
1990).
Underfeeding
has
been
shown
to
induce
endocrine
responses,
including
reduced
levels
of
circulating
hormones
and
gonadal
weight
(
Mobarak
1990).
Bennett
and
Ganio
(
1991)
reviewed
the
relationship
between
food
consumption
and
reproductive
success
in
avian
reproduction
studies.
Using
pair­
fed
controls,
some
researchers
demonstrated
equivalent
reductions
in
reproductive
capacity
in
birds
treated
with
OP
pesticides
(
Stromborg
1986a,
1986b;
Bennett
and
Bennett
1990).
Other
OP
pesticides
appear
to
induce
reproductive
deficits
above
those
attributable
to
reduced
caloric
intake
alone.
Battelle
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Growth
rate
and
food
consumption
have
a
long
history
as
endpoints
for
assessing
the
impact
of
endocrine
agents
in
the
poultry
industry.
Estrogenic
compounds
were
used
in
the
poultry
industry
for
many
years
as
a
substitute
for
surgical
caponization
and
were
found
to
markedly
increase
body
weight.
Because
estrogens
are
hyperlipidemic
in
avian
species,
much
of
the
weight
gain
is
in
the
form
of
abdominal
and
muscle
fat
(
Snapir
et
al.
1983).
In
support
of
this
weight
gain,
food
consumption
is
increased.
In
contrast,
androgenic
compounds
appear
to
inhibit
growth
in
birds
prior
to
sexual
maturity
by
terminating
bone
growth.
However,
androgens
are
anabolic
after
sexual
maturity
and
epiphyseal
closure
(
Weppleman
1984).
Antiandrogenic
compounds
also
have
the
potential
to
alter
normal
rates
of
growth
and
food
consumption,
as
do
thyroidogenic
compounds.
Regulation
of
the
metabolic
rate
of
poultry
as
a
means
to
affect
growth
has
involved
various
methods
of
controlling
thyroid
function,
such
as
dietary
supplements
with
thiouracil,
for
example
(
Moreng
and
Avens
1985;
Marks
1992).
These
studies
employed
body
weight
or
a
body
weight/
food
consumption
index
as
an
indicator
of
thyroidogenic
or
antithyroidogenic
effects.
Body
weight
measurements
of
the
female
and
male,
if
cohabiting,
should
not
be
obtained
during
the
egg­
laying
period.
Fearfulness
has
been
positively
correlated
to
the
numbers
of
abnormal,
cracked,
and
body­
checked
eggs
in
poultry
(
Ouart
and
Adams
1982;
Jones
and
Huges
1986;
Mills
et
al.
1991).

Quail
feeding
behaviors
result
in
a
great
deal
of
food
scattering,
even
when
screens
and
other
devises
are
used
to
reduce
wastage;
therefore,
food
consumption
data
can
be
quite
variable
and
should
best
be
considered
an
estimate
rather
than
a
absolute
value.
However,
food
consumption
data
are
essential
in
the
interpretation
of
body
weight
changes
to
assess
effects
of
treatment
on
eating
and
feed
efficiency
(
feed
consumed/
grams
weight
gain),
and
to
calculate
consumed
dosages
(
milligrams
of
test
substance/
kilograms
body
weight).

6.2
Measures
of
Reproductive
Performance
6.2.1
Fecundity
As
a
measure
of
reproductive
success,
fecundity
is
widely
used
in
oviparous
animals
to
provide
insight
into
potential
effects
of
xenobiotics
at
the
population
level.
In
avian
reproduction
toxicity
tests,
fecundity
is
measured
by
the
number
of
eggs
laid
per
pen
within
treatment
groups.
It
was
highly
correlated
with
adult
body
weight
and
food
consumption
in
past
avian
reproductive
toxicity
studies
(
Mineau
et
al.
1994)
and
is
affected
by
environmental
conditions,
age,
and
intraspecific
genetic
variation
(
McNabb
et
al.
1993).
In
females,
the
number
of
eggs
laid
is
the
difference
between
oocyte
recruitment
and
atrecia
(
oocyte
degeneration)
in
the
ovarian
hierarchy
of
oocyte
maturation
(
Ryan
1981).
In
males,
fecundity
is
function
of
semen
quality
and
the
number
of
sperm
that
have
the
potential
to
successfully
complete
all
steps
in
the
fertilization
process:
sperm
movement,
storage
in
the
females
sperm
storage
tubules,
binding
and
penetrating
the
perivitelline
layer,
and
fertilization
(
Donoghue
1999).

Indeterminate
layers,
such
as
Japanese
quail
and
bobwhite,
are
used
in
reproductive
studies,
because
they
can
provide
a
large
number
of
eggs
over
a
long
laying
period.
However,
using
the
total
number
of
eggs
produced
per
pen
over
the
entire
course
of
a
test
as
an
endpoint
is
problematic.
Maximum
egg
production
occurs
about
3
or
6
weeks
after
the
onset
of
laying
for
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April
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the
Japanese
quail
and
northern
bobwhite,
respectively.
Egg
production
prior
to
the
peak
laying
period
for
these
species
is
low,
highly
variable,
and
likely
to
result
in
a
loss
of
statistical
power
when
incorporated
with
data
from
the
subsequent
higher,
more
stable
period
of
maximum
egg
production
(
Springer
and
Collins
1999).
Issues
of
attaining
chemical
equilibrium
in
the
tissues
of
the
bird
and
delayed
toxic
response
after
one
or
two
gamete
cycles,
also
impose
restrictions
on
using
early
eggs
in
a
single
number
endpoint.
Similarly,
fecundity
data
collected
for
10
weeks
after
the
onset
of
lay
will
increase
in
variability
as
the
aging
breeders
terminate
egg­
laying.
A
compromise
between
having
a
large
sample
and
reducing
intratreatment
variability
is
to
collect
for
fecundity
measurement
only
those
eggs
produced
during
peak
production
and
for
only
as
long
as
maximum
production
is
maintained.
This
alternative
approach
was
suggested
in
the
ASTM
(
1990)
guideline,
wherein
eggs
could
be
collected
for
6
weeks
after
50%
of
the
control
hens
have
laid
one
egg
or
until
all
control
pens
produced
25
eggs.
The
latter
accounts
for
the
number
of
eggs
a
quail
or
mallard
would
lay
in
two
clutches
in
the
wild.
This
collection
period
corresponds
to
the
optimum
period
for
egg
production
and
data
collection,
which
is
the
fifth
to
tenth
week
after
onset
of
egg­
laying
(
Springer
and
Collins
1999).

6.2.2
Gamete
Viability
and
Fertilization
Rate
Fertility
is
under
direct
regulation
of
the
reproductive
axis
and
is
measured
by
the
number
of
eggs
set
that
have
viable
embryos
at
first
candling.
It
indicates
the
impact
of
xenobiotics
on
parental
reproductive
function
and
is
therefore
a
key
endpoint.
However,
this
endpoint
provides
information
only
on
the
integrated
effects
of
both
male
and
female
gametic
function.
To
evaluate
gender­
specific
effects
on
gamete
viability
when
decreased
fertility
is
observed,
other
methods
must
be
used.
For
example,
the
treated
birds
could
be
mated
with
nonexposed
birds
following
the
termination
of
exposure
as
a
fertility
trial.
This
approach
is
recommended,
because
the
many
assays
of
spermatozoal
function,
such
as
sperm
concentration
and
sperm
motility,
for
example,
have
been
poor
predictors
of
fertility
in
birds
(
Wishart
and
Staines
1999;
Donoghue
1999).

A
limited
natural
mating
fertility
trial
has
been
designed
for
Japanese
quail
(
Reddish
et
al.
1996).
In
addition
to
validating
the
ability
of
this
modified
fertility
trial
to
evaluate
individual
male
reproductive
performance,
Reddish
et
al.
(
1996)
also
demonstrated
the
efficacy
of
iterative
least
squares
analysis
proposed
by
Kirby
and
Froman
(
1990,
1991)
to
examine
fertility
in
naturally
mating
populations
of
Coturnix.
In
the
limited
natural
mating
fertility
trial,
the
male
is
mated
with
an
untreated
female
for
48
h
and
removed.
Eggs
produced
by
the
female
are
then
opened
each
day
and
examined
for
fertility.
Because
sperm
in
the
sperm
storage
tubules
of
the
female
Japanese
quail
deplete
to
50%
by
the
fourth
day
after
copulation
and
are
removed
completely
by
the
ninth
day,
male­
specific
fertility
data
can
be
gathered
in
a
few
days
(
Reddish
et
al.
1996;
Schom
and
Abbot
1974).
In
the
northern
bobwhite,
50%
depletion
of
sperm
occurs
after
9
days
and
is
complete
by
13
days
(
Schom
and
Abbot
1974).
A
similar
fertility
trial
could
be
conducted
for
treated
females
using
unexposed
males,
once
the
females
no
longer
produce
fertile
eggs
following
removal
of
the
original,
treated
male
at
4
to
9
days.
Gender­
specific
effects
on
sexual
behavior,
sensitive
indicators
of
endocrine
disruption
(
Section
6.2.3),
could
also
be
measured
during
these
fertility
trials.
When
conducting
both
fertility
and
behavior
trials,
it
is
important
to
the
interpretation
of
results
that
birds
with
reproductive
experience
be
used
(
Reddish
et
al.
1996;
Battelle
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April
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2003
Halldin
et
al.
1999);
however,
current
guidelines
require
first­
year
breeders
(
Section
11.0).
Although
these
trials
are
relatively
short
and
easily
conducted,
there
is
cost
added
to
maintain
the
untreated
birds
during
a
quarantine/
acclimation
period
and
the
fertility
trial,
and
to
maintain
the
treated
birds
for
up
to
an
additional
18
days.
There
is
additional
cost
of
labor
for
collecting
and
inspecting
the
eggs.
Recent
development
of
measures
that
successfully
quantify
sperm
function
in
birds
may
provide
a
more
economical
means
of
determining
gender­
specific
effects
of
xenobiotics
on
fertility
(
Section
6.2.2.1).

Selection
of
pairs
for
exposure
regimens
that
start
after
egg
production
has
begun
requires
that
the
pair
are
producing
fertilized
eggs.
The
fertility
status
of
the
male
is
poorly
characterized
by
this
method,
because
with
continuous
cohabitation
and
opportunity
for
multiple
copulations,
low
fertility
could
be
masked.
Therefore,
allocation
of
males
to
the
test
should
be
based
on
tests
of
gamete
viability.
Collection
of
pretreatment
data
on
male
fecundity
should
also
be
conducted
for
covariate
analysis.

6.2.2.1.
Sperm
Motility
and
Morphology,
and
Fertilization
Success.
Male
fertility
has
been
assessed
in
avian
reproduction
toxicity
studies
by
the
production
of
fertilized
eggs.
Although
this
endpoint
incorporates
all
the
reproductive
functions,
it
is
a
relatively
insensitive
endpoint
upon
which
to
evaluate
the
impact
of
a
chemical
on
the
male
reproductive
system.
As
discussed
above
(
Section
6.2.2),
gender­
specific
effects
cannot
be
separated
in
this
integrated
measure,
and
therefore,
little
use
of
fertility
trials,
which
are
considered
to
be
the
ultimate
test
of
fertility
in
poultry
(
Reddish
et
al.
1996),
has
been
made
in
avian
toxicity
studies.
Other
traditional
measures
of
semen
quality
have
been
less
useful
in
predicting
the
fertilizing
ability
of
avian
spermatozoa,
including
adenosene
triphosphate
content
of
sperm,
lipid
peroxidation
of
sperm,
sperm
morphology,
plasma
membrane
integrity,
and
cell
viability
(
Reddish
et
al.
1996;
Wishart
1995;
Donoghue
1999;
Wishart
and
Staines
1999).
Evaluation
of
sperm
quality
using
measures
of
sperm
number,
sperm
motility,
and
sperm
morphology
to
enhance
the
interpretation
of
results
of
avian
reproductive
toxicity
tests
and
provide
information
on
possible
mechanisms
of
action
have
been
used
only
sporadically
(
e.
g.,
Damron
and
Wilson
1975)
and
seldom
in
tests
for
pesticide
registration
(
Mineau
et
al.
1994).
Common
methods
for
determining
sperm
concentration
(
number
of
sperm
per
milliliter
semen)
and
sperm
volume
(
sperm
concentration
times
volume
of
ejaculate)
involve
counting
the
spermatozoa
in
a
known
amount
of
semen
using
a
spermatocrit
or
direct
counting
hemocytometer,
by
flow
cytometry,
or
by
mounting
semen
on
a
hanging
drop
slide.
In
addition
to
ejaculate
samples,
sperm
concentration
can
be
derived
from
the
distal
deferent
duct
at
necropsy
or
from
testicular
histology.
The
distal
portion
of
the
deferent
duct
contains
92%
of
the
extragonadal
sperm
reserve,
which
is
equivalent
to
the
number
of
sperm
produced
daily
by
the
testis
(
Clulow
and
Jones
1982).
Daily
sperm
production
in
Japanese
quail
is
about
9.25
x
106
sperm/
g
testis/
day
(
Lin
et
al.
1990).
Normal
sperm
concentration
is
2.3
x
106/
mL
in
the
distal
deferent
duct
and
2.3
x
106/
mL
in
the
seminferous
tubule
of
the
testis
(
Kirby
et
al.
1990).
Routine
evaluation
of
avian
sperm
motility
and
morphology
is
performed
by
either
phase
contrast
or
differential
interference
contrast
microscopy
(
Bakst
and
Cecil
1991).
Scanning
and
transmission
electron
microscopy
can
also
be
used
to
examine
the
ultrastructure
of
sperm
(
Thurston
and
Hesa
1987);
however,
equipment
and
supply
costs
are
high
for
electron
microscopy
evaluations,
and
specimen
preparation
time
can
extend
over
several
days.
Fixation
and/
or
dehydration
processes
introduce
artifacts
that
also
limit
the
usefulness
of
electron
Battelle
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April
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microscopy
in
detecting
abnormalities
in
the
ultrastructure
of
sperm
before
and
after
treatments
(
Bakst
1993).
Histological
quantification
of
sperm
is
discussed
further
in
Section
7.1.2.

Percentage
of
motile
sperm
and
characteristics
of
sperm
motion
have
been
used
by
investigators
to
evaluate
potential
fertility
in
poultry
(
e.
g.
Wilson
et
al.
1979;
Wishart
1995;
Wishart
and
Palmer
1986;
Froman
and
McLean
1996).
Decreased
motility
of
sperm
can
result
form
structural
abnormalities,
loss
of
mitochondrial
function,
cytological
damage
from
direct
exposure
to
xenobiotics
or
hormones
during
development,
or
from
the
effects
of
abnormal
testicular
development
in
response
to
chemical
exposure
(
Kime
et
al.
2001).
In
birds,
sperm
are
immotile
prior
to
ejaculation,
limiting
collection
of
sperm
for
motility
samples
to
those
obtained
in
ejaculated
semen
(
Ashizawa
and
Sano
1990).
Sperm
motility,
the
swirling
movement
of
sperm
(
swirl
method)
or
the
percentage
of
spermatozoa
moving
in
a
forward
motion
when
viewed
under
high
magnification
are
historically
measured
by
direct
count
(
Wilson
et
al.
1979).
Computer­
based
measurement
of
spermatozoa
swimming
speed
as
straight­
line
velocity
is
a
more
quantitative
and
objective,
less
variable
method
developed
in
recent
years.
Such
videographic
techniques
also
provide
a
permanent
record
of
motility.
However,
they
require
substantial
technical
expertise,
and
the
equipment
is
expensive.
Videographic
sperm
analysis
equipment
ranges
from
about
$
25,000,
without
an
external
negative
phase
microscope,
to
$
35,000
with
a
microscope.
Basic
software
for
morphology
and
motility
assessments
ranges
between
about
$
5,000
and
$
10,000.
Other
motility
measures
that
are
highly
correlated
with
fertility
include
spectrophotometer
techniques
based
on
the
rheotactic
properties
of
sperm.
For
example,
in
an
assay
developed
by
Wishart
and
Ross
(
1985),
sperm
align
in
parallel
under
induced
flow
in
a
tube,
and
when
the
flow
is
stopped,
the
light
scatters
from
the
sperm
changes
as
the
sperm
reorient.
This
change
in
light
scatter
is
related
to
the
percentage
of
motile
sperm
present
and
the
velocity
of
sperm
motion.
Another
of
these
methods
determines
a
sperm
motility
index
based
on
the
frequency
that
sperm
within
a
capillary
tube
alter
a
light
path
in
a
Sperm
Quality
Analyzer
(
Introtech,
San
Diego,
California).
These
methods
are
objective,
simple,
and
rapid
(
McDaniel
et
al.
1998).
However,
like
most
traditional
semen
assays,
they
measure
one
characteristic
of
sperm
(
Amann
and
Hammerstedt
1993)
and
do
not
account
for
the
physiological
conditions
and
complexity
of
fertilization.
Motility
may
not
be
very
predictive
of
fertility,
because
oviduct
factors
also
influence
sperm
movement.

A
relatively
new
approach
to
analyzing
sperm
motion
simulates
a
critical
step
for
internal
fertilization
in
the
female
(
Froman
and
McLean
1996).
In
contrast
to
motility
measures
that
determine
the
percentage
of
sperm
that
are
moving,
this
sperm
mobility
test
measures
the
net
movement
of
a
sperm
population
against
resistance,
a
condition
sperm
must
overcome
in
the
oviduct
(
Donoghue
1999;
Froman
et
al.
1999).
A
sperm
suspension
is
placed
on
top
of
Accudnez,
a
dense,
nonionic,
biologically
inert
material
commonly
used
in
density
gradient
centrifugation.
The
absorbance
of
the
Accudenz
is
recorded
after
5
min
of
incubation
at
body
temperature
(
41/
C),
at
which
time
the
absorbance
is
proportional
to
the
number
of
sperm
that
have
penetrated
the
Accudenz
layer.
The
sperm
that
swim
into
the
Accudenz
are
highly
mobile,
directional
swimmers.
Subfertile
males
may
have
a
normal
semen
concentration,
but
a
low
number
of
highly
mobile
sperm
and
a
high
number
of
immobile
sperm,
even
though
they
may
be
technically
motile;
in
motility
tests,
motile
often
means
"
not
immotile."
This
method
has
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repeatedly
been
shown
to
be
a
primary
determinant
of
fecundity
and
highly
predictive
of
fertility
and
male
fitness
in
poultry
(
Froman
and
McLean
1996;
Holbserger
et
al.
1998;
King
and
Donoghue
1998;
Rhoads
et
al.
1998).
The
method
is
inexpensive,
objective,
accurate,
and
requires
little
expertise.
Interassay
CVs
for
this
assay
range
from
2.6%
to
9.2%
(
Froman
and
McLean
1996).
This
procedure
has
the
potential
to
be
a
sensitive
and
economical
endpoint
in
avian
reproduction
toxicity
tests,
but
needs
to
be
adapted
to
and
validated
in
quail
and
under
conditions
of
endocrine
and
toxin
challenge.

The
process
of
fertilization
involves
not
only
the
transport
of
sperm,
but
its
storage
in
the
sperm
storage
tubules
of
the
oviduct,
the
binding
of
the
sperm
to
the
outer
investment,
or
perivitelline
layer,
of
the
ovum
at
ovulation,
and
penetration
of
the
ovum
(
Bakst
et
al.
1994;
Robertson
et
al.
1998).
There
have
been
a
number
of
assays
developed
and
evaluated
in
recent
years
to
quantify
these
functions
of
sperm
in
an
attempt
to
replace
the
traditional
measures
of
semen
quality
which
have
proven
to
be
poor
predictors
of
male
fertility.
These
new
assays
quantify
the
number
of
sperm
that
interact
with
the
perivitelline
layers
of
the
egg
in
the
infundibulum.
Systems
measuring
the
interaction
of
sperm
with
the
perivitelline
layers
have
proven
to
be
highly
predictive
of
individual
fertility
in
poultry
under
a
variety
of
environmental
stresses.
Most
of
these
assays
are
simple,
quick,
and
noninvasive.

One
of
the
more
involved,
but
automated
methods
for
measuring
sperm
interaction
with
the
perivitelline
layers
is
an
in
vitro
sperm­
binding
assay
(
Barbato
et
al.
1998)
that
uses
microwell
plates
coated
with
a
solubilized
extract
of
the
perivitelline
layer
from
chicken
eggs.
Sperm
suspensions
of
known
concentration
are
incubated
on
a
microwell
plate,
the
unbound
sperm
are
removed
by
washing,
and
the
remaining
bound
sperm
are
counted.
So
far
,
sperm
binding
has
been
shown
to
be
predictive
of
fertility
in
several
strains
of
chickens
(
Barbato
et
al.
1998;
Barbato
1999).
A
commercial
form
of
the
assay
(
BioPore,
Inc.,
State
College,
Pennsylvania)
is
available.
The
assay
requires
technical
skill
and
specialized
equipment
for
analysis
and
ranking
of
male
fertility.
Although
the
method
has
not
been
adapted
to
quail,
it
has
been
simplified
for
use
in
large­
scale
poultry
flocks
(
Gill
et
al.
1998).

The
remaining
systems
that
measure
the
fertilizing
capacity
of
sperm
are
in
vivo
assays
based
on
1)
the
number
of
sperm
that
bind
to
the
inner
perivitelline
layer
(
IPVL)
and
undergo
an
acrosome
reaction
at
fertilization;
or
2)
the
number
of
sperm
that
become
trapped
in
the
outer
perivitelline
layer
(
OPVL)
as
it
is
laid
down
around
the
IPVL
within
a
few
minutes
after
fertilization.
All
of
the
assays
involve
excising
a
1
cm2
to
2
cm2
piece
of
the
perivitelline
layer
from
a
fresh
egg,
rinsing
the
tissue
to
remove
any
yolk,
and
placing
the
tissue
on
a
slide.
To
count
sperm
trapped
in
the
OPVL,
indicative
of
the
number
of
sperm
present
in
the
oviduct
during
fertilization,
the
tissue
layer
is
stained
with
DNA­
binding
fluorochrome
and
viewed
under
400X
magnification
using
a
fluorescence
microscope.
This
method
requires
use
of
special
equipment,
and
the
counting
of
many
fields
at
high
magnification.

For
sperm
that
have
bound
with
the
IPVL,
released
acrosomal
enzymes,
and
hydrolyzed
a
small
hole
through
which
they
have
passed
to
reach
the
oocyte,
the
tissue
is
either
fixed,
dried,
and
stained
with
Schiff's
reagent
(
Bramwell
et
al.
1995;
Howarth
and
Donoghue
1997),
or
viewed
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wet
and
unstained
with
darkfield
optics
(
Birkhead
et
al.
1993).
The
unstained
method
involves
fewer
steps,
but
must
be
read
within
24
to
48
h.
The
staining
method
allows
for
storage
of
the
tissue
and
has
been
shown
to
result
in
the
identification
of
more
holes
(
Fairweather
1998).
Sperm
holes
can
be
counted
from
any
area
of
the
IPVL,
though
the
area
directly
over
the
germinal
disk
has
the
highest
density.
Holes
over
the
germinal
disk
are
more
easily
enumerated
and
provide
data
more
directly
linked
to
egg
fertility
(
Wishart
and
Staines
1999).
This
method
takes
only
a
few
minutes
per
slide
to
prepare
and
count.
It
has
been
evaluated
against
several
sperm
quality
tests
and
validated
in
poultry
fertility
tests
(
Robertson
et
al.
1998;
Wishart
1985).
The
IPVL
method
also
has
been
successfully
used
to
evaluate
the
effects
of
dietary
energy
on
sperm
function
(
Bramwell
et
al.
1996)
and
the
effects
of
heat
stress
on
fertility
of
breeder
males
(
McDaniel
et
al.
1996).
Of
the
male
fertility
measures
available,
the
IPVL
method
is
the
least
invasive,
most
economical
in
labor
and
equipment,
requires
minimal
expertise,
provides
among
the
most
reliable
assessments
of
fertility,
and
has
been
used
with
success
to
discriminate
between
fertility
of
males
subjected
to
different
dietary
or
environmental
conditions.
Additional
advantages
of
this
assay
include
1)
the
ability
to
detect
differences
in
fertility
between
groups
of
birds
before
it
is
detectable
in
the
proportion
of
fertilized
eggs
laid
(
Donoghue
et
al.
1995);
2)
a
more
accurate
measure
of
fertility
status
of
unincubated
eggs
than
attainable
by
morphology
of
the
germinal
disk
(
Wishart
and
Staines
1999);
3)
ability
to
store
eggs
for
several
weeks
before
examination;
and
4)
ability
to
substantially
shorten
standard
fertility
trials
by
assessing
fertility
of
the
second
mating
before
infertile
eggs
are
laid.

6.2.2.2
Egg
Quality.
Of
the
various
interrelated
functions
that
contribute
to
reproductive
success
in
birds,
formation
of
a
robust
eggshell
to
protect
the
developing
embryo
has
been
one
of
the
most
frequently
measured
parameters
in
avian
reproductive
toxicology.
Eggshell
quality
emerged
as
a
key
indicator
of
reproductive
impairment
in
natural
populations
of
birds
from
the
eggshell
thinning
phenomenon
and
associated
reproductive
failure
of
several
sensitive
bird
species
exposed
to
OC
pesticides
in
the
mid­
1900s
(
Cooke
1973).
The
mechanisms
underlying
the
daily
fabrication
of
eggshells
are
not
completely
understood,
but
appear
to
be
susceptible
to
a
variety
of
contemporary
environmental
chemicals
(
Haegele
and
Tucker
1974;
Fleming
et
al.
1983;
Ormerod
et
al.
1988).
Since
the
introduction
of
the
EPA
test
guideline
for
avian
reproduction
studies
in
1975,
egg
quality
endpoints,
particularly
eggshell
thickness,
have
continued
to
be
useful
tools
in
hazard
assessment.
In
a
review
of
134
avian
reproduction
studies
conducted
in
support
of
pesticide
registration,
eggshell
thickness
was
among
those
variables
shown
to
be
effective
in
indicating
a
response
to
chemical
exposure
(
Mineau
et
al.
1994).
Eggshell
effects
were
observed
in
17%
of
the
studies.

In
addition
to
providing
mechanical
protection,
a
well­
formed
eggshell
prevents
water
loss,
protects
against
infection,
and
is
a
major
source
of
calcium
for
the
developing
embryonic
skeleton
(
Lavelin
et
al.
2000).
The
functional
qualities
of
the
eggshell
that
provide
a
protective
environment
for
the
developing
embryo
are
its
size
and
shape,
thickness,
and
ultrastructure
of
mineral
and
protein.
Measures
that
are
used
to
assess
these
qualities
include
shell
thickness,
breaking­
strength,
and
the
number
of
cracked
or
soft
shells
and
of
eggs
without
shells.
Electron
microscopy
can
be
used
for
detailed
examination
of
the
shell.
These
measures
are
used
to
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provide
information
on
the
potential
for
successful
development
of
the
embryo
and
hatch
of
the
chick.

One
of
the
most
common
assays
of
eggshell
quality,
called
candling,
is
the
examination
of
the
exterior
soundness
of
the
shell.
Candling
is
accomplished
by
turning
the
egg,
as
it
is
held
near
a
light
source,
and
observing
the
shell
for
checks,
cracks,
and
texture
(
thin
spots,
ridges).
Although
it
is
a
simple,
inexpensive
technique,
it
is
dependent
on
observation
criteria
that
are
undefined
in
avian
reproductive
tests.
Features
such
as
body
checks,
which
are
cracks
that
occur
while
the
egg
is
in
the
uterus
or
shell
gland
and
that
have
been
repaired
with
a
layer
or
ridge
of
deposited
calcium,
could
be
scored
differently
by
different
laboratories,
and
by
different
individuals
within
laboratories.
Depending
on
the
extent
of
the
cracking,
checked
shells
can
be
weaker
than
those
that
are
unchecked
(
Moreng
and
Avens
1985).
Because
cracked
eggs
are
removed
from
the
population
before
incubation,
the
potential
impact
of
inaccurate
or
inconsistent
scoring
on
subsequent
fitness
parameters
can
be
marked.
Therefore,
a
uniform
scoring
system
among
laboratories
is
needed.
Other
factors,
such
as
handling
of
the
birds,
access
of
the
birds
to
the
eggs,
housing
materials,
and
slope
of
the
cage
floor,
can
confound
the
sensitivity
of
this
egg
quality
endpoint
by
introducing
cracks
not
caused
by
treatment.

In
an
interlaboratory
comparison
among
five
separate
testing
laboratories,
quail
from
the
same
supplier
were
used,
but
the
birds
were
maintained
under
different
husbandry
methods.
The
background
proportion
of
cracked
eggs
in
the
controls
varied
greatly
between
laboratories,
as
did
the
sensitivity
of
identifying
treatment­
related
effects
(
Schlatterer
et
al.
1993).
It
has
also
been
observed
that
some
quail
hens
produce
clutches
with
a
large
number
of
cracked
eggs
(
Bennett
and
Ganio
1991).
Bennett
and
Ganio
(
1991)
demonstrated
that
the
distribution
of
such
hens
within
groups
and
the
rate
of
cracking
per
hen
could
mask
chemical­
induced
effects.
Using
a
typical
pattern
of
cracking
in
a
hypothetical
study,
in
which
the
same
number
of
pens
in
each
group
had
the
same
pen­
wide
cracking
rate,
they
showed
that
one­
way
ANOVA
could
not
detect
a
true
chemical
effect
in
the
number
of
14­
day­
old
chicks
in
the
low­
dose
group
of
their
hypothetical
study.
Because
of
the
potential
for
masking
of
chemical­
related
effects,
it
is
important
to
the
outcome
of
avian
reproduction
studies
not
only
to
properly
identify
cracked
eggs,
but
also
to
evaluate
their
distribution
among
hens
within
groups.
Some
laboratories
use
the
frequency
of
cracking
as
an
indirect
measure
of
shell
strength
(
Schlatterer
et
al.
1993).
However,
the
frequency
of
cracked
eggs
is
considered
to
be
a
poor
measure
of
egg
quality
and
should
be
used
mainly
to
assure
that
background
cracking
rates
are
within
an
acceptable
range
and
uniform
pattern
(
Bennett
and
Ganio
1991).

Shell
damage
is
directly
related
to
shell
strength,
and
shell
strength
is
determined
by
the
organization
of
the
organic
matrix,
and
by
the
thickness
of
the
shell,
measured
as
calcium
carbonate
content,
particularly
on
the
palisade
layer.
Thinning
of
the
eggshell
can
result
in
egg
breakage,
egg­
eating
behavior,
or
disappearance
of
eggs
from
the
nest;
therefore,
measures
of
eggshell
thickness
are
commonly
used
to
monitor
eggshell
quality
in
the
wild
and
in
laboratory
studies.
Cooper
(
1991)
reviewed
the
relationship
of
eggshell
thinning,
eggshell
breakage,
and
reproductive
failure
in
natural
populations
of
birds.
He
found
that
eggshell
thinning
of
10%
leads
to
cracking
of
the
shells
and
increased
embryonic
mortality,
that
extensive
egg
breakage
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occurs
when
eggshell
thinning
is
greater
than
15%
to
20%,
and
that
in
nature,
the
latter
is
associated
with
population
decline
(
Anderson
and
Hickey
1972;
Lincer
1975;
Risebrough
et
al.
1968;
Stoewasand
et
al.
1971;
Spann
et
al.
1972;
Cooper
1991).
Cooper
(
1991)
also
concluded
that
thinning
of
2%
to
5%
could
cause
some
loss
of
eggs,
but
that
it
was
difficult
to
discern
from
background
loss
levels.
There
is
considerable
species
variation
in
the
amount
of
egg
thinning
observed
from
similar
exposures
to
chemicals.

Several
techniques
have
been
developed
to
measure
eggshell
thickness.
The
three
methods
in
common
practice
are
as
follows:
1)
determination
of
the
specific
gravity
of
the
egg,
2)
calculation
of
a
thickness
index
from
the
ratio
of
the
weight
and
surface
area
of
the
shell,
and
3)
a
direct
measure
of
the
thickness
of
the
shell.
All
three
methods
are
relatively
simple
and
inexpensive
to
perform.
The
specific
gravity
method
is
a
noninvasive
procedure
based
on
determining
the
egg's
density
relative
to
water
at
the
same
temperature.
Because
the
specific
gravity
of
a
shell
is
more
than
twice
that
of
the
other
parts
of
the
egg
(
yolk,
albumen,
and
membranes),
it
has
a
major
influence
on
the
specific
gravity
of
the
whole
egg.
Specific
gravity
and
eggshell
thickness
are
highly
positively
correlated
(
Bennett
1992),
as
are
specific
gravity
and
incidence
of
cracks
and
breaks.
In
chickens,
hatchability
of
eggs
with
a
specific
gravity
of
<
1.080
was
at
least
2%
less
than
the
hatchability
of
thicker­
shelled
eggs.
The
incidence
of
embryonic
death
was
also
higher
in
the
thin­
shelled
eggs
(
Wells
1967).
Specific
gravity
values
for
Coturnix
range
between
about
1.066
and
1.068
(
Marks
and
Britton
1972;
Goodman
1965).
Similar
values
are
reported
for
bobwhite
eggshells
(
Mahmound
and
Coleman
1967).

This
method
typically
involves
the
preparation
of
18
or
more
salt
solutions
ranging
in
specific
gravity
from
1.030
to
1.090,
typically
at
intervals
of
0.0025.
The
solutions
are
verified
with
a
hydrometer.
Eggs
are
immersed
first
in
pure
water,
then
into
each
of
the
salt
solutions,
and
rinsed
in
pure
water
in
between
test
solutions.
The
specific
gravity
of
the
solution
in
which
the
egg
first
floats
is
recorded.
Because
this
method
is
accurate
only
when
the
egg
has
small
air
cells
(
moisture
loss
reduces
specific
gravity
measurements),
eggs
must
be
measured
within
24
h
after
collection,
and
consistent
timing
of
the
specific
gravity
determinations
are
important
for
reliable
results.
The
specific
gravity
method
has
the
advantage
of
not
having
to
sacrifice
eggs
and
thereby
reduce
the
number
available
for
hatching
rate
measurements.
However,
the
collection
time
and
storage
restrictions
are
inconvenient
and
result
in
increased
labor,
particularly
on
weekends.
Although
the
specific
gravity
method
could
be
used
in
the
field
(
it
is
used
on
poultry
farms)
to
sample
wild
populations
noninvasively,
it
is
relatively
cumbersome
for
fieldwork.

Shell
thickness
indices
have
been
and
continue
to
be
used
to
monitor
eggshell
quality
of
a
wide
variety
of
wild
bird
species.
Changes
in
shell
thickness
of
field­
collected
eggs
usually
have
been
detected
by
one
of
several
indices
derived
from
the
weight
of
the
egg
and
some
estimate
of
its
surface
area.
A
small
hole
is
drilled
in
the
egg,
the
contents
are
blown
out,
leaving
the
membranes
intact,
and
the
shell
is
dried.
The
eggshell
is
weighed,
and
the
length
and
maximum
width
of
the
egg
are
measured.
The
weight
of
the
shell
is
then
divided
by
a
function
of
the
length
and
width
that
is
proportional
to
the
surface
area
of
the
egg.
The
oldest
index
and
the
one
most
widely
used
is
the
Ratcliffe
or
Shell
Index
(
Ratcliffe
1967):
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Wt/
LW
(
1)

where
Wt
is
the
weight
of
the
shell,
L
is
the
length
of
the
shell,
and
W
is
the
width
of
the
shell
at
its
widest
point.
Other
indices
of
thickness
include
the
Nybo­
Green
index,
which
is
based
on
early
assumptions
by
Romanoff
and
Romanoff
(
1949)
and
Peakall
et
al.
(
1973),
and
on
later
empirical
data
by
Pagnaelli
et
al.
(
1974)
and
Hoyt
(
1979)
that
the
surface
area
is
proportional
to
L2/
3
W4/
3
(
Nybo
et
al.
1997;
Green
1998),
and
the
Moriarty­
Nygard
index,
which
assumes
the
surface
area
of
the
egg
is
a
prolate
spheroid
with
semi­
axes
L/
2
and
W/
2
(
Moriarty
et
al.
1986;
Nygard
1999).
Green
(
2000)
compared
the
three
thickness
indices
using
museum
collections
of
eggs
for
several
species
of
thrushes
and
found
that
the
difference
in
coefficient
of
variation
among
the
indices
was
very
small
(
e.
g.,
7.393­
7.597
for
the
European
blackbird,
5.872­
5.909
for
the
Mistle
thrush),
indicating
that
the
ability
of
these
indices
to
detect
a
decrease
in
shell
thickness
was
about
equivalent,
at
least
for
the
species
measured.
The
preparation
time
for
this
egg
quality
assay
is
about
equivalent
to
that
of
the
direct
method
discussed
below,
but
the
actual
measurement
time
is
much
less
labor­
intensive
and
not
as
subject
to
fatigue
effects
and
individual
variation
in
sample
point
selection
and
measurements.

Current
guidelines
for
avian
reproduction
tests
(
EPA
540/
9­
82­
024;
OECD
206)
support
the
use
of
direct
eggshell
thickness
measurements
to
evaluate
the
effect
of
a
chemical
on
the
hormone­
mediated
process
of
shell
mineralization.
This
method
consists
of
cutting
an
egg
open
around
its
equator,
emptying
the
contents,
and
drying
the
eggshell
with
its
membranes,
after
which
the
shell
thickness
is
measured
with
a
micrometer.
It
is
important
to
consistently
obtain
thickness
measurements
from
one
specified
region
of
an
egg,
because
shell
and
membrane
thickness
can
vary
significantly
along
the
egg
(
Dirksen
et
al.
1991).
The
equator
of
the
egg
is
selected
as
the
area
from
which
the
measurements
are
taken,
because
it
represents
a
wide
band
of
uniform
thickness.
Also
this
area
is
the
traditional
measurement
site
used
by
early
investigators
of
egg
quality
effects
of
dichlorodiphenylethylene
(
DDE)
and
other
environmental
contaminants
(
e.
g.,
Koeman
et
al.
1972;
Newma
1979).
Drying
times
vary
from
1
day
to
1
week
under
ambient
conditions,
although
48
h
of
drying
appears
to
be
used
most
often.
A
number
of
measurements
between
three
and
nine
should
be
selected
as
standard
per
shell
for
each
test.
Although
no
formal
study
has
been
conducted,
several
researchers
believe
that
there
can
be
substantial
differences
in
values
obtained
by
different
staff
for
the
same
shell,
and
that
less
variable
data
are
obtained
when
a
single
staff
member
conducts
the
measurements
throughout
the
course
of
the
study.
Variation
is
thought
to
arise
from
the
way
in
which
a
micrometer
is
held
or
sample
areas
on
the
shell
are
selected.
For
example,
it
has
been
noted
that
areas
where
the
underlying
membranes
have
been
disrupted
during
preparation
of
the
egg
differ
greatly
in
thickness
from
surrounding
areas
with
intact
membranes.
Because
fatigue
of
the
staff
can
affect
the
quality
of
data
during
a
long
session
of
measuring
shell
thickness,
some
laboratories
begin
each
test
lot
with
control
eggs.
Even
small
improvements
in
data
quality
are
important
in
light
of
the
relatively
few
eggs
that
are
tested
for
eggshell
thickness
during
reproductive
toxicity
tests.

Although
thickness
contributes
to
the
overall
strength
of
the
shell,
it
accounts
for
only
a
small
fraction
of
the
shell
resistance
to
mechanical
stress
(
Lavelin
et
al.
2000).
Researchers
have
used
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two
assays
to
measure
the
resistance
of
eggs
to
mechanical
stress.
One
technique
measures
the
shear
fracture
force
required
to
puncture
the
shell
(
Stevenson
et
al.
1981).
This
puncture
can
be
repeated
several
times
on
each
egg
to
obtain
an
average
puncture
force
value.
However,
it
has
the
drawback
that
it
does
not
represent
the
resistance
to
forces
encountered
in
nests
in
the
field
and
is
therefore
seldom
used
in
avian
reproduction
studies
to
evaluate
shell
strength.
The
second
egg
strength
assay
measures
the
compression
strength
of
the
shell
that
more
directly
represents
the
mechanical
stress
encountered
during
natural
incubation.
In
this
method,
the
tensile
fracture
force
is
measured
with
a
universal
testing
instrument,
wherein
an
egg
is
compressed
at
the
equator
between
two
stainless
steel
surfaces.
The
compression
on
the
egg
and
the
load
at
which
the
egg
failed
are
recorded.
Studies
comparing
the
eggshell
thickness
and
compression
strength
methods
with
both
laboratory­
treated
and
field­
collected
eggs
indicate
that
the
compression­
breaking­
strength
method
can
detect
shell
damage
at
lower
exposure
concentrations
than
the
shell
thickness
assay
(
Cooke
1979;
Snyder
et
al.
1973;
Carlisle
et
al.
1986;
Bennett
et
al.
1988;
Henny
and
Bennett
1990).
Scanning
electron
microscopy
has
been
employed
to
examine
the
integrity
of
shell
ultrastructure
in
eggshells
that
were
evaluated
by
both
shell­
thickness
and
breaking­
strength
assays.
Ultrastructure
defects
were
observed
in
shells
that
were
of
normal
thickness
but
were
identified
as
weak
by
the
compression­
strength
assay
(
Bennett
et
al.
1988,
Henny
and
Bennett
1990).
In
these
eggshells,
the
mammallae,
an
array
of
rounded
cones
that
forms
the
foundation
layer
of
the
shell
on
which
the
remaining
crystal
growth
occurs,
were
poorly
formed
and
irregular,
indicating
that
resistance
to
breakage
is
a
direct
function
of
eggshell
integrity
rather
than
of
than
thickness,
and
that
compression­
breaking
strength
can
provide
an
indirect
measure
of
ultrastructure
integrity.

Although
more
sensitive
than
the
thickness
assay
in
identifying
treatment
effects,
the
breaking­
strength
test
appears
more
variable
than
shell
thickness.
The
coefficient
of
variation
for
the
compression
test
data
in
the
Bennett
et
al.
(
1988)
study
was
12%
to
16%
for
pretreatment
eggs
compared
with
9%
for
thickness
measurements
on
the
same
shells.
Another
drawback
of
the
compression
test
is
that
it
cannot
be
performed
on
cracked
or
soft­
shelled
eggs,
whereas
the
thickness
assay
can
be
used
on
eggs
in
those
conditions.
The
use
of
both
methods
has
been
encouraged
by
a
number
of
investigators
to
better
assess
the
potential
damage
to
eggshell
quality
from
dietary
exposure
to
xenobiotic
chemicals,
in
general
(
Carlisle
et
al.
1986;
Bennett
et
al.
1988;
Henny
and
Bennett
1990).
It
is
particularly
important
in
the
context
of
an
avian
reproduction
toxicity
test
to
maximize
the
shell
quality
information
obtained
from
the
relatively
few
eggs
tested
relative
to
the
number
produced.

Although
the
compression­
breaking­
strength
method
is
more
sensitive
than
the
thickness
assay,
the
high
up­
front
cost
of
the
equipment
to
perform
this
test
has
limited
its
use.
However,
once
equipment
is
established,
there
are
labor
savings
in
using
the
assay.
Cost
of
universal
materials­
testing
instruments
ranges
from
$
10,000
to
$
30,000,
depending
on
manufacturer,
computer
and
software
options,
and
accessories.
Labor
hour
estimates
from
two
commercial
testing
laboratories
that
conduct
both
eggshell
thickness
and
shell
strength
are
about
8
h
per
200
eggs
plus
an
additional
4
h
of
preparation
time
per
week
for
shell
thickness
measurements.
In
contrast,
about
4
h
of
labor
are
required
to
measure
the
breaking­
strength
of
200
eggs,
with
little
to
no
preparation
time.
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Recent
studies
indicate
that
shell
strength
is
positively
correlated
with
the
matrix
proteins
that
pervade
the
mineral
phase
of
the
avian
eggshell
and
influence
mineralization.
Several
eggshell
matrix
proteins
that
have
the
potential
to
be
biomarkers
of
shell
quality
have
been
identified
(
Hike
et
al.
2000).
These
researchers
have
clearly
identified
a
high
correlation
between
expression
of
one
of
the
proteins
in
the
mammillae,
and
shell
strength
and
ultrastructure
integrity.
It
is
likely
that
immunochemical
analysis
of
the
eggshell
matrix
will
ultimately
provide
a
rapid,
simple,
and
highly
sensitive
measure
of
eggshell
quality
in
the
future.

6.2.2.3
Hatching
Success.
Hatching
is
a
critical
developmental
stage
that
is
vulnerable
to
disruption.
It
has
been
shown
to
be
sensitive
to
a
wide
array
of
chemicals
(
Hoffman
and
Heinz
1988;
Heinz
et
al.
1989;
Rice
and
O'Keefe
1995;
Hoffman
1978;
Coon
et
al.
1979;
Hoffman
1990)
with
consequent
increases
in
mortality.
Therefore,
hatching
success
is
a
key
endpoint
in
avian
reproduction
toxicity
tests.
There
are
a
number
of
factors
that
reduce
hatchability.
In
addition
to
handling
trauma,
incubation
conditions,
and
other
husbandry
issues,
hatchability
is
affected
by
poor
eggshell
quality,
nutrition
of
the
hen,
which
corresponds
to
nutrient
exhaustion
of
the
embryo,
and
teratogenic
factors.
Embryo
viability
and
subsequent
hatching
success
also
provide
information
on
the
effects
of
in
ovo
deposition
of
chemicals
on
embryo
development.
These
measures
are
highly
correlated
in
reproductive
toxicity
tests
with
chick
survival
to
Day
14
(
Mineau
et
al.
1994).
Typically,
embryo
viability
is
evaluated
at
both
early
and
late
periods
of
development,
providing
information
on
fertility
and
embryo
toxicity,
respectively.
Viability
at
these
two
time
periods
is
generally
reported
separately;
however,
a
combination
of
the
early
and
late
embryo
viability
variables
is
a
more
effective
indicator
of
effect
(
Mineau
et
al.
1994).

Embryo
viability
and
the
corresponding
hatching
success
are
also
related
to
eggshell
porosity,
which
affects
gas
exchange
and
rate
of
water­
loss
in
the
egg.
Peebles
and
Marks
(
1991)
demonstrated
that
increased
eggshell
permeability
was
associated
with
embryonic
death
and
decreased
hatchability
in
Japanese
quail
selected
for
meat­
production
growth.
Altered
eggshell
quality
could
be
one
of
the
factors
contributing
to
differences
in
reproductive
parameters
of
different
strains
of
Japanese
quail,
and
underscores
the
need
to
standardize
strain
selection.

6.2.2.4
F1
and
F2
Post­
Hatch
Survivorship.
Post­
hatch
survivorship
is
a
primary
production
endpoint
that
integrates
the
following:
°
fecundity
number
of
eggs
laid
°
fertility
proportion
of
eggs
laid
or
set
that
are
fertile
°
embryogenesis
proportion
of
live
embryos
of
those
fertile
°
hatchability
proportion
of
eggs
with
embryos
that
hatch
°
chick
viability
proportion
of
hatchlings
that
survive
to
14
days
old.

Although
this
endpoint
provides
an
overall
measure
of
reproductive
capacity/
success
per
pair
or
per
pen,
CVs
for
this
parameter
are
high,
and
the
power
of
tests
to
detect
20%
reduction
in
the
number
of
14­
day
survivors
of
quail
is
lower
than
that
for
the
other
reproduction
endpoints
(
Springer
and
Collins
1999).
To
attain
adequate
power
to
detect
effects
on
this
endpoint,
a
significant
increase
in
the
number
of
replicates
would
have
to
be
incorporated
into
the
test
design,
if
ANOVA
methods
were
used.
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6.2.3
Changes
in
Breeding
Behavior
Appropriate,
timely
breeding
behavior
is
an
important
component
of
reproductive
success
in
birds.
Evidence
of
altered
breeding
behavior
in
wild
birds
exposed
to
environmental
pollutants
has
been
reported
(
Peakall
and
Fox
1987;
Fox
1993;
Thaxton
and
Parkhurst
1973).
Recent
studies
with
endogenous
and
exogenous
steroids
indicated
that
exposure
to
ecosteroids
at
critical
periods
of
a
bird's
life
can
cause
profound,
irreversible
changes
in
these
critical
behaviors
(
Berg
et
al.
1999;
Halldin
et
al.
1999;
Eroschenko
et
al.
2002
;
Balthazart
and
Surelemont
1990a;
Panzica
et
al.
1996;
Panzica
et
al.
1999).
Sexual
behavior
in
the
Japanese
quail
has
been
well
described
(
Schein
and
Carter
1972;
Sefton
and
Siegel
1973),
and
it
includes
courtship
behavior,
such
as
crowing
and
strutting,
and
mating
behaviors,
such
as
copulation.
In
male
birds,
sexual
behaviors
are
androgen­
dependent
(
Adkins
and
Adler
1972;
Adkins
1977),
and
there
appear
to
be
varying
thresholds
of
hormonal
stimulation
(
Ottinger
and
Brinkley
1978).
For
example,
onset
of
crowing
occurred
at
serum
testosterone
levels
of
3.3
ng/
mL,
whereas
4.7
ng/
mL
was
required
for
the
initiation
of
mating
behavior.

Testosterone
has
been
shown
to
affect
behavior
by
its
action
on
specific
regions
of
the
brain.
The
lateral
septum,
medial
preoptic
nucleus
(
POM),
and
bed
nucleus
of
the
stria
terminalis
(
BST)
are
sexually
dimorphic
structures
in
the
quail
brain
that
are
involved
in
the
activation
of
breeding
behaviors
(
Halldin
et
al.
1999;
Balthazart
and
Surelemont
1990a,
1990b;
Panzica
et
al.
1996;
Panzica
et
al.
1999).
Hormonal
manipulation
of
juveniles
and
adults
results
in
profound
morphological
changes
in
the
POM
(
Panzica
et
al.
1994)
and
subsequent
changes
in
copulatory
behavior
in
the
Japanese
quail
(
Halldin
et
al.
1999).
During
embryogenesis,
exposure
to
estrogens
or
estrogen
agonists
organizes
areas
of
the
POM
in
male
birds
in
a
nonmasculine
manner,
resulting
in
irreversible
depression
of
copulatory
behavior
in
the
adult.
Such
in
ovo
exposure
has
been
shown
to
cause
significant
depression
of
male
sexual
activity
at
concentrations
well
below
those
that
affect
plasma
testosterone
levels,
body
weight,
or
the
gonadosomatic
index
(
Berg
et
al.
1999).
Eroschenko
et
al.
(
2002)
demonstrated
that
at
maturity,
male
Japanese
quail
that
had
been
exposed
to
methoxychlor,
a
metabolite
of
methoxychlor,
or
to
17
$­
estradiol
in
ovo,
displayed
significantly
altered
sexual
behavior,
such
as
decreased
copulation
attempts
and
lengthened
mounting
latency,
although
cloacal
gland
size,
and
the
weight
and
histological
morphology
of
their
testes
were
normal.
These
data
indicated
that
prenatal
exposure
to
even
weakly
estrogenic
compounds
could
result
in
a
permanent
feminization
of
the
brain
of
male
Japanese
quail.

Measurement
of
copulatory
behavior
is
well
studied
in
Japanese
quail,
because
this
species
has
been
used
for
many
years
as
a
model
for
studies
of
testosterone
control
of
breeding
behavior
(
Mill
et
al.
1997).
Copulatory
behaviors
are
governed
by
the
aromatization
of
testosterone
to
estrogen
(
Balthazart
et
al.
1995).
The
behavior
is
well
defined
and
follows
a
particular
sequence
of
neckgrab,
mount
attempt,
mount,
and
cloacal
contact
movement
(
Halldin
et
al.
1999).
It
is
easily
measured
in
the
laboratory
by
housing
male
birds
singly
and
then
recording
specific
responses
following
introduction
of
a
receptive
female
into
the
cage.
These
measured
responses
are
indicative
of
sexual
motivation
and
the
ability
to
exhibit
reproductive
behavior.
They
include
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April
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latency
to
mount,
mating
attempts,
and
completed
matings.
Tests
that
can
be
completed
in
2
to
5
min
for
each
bird
on
as
few
as
2
to
5
consecutive
days
have
been
shown
to
accurately
reflect
male
reproductive
behavior
(
Halldin
et
al.
1999).
However,
size
of
the
test
arena
can
affect
the
results:
small
arena
size
(
50
cm
x
60
cm)
greatly
enhances
response
(
Riters
et
al.
1998),
probably
as
a
result
of
the
proximity
of
the
female
to
the
male
bird.
Care
must
be
taken
to
reduce
observer
bias
by
conducting
the
tests
such
that
the
observer
is
unaware
of
the
treatment
associated
with
the
test
subjects.
Further,
it
could
be
important
to
select
behaviors
for
measurement
that
are
less
dependent
on
the
receptivity
of
the
female,
such
as
mount
attempts
and
cloacal
contact
movement;
they
are
better
measures
of
male
consummatory
sexual
behavior
(
Halldin
et
al.
1999).

Courtship
has
also
been
studied
(
Ottinger
et
al.
1982).
Like
other
sexual
behaviors,
it
appears
to
follow
a
cyclic
pattern
(
Wada
1982;
Ottinger
et
al.
1982).
When
behavioral
tests
are
used
as
endpoints
for
reproduction
toxicity
studies,
the
cyclicity
of
the
behavior
must
be
taken
into
account
so
that
behaviors
are
measured
within
the
appropriate
and
same
activity
period
during
the
day
(
Halldin
et
al.
1999).

Appetitive
sexual
behavior
and
copulation
behavior
of
the
male
quail
appear
to
be
under
similar
endocrine
control:
aromatization
of
testosterone
in
the
brain
(
Balthazart
et
al.
1997,
1995).
These
behaviors
indicate
male
interest
in
the
female,
and
they
include
precopulatory
responses,
such
as
approaching
and
remaining
near
a
female.
Two
methods
have
been
used
by
a
number
of
researchers
to
measure
appetitive
sexual
behavior
in
Japanese
quail
treated
with
steroids.
One
index
of
response
measures
the
duration
and
number
of
times
a
male
quail
stands
in
front
of
a
narrow
window
that
allows
him
to
see
a
female
in
an
adjoining
cage.
This
proximity
response
is
learned
behavior
and
is
only
displayed
in
males
that
have
copulated
with
a
female
(
Domjan
and
Hall
1986;
Riters
et
al.
1998).
The
second
method
is
independent
of
sexual
learning,
although
Riters
et
al.
(
1998)
showed
a
stronger
response
in
experienced
males.
It
measures
the
rhythmic
contraction
of
the
cloacal
sphincter
of
a
male
provided
visual
access
to
a
female.
Cloacal
sphincter
contractions
occur
during
the
production
of
foam
that
is
transferred
to
the
female
cloaca
during
copulation
(
Cheng
et
al.
1989a,
1998b).
Simple
visual
counts
of
contractions
are
made
by
an
observer
who
views
the
bird
through
a
mirror
positioned
below
a
glass
floor,
such
as
that
of
an
aquarium
as
test
chamber.

To
date,
breeding
behavior
appears
to
be
among
the
most
sensitive
endpoints
measured
for
birds
exposed
to
estrogen
or
estrogen­
agonists
(
Halldin
et
al.
1999).
Male
copulatory
behavior
has
been
shown
to
be
a
highly
sensitive
indicator
of
in
ovo
endocrine
disruption
and
is
the
most
commonly
tested
behavioral
endpoint
in
endocrine
effects
tests.
Currently,
the
endpoint
of
copulatory
behavior
is
being
compared
in
both
bobwhite
and
quail
at
the
University
of
Maryland
(
M.
A.
Ottinger,
personal
communication,
2002).
Given
the
sensitivity
of
this
endpoint
and
its
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April
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ecological
relevance,
male
copulatory
behavior
should
be
incorporated
into
avian
reproductive
toxicity
tests
to
evaluate
EDCs
in
F1
males.

6.3
Neurological/
Central
Nervous
System
Impairment
Tests
Endpoints
that
are
indicative
of
central
nervous
system
(
CNS)
effects
and/
or
hormonally­
mediated
toxic
responses
measure
behaviors,
such
as
open­
field
motor
ability,
panic
and
separation
responses,
and
hearing
and
location
skills.
These
behaviors
reflect
integrated
response
to
toxic
or
hormone
insult.
Open­
field
tests
provide
indication
of
motor
problems
associated
with
neural
or
CNS
impairment
and
can
be
conducted
at
any
stage
of
maturation,
from
juvenile
to
adult.
Runway
tests
are
often
conducted
with
young
chicks
to
take
advantage
of
the
chicks'
need
to
group
with
conspecifics.
An
individual
chick
is
placed
on
one
end
of
a
runway
opposite
an
open
cage
containing
conspecifics.
The
course,
the
time
to
rejoin
conspecifics,
and
behaviors
such
as
defecation,
inquisitiveness,
and
tonic
immobility,
are
recorded.
These
tests
are
repeated
as
the
chicks
age
to
track
performance
improvement
and
maturity,
with
less
need
to
seek
conspecifics.
In
a
recent
test
using
Japanese
quail,
chicks
hatched
from
parents
fed
an
endocrine
disrupting
chemical,
methoxychlor,
displayed
immature
behaviors
longer
than
did
controls,
suggesting
a
slower
maturation
in
response
to
parental
exposure
(
M.
A.
Ottinger,
personal
communication,
2002).
The
test
was
ineffective
in
measuring
the
response
of
northern
bobwhite
chicks,
because
they
did
not
adapt
well
to
the
test
paradigm.
In
runway
tests
using
maternal
calls
as
the
approach
incentive,
ducklings
treated
with
DDE
were
more
responsive
than
control
ducklings
to
the
calls
(
Heinz
1976),
whereas
treatment
with
methylmercury
resulted
in
hyporesponsive
ducklings
(
Heinz
1979).
The
opposite
was
found
in
runway
avoidance
tests
with
a
fright
stimulus.
In
the
avoidance
tests,
methylmercury
treatment
resulted
in
hyper­
responsive
ducklings,
whereas
ducklings
treated
with
DDE
were
hyporesponsive
to
the
stimulus
(
Heinz
1976,
1979).
In
contrast,
activity
level
and
exploratory
behavior
in
an
open­
field
test
were
unaffected
by
DDE
exposure
(
Heinz
1979).
Runway
tests
appear
to
be
sensitive
in
some
species
and
are
relatively
simple
to
perform,
but
require
expertise
in
interpreting
results.
Additional
work
is
required
to
verify
which
behavior,
or
set
of
behaviors
or
stimuli,
are
most
effective
and
constitute
an
adequate
endpoint
for
a
wide
variety
of
chemicals.
The
most
convenient
age
to
test
the
chicks
is14
days,
when
the
F1
and
F2
survivors
are
terminated.
The
determination
of
the
sensitivity
of
the
behavioral
assays
at
this
age
should
be
emphasized.

7.0
ASSAY
ENDPOINTS:
PHYSIOLOGICAL
This
section
describes
the
physiological
endpoints
that
can
serve
as
direct
measurements
of
endocrine
activity
in
an
avian
two­
generation
reproduction
toxicity
test.
Emphasis
is
given
to
endpoints
recommended
by
EDSTAC
(
EPA
1998)
and
those
compiled
by
OECD.
Additional
endpoints
were
added
from
the
literature
review.
The
physiological
endpoints
include
morphological
and
histological
evaluations,
brain
chemistry,
and
plasma
and
fecal
steroid
analysis.
These
endpoints
are
listed
in
Table
7­
1
in
relation
to
the
life
stage
at
which
the
endpoint
can
be
measured.
The
potential
underlying
endocrine
mechanisms
(
estrogenic,
androgenic,
and
thyroidogenic)
are
also
provided.
Battelle
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April
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2003
Table
7­
1.
Physiological
Endpoints
Specific
to
Endocrine
Active
Substances(
a)

Physiological
Endpoints
Critical
Life
Stages
for
Evaluation
of
Impact
Type
of
Endocrine
Activity
Embryo
genesis
Early
development
Sexual
maturation
Egg­
laying
Estrogenic
Androgenic
Thyroidogenic
For
Parents
and
Adult
F1
GROSS
MORPHOLOGY
AND
HISTOLOGY
size
and
weight
of
testes,
ovaries,
thyroid,
adrenals,
brain,
oviduct
yes
yes
yes
+/
 
(
b,
c)
+/­­
+/­­

histology
of
thyroid,
adrenals,
gonads,
and
brain
yes
yes
yes
+/­­
+/­­
+/­­

testicular
spermatid
counts
and
morphology,
sperm
mobility
test,
perivitelline
layer
sperm
yes
+/­­

gross
anomalies
of
the
genital
tract
yes
yes
yes
+/­­
+/­­

cloacal
gland
area
yes
yes
+/­­

PLASMA
AND
FECAL/
URATE
HORMONES
steroid
hormones
(
estradiol,
testosterone,
corticosterone)
yes
yes
yes
+/­­
+/­­

vitellogenin
(
males
only)
yes
yes
yes
+/­­

thyroid
hormones
and
TSH
yes
yes
yes
+/­­

BRAIN
CHEMISTRY
GnRH
yes
yes
yes
+/­­
+/­­

catecholamine
yes
yes
yes
+/­­
+/­­

aromatase
yes
yes
yes
+/­­

foam
gland
test
yes
+/­­

presence
of
medullar
bone
yes
yes
+/­­

For
F1
and
F2
Chicks
gonad
weights
yes
yes
+/­­
+/­­

oviduct
weights
and
differentiation
yes
+/­­

wing
and
bone
length
yes
+/­­
+/­­

skeletal
X­
ray
yes
+/­­
+/­­

thyroid
weight
and
histology
yes
+/­­

plasma
sex
steroids
yes
+/­­
+/­­
+/­­

a)
Table
from
Bennett
et
al.
(
2001).
b)
+
indicates
positive
activity
(
e.
g.,
estrogenic,
androgenic,
thyroidogenic).
c)
 
indicates
negative
activity
(
e.
g.,
antiestrogenic,
antiandrogenic,
antithyroidogenic).
Battelle
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2003
7.1
Organ
Growth
and
Morphological
Changes
Measures
of
growth
and
morphological
changes
of
reproductive
organs
and
their
accessory
structures
are
primary
endpoints
of
xenobiotic
impact
on
the
development
and
integrity
of
reproductive
tract.
The
gross
measures
of
organ
weight
and
gross
morphology
are
among
the
most
economical
of
the
endocrine­
sensitive
endpoints.
Microscopic
determination
of
gonadal
abnormalities,
gamete
production,
and
steroidogenic
capability
are
among
the
most
sensitive
measures
that
can
give
insight
into
the
mechanism
of
action
or
target
structures
of
chemical­
induced
injury
or
endocrine
imbalance.

7.1.1
Development
of
Gonadal
and
Accessory
Structures
Gonad
weight
is
a
rapid
quantitative
index
that
reflects
the
effect
of
a
test
substance
on
gonadal
development
and
reproductive
condition.
For
example,
weights
of
testes
and
epididymes
are
often
used
as
indicators
of
possible
alteration
in
androgen
status
and
a
correlation
between
testis
weight
and
the
number
of
germ
cells
in
the
testis
has
been
demonstrated
(
Zenic
and
Clegg
1989).
Yet,
testicular
weights
are
not
as
sensitive
as
sperm
counts
in
assessing
reproductive
capacity
and
are
compromised
by
edema
(
Thomas
and
Thomas
2001).
Ovary
weight
in
growing
and
mature
birds
is
a
good
measure
of
endocrine
and
reproductive
condition.
A
companion
measure
of
reproductive
status
in
female
quail,
considered
by
some
authors
to
be
a
better
predictor
of
female
maturation
than
ovary
weight,
is
oocyte
diameter.
One
version
of
this
endpoint
is
the
measurement
of
the
diameter
of
the
largest
ovarian
follicle
prior
to
first
egg
laid
(
Phillips
et
al.
1997);
another
is
the
mean
number
of
oocytes
per
female
that
show
initiation
of
rapid
growth
 
that
is,
oocytes
greater
than
4
mm
in
diameter
and
yellow
in
color
(
Thanton
and
Parkhurst
1973).
A
pattern
of
normal
ovarian
development
in
Japanese
quail
has
been
described
from
hatch
to
adulthood
by
several
investigators
(
e.
g.,
Pageaux
et
al.
1984;
Lucy
and
Harshan
1999);
however,
the
reported
timing
of
growth
events
varies
significantly
among
them.

For
example,
Lucy
and
Harshan
(
1999)
reported
that
rapid
growth
of
the
oviduct
of
Japanese
quail
begins
between
30
and
40
days
of
age
and
attains
adult
weight
and
functional
maturity
between
50
and
60
days
of
age.
By
comparison,
Pageaux
et
al.
(
1984)
reported
that
rapid
growth
of
the
oviduct
of
Japanese
quail
began
between
21
and
28
days
of
age,
reaching
adult
weight
by
45
days.
Such
differences
underscore
the
variation
management
practices
between
laboratories
and/
or
in
sexual
maturation
rates
found
among
the
various
breeding
stock
of
Japanese
quail.
The
characteristic
response
of
the
oviduct
to
estrogen
is
an
increase
in
wet
weight,
water
content,
and
glycogen
(
Bitman
et
al.
1968).
Differentiation
of
oviduct
segments
(
infundibulum,
magnum,
isthmus,
shell
gland,
and
vagina)
can
be
used
as
a
measure
of
the
maturation
(
Lucy
and
Harshan
1999)
and
can
be
used
as
another
easily
obtained
biomarker
for
delayed
or
accelerated
maturation
at
necropsy.
Incidence
rates
of
right
oviducts
in
F1
and
F2
chicks
indicate
abnormal
endocrine
signal,
and
in
adult
birds,
is
important
to
measure,
because
it
is
a
condition
that
is
associated
with
reduced
fertility
in
Japanese
quail
(
Rissman
et
al.
1984).

A
characteristic
feature
of
sexual
maturation
in
birds
is
the
asymmetry
of
their
gonads.
In
most
species,
the
right
ovary
does
not
develop,
and
the
right
testis
is
often
smaller
than
the
left
(
Perrin
Battelle
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2003
et
al.
1995).
A
ratio
of
testis
weights,
left
divided
by
right,
is
often
used
to
characterize
the
effect
of
xenoestrogens
in
reducing
the
size
of
the
right
testis
(
Rissman
et
al
1984).

Organ
weights
in
toxicity
studies
are
typically
recorded
in
one
of
three
formats,
as
the
absolute
or
wet
tissue
weights,
or
as
somatic
or
brain
indices.
An
organosomatic
index
is
the
absolute
weight
of
the
organ
normalized
to
the
weight
of
the
animal.
It
provides
a
general
measure
of
maturity
and
reproductive
condition.
However,
this
approach
assumes
that
the
ratio
of
organ
weights
to
body
weight
describes
the
entire
relationship
between
the
two
variables
and
does
not
account
for
positive
or
negative
growth
of
an
organ
with
respect
to
the
overall
body
weight
(
Salsburg
1986).
Therefore,
normalizing
organ
mass
to
body
weight
if
changes
in
body
weight
occur,
for
example,
could
mask
the
effect
of
a
toxicant
on
the
size
of
the
gonads
(
Ballantyne
1999).
If
the
strain
of
quail
experiences
photoperiod
drift
and
if
some
individuals
within
groups
therefore
cannot
be
synchronized,
then
a
large
variability
will
be
manifested
in
this
endpoint
(
Grossmen
et
al.
1982).
Statistically,
a
better
measure
of
organ
growth
is
multivariate
analysis
of
covariance
of
the
gonad
and
body
weights
(
Salsburg
1986;
Weatherly
1990).
Regression
analysis
is
performed
on
the
body
weight
and
gonad
weight
data
of
each
group.
A
weighted
average
is
calculated
from
the
quotient
of
the
gonad
weight
and
the
slopes
for
each
treatment
group.
Gonad
weight
is
then
adjusted
by
the
average
body
weight.

Because
of
the
potential
problems
with
normalizing
organ
weight
by
body
weight
of
the
animal,
some
investigators
report
gonadal
weights
as
a
ratio
of
the
organ
weight
to
the
brain
weight.
Brain
weight
is
used
in
the
index,
because
this
measure
is
usually
very
stable,
and
chemicals
that
cause
a
change
in
body
weight
typically
do
not
affect
the
brain
weight
of
the
animal
(
Ballantyne
1999).
Reporting
gonad
or
other
organ
weights
relative
to
brain
weight
requires
that
the
brain
be
removed
with
precision.
In
particular,
the
point
at
which
the
brain
is
severed
from
the
brain
stem
must
be
clearly
established
in
the
necropsy
protocol
and
then
strictly
followed.

Developmental
landmarks
in
F1
and
F2
chicks
may
be
used
to
assess
physiological
age
of
a
growing
animal.
Morphological
features
can
be
evaluated
during
early
weeks
of
growth,
and
the
developmental
landmarks
must
be
clearly
identifiable
and
directly
related
to
normal
ages
or
stages
of
maturation.
Potential
avian
developmental
landmarks
include
hatch
date,
age
of
female
when
first
egg
is
laid,
age
of
male
when
foam
gland
function
begins
and
gland
size
increases,
and
age
at
which
sexually
dimorphic
plumage
develops.
A
summary
of
developmental
landmarks
and
approximate
ages
is
provided
in
Table
7­
2.
Histological
examination
of
major
tissues
can
also
provide
information
on
toxic
effects
of
test
substances
on
maturing
organs
and
tissues
(
Section
7.1.4).
Embryonic
histology
relative
to
gonadal
development
is
discussed
in
this
section,
because
it
is
well
described
for
the
perihatch
period,
which
is
usually
2
days
before
hatch,
it
has
application
to
field
exposure
assessment,
and
it
decreases
with
age
posthatch.
Battelle
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Table
7­
2.
Gross
Landmarks
of
Sexual
Differentiation
in
Japanese
Quail
and
Age
of
Appearance(
a)

Parameter
Mean
Day
of
Onset
Maximum
(
day)

testes
weight
(
mg)
22
49­
57
sexually
dimorphic
plumage(
b)
21­
25
 
crowing
(
per
30
min)
33
50
age
of
male
when
foam
gland
matures
32
50
(
maximum
area)

age
of
onset
of
egg
production
42
77(
c)

mating
attempts
(
per
5
min)
34
52
cloacal
contacts
(
per
5
min)
37
81
a)
Data
from
Ottinger
and
Brinkley
(
1979b).
b)
Estrogen­
dependent.
c)
About
3­
5
weeks
after
first
egg
laid.

7.1.2
Histopathology
in
Juveniles
Light
and
electron
microscopy
of
immature
gonadal
tissue
can
be
used
to
detect
changes
in
gonadal
morphology
and
ultrastructural
sterodiogenic
capability
(
Abdelnabi
et
al.
2000).

Because
the
avian
female
is
heterogametic
(
Z/
W),
estrogen
is
important
for
differentiation
to
a
phenotypic
female
(
Andrews
et
al.
1997),
and
inhibition
of
estrogen
synthesis
will
result
in
genetic
females
with
male
phenotype
(
Elbrecht
and
Smith
1992).
Exposure
in
ovo
to
estrogenic
compounds
gives
rise
to
males
with
feminized
sex
organs,
such
as
the
presence
of
Müllerian
ducts
and
ovary­
like
tissue
in
the
left
testes,
and
females
with
Müllerian
duct
malformations,
such
as
the
persistence
of
a
right
Müllerian
duct
or
hypertrophied
ducts.
The
most
sensitive
of
these
endpoint
malformations
is
the
transformation
of
the
left
male
gonad
into
an
ovotestis.
Significantly
increased
incidence
of
male
embryos
exhibiting
an
ovotestis
was
observed
at
doses
as
low
as
0.7
ng/
g
egg
for
endogenous
or
exogenous
estrogens
(
Berg
et
al.
1999).
The
ovotestis
results
from
the
juxtaposition
of
the
male
medulla
and
an
ovary­
like
cortex,
each
characterized
by
its
own
histological
features.
The
scoring
criterion
for
an
ovotestis
is
the
appearance
of
oocyte­
like
cells
in
meiotic
prophase
in
the
male
testicular
cords
(
Berg
et
al.
1999).
The
degree
of
feminization
of
the
left
testis
has
been
quantified
by
comparing
the
abnormal
area
with
the
total
area
using
image
analysis.
The
right
testis
in
treated
male
embryos
is
markedly
reduced
(
Perrin
et
al.
1995).
Genetic
females
treated
in
ovo
with
estrogens
have
enhanced
cortical
proliferation
of
the
ovary
and
a
severe
reduction
of
the
rudimentary
right
gonad.
Gonadal
dissymmetry
is
not
conserved
after
hatching,
but
decreases
with
age,
disappearing
prior
to
sexual
maturation.
After
hatch
the
germ
cells
of
the
ovotestes
enlarge
and
are
enclosed
in
follicles,
but
ultimately
are
resorbed
between
3
and
5
weeks
of
age
(
Scheib
and
Reyss­
Brios
1979).
Boss
and
Witschi
(
1947)
reported
that
in
ovo
exposure
of
herring
gulls
(
Laras
argentatus)
to
stilbesterol
resulted
in
ovo
testes
that
persisted
for
4
years,
but
the
gulls
were
injected
with
stilbesterol
Battelle
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weekly
after
hatch.
Halldin
et
al.
(
1999)
also
found
that
embryonic
exposure
to
diethylstilbestrol
or
ethinyletradiol
did
not
result
in
observable
histological
changes
in
the
testes
of
8­
week­
old
male
quail,
but
pointed
out
that
persistence
of
ovotestes
into
adulthood
could
be
dose­
dependent.

It
should
be
noted
that
transformation
of
the
left
testis
into
an
ovotestis
has
been
observed
in
untreated
control
embryos.
In
a
study
conducted
by
Berg
et
al.
(
1999),
about
18%
of
the
controls
had
ovary­
like
tissue
in
the
left
testis.
In
female
quail,
Müllerian
duct
malformations
from
in
ovo
exposure
to
estrogenic
compounds
appear
to
be
conserved
into
adulthood
(
Rissman
et
al.
1984;
Adkins­
Regan
et
al.
1995).

Because
ovotestes
and
Müllerian
duct
malformations
are
sensitive
measures
of
endocrine
disruption
from
in
ovo
exposure
and
are
likely
to
persist
in
14­
day­
old
chick,
these
tissues
should
be
collected
from
at
least
one
of
the
weekly
batches
of
the
F1
and
F2
surviving
chicks.
Weights
of
the
gonadal
tissue
and
its
appearance
at
necropsy
should
be
recorded.
The
tissue
should
be
preserved
for
histological
examination
and
the
gonadal
tissues
from
control
chicks
and
chicks
of
the
high
dosed
parents
evaluated
histologically.
Additional
dose
groups
can
be
examined
if
warranted
by
the
presence
of
abnormal
gonadal
tissue
in
the
high­
dose
chicks.

Having
information
on
the
dose
and
embryonic/
chick
histological
response
to
the
chemical
would
provide
exposure
information
and
comparison
for
field
studies
or
cases
in
which
egg
samples
of
wild
birds
can
be
collected.

7.1.3
Histopathology
in
Sexually
Mature
Individuals
Histological
methods
are
sensitive
indicators
of
gonadal
function
and
pathological
processes
in
animals
and
can
provide
information
on
the
site
of
action
of
a
xenobiotic.
However,
usefulness
of
these
observer­
based
methods
to
quantify
effects
in
avian
reproduction
toxicity
tests
is
limited
by
lack
of
a
uniform
classification
system
and
codification
of
evaluation
criteria.
Standardization
of
structural
measurements,
such
as
tubule
lumens,
randomization
of
selected
structures,
such
as
tubules,
for
measurement,
and
scoring
criteria
would
provide
repeatable
interpretation
of
tissue
changes
induced
by
xenobiotics
and
endogenous
hormones
in
birds
that
can
be
verified
by
different
investigators.
For
example,
routine
histological
evaluations
detect
testicular
damage
only
when
there
is
severe
depletion
of
the
seminiferous
epithelium,
obvious
cellular
degeneration,
or
sloughed
cells
in
the
seminiferous
tubule
lumen.
Less
prominent,
though
reproductively
important
lesions
could
be
identified
by
morphometric
measurements
of
the
testicular
structures.
Of
particular
importance
in
the
evaluation
of
testicular
injury
is
the
use
of
seminiferous
tubule
stages
(
Creasy
1997;
Lin
and
Jones
1993).
Quantification
of
cell
staging
involves
examining
cross­
sections
of
seminiferous
tubules
to
determine
the
frequency
distribution
of
distinct,
sequential
associations
of
developing
germ
cells,
which
could
be
identified
as
stages
of
proliferation
and
differentiation.
In
the
Japanese
quail,
these
stages
have
been
well
characterized
and
are
arranged
in
a
helix
that
extends
along
the
length
of
the
seminiferous
tubule
(
Lin
and
Jones
1990).
The
duration
of
the
stages
is
a
time­
specific
process
that
ranges
from
2.5
h
to
15.5
h
in
Japanese
quail
(
Lin
et
al.
1990).
Therefore,
the
abundance
of
any
given
stage
is
directly
proportional
to
the
duration
of
the
stage.
Quantitative
and
semiquantitative
methods
devised
for
the
assessment
of
the
effects
of
xenobiotics
on
testicular
Battelle
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April
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2003
germ
cells
involve
measurement
of
the
cross­
sectional
area
of
semiferous
tubules
and
the
ratio
of
the
germ
cell
to
the
Sertoli
cell,
and
counting
the
number
of
spermatids
per
seminferous
tubule
(
Creasy
1997).

In
addition
to
the
codification
of
lesions
or
structural
measurements,
components
of
histological
examinations
that
need
to
be
standardized
to
avoid
artifacts
that
can
interfere
with
the
evaluations
are
1)
handling
of
unfixed
reproductive
tissues;
2)
the
tissue
embedding
and
sectioning
processes;
3)
selection
of
the
chemical
fixative
protocol
and
stains;
4)
sectioning
procedures;
and
5)
the
codification
of
lesions
or
structural
measurements.
Handling­
artifacts
in
testes
are
common
and
result
from
dropping
testes
on
weighing
scales
or
from
excessive
manipulation
of
the
tissue
with
forceps
(
Foley
2001).
The
common
fixatives
used
in
histology
of
reproductive
tissues
are
10%
neutral
buffered
formalin
and
Bouin's
solution.
Tissue
shrinkage
and
staining
properties
are
different
for
these
two
fixatives.
Formalin
fixation,
though
routinely
used,
results
in
artifacts
in
the
germinal
epithelium
of
the
testis
when
used
prior
to
embedding
in
paraffin.
Bouin's
fixative
provides
superior
preservation
of
cellular
detail
in
tissues
such
as
the
testis
(
Foley
2001).
There
are
a
number
of
disadvantages
of
using
Bouin's
fixative:
critical
timing
of
tissue
trimming
and
processing,
and
storage
of
fixed
tissue,
for
example.
Therefore,
protocols
using
a
combination
of
initial
fixation
of
the
testes
in
formalin
followed
by
immersion
of
trimmed
sections
in
Bouin's
solution
have
been
developed
to
minimize
the
use
of
Bouin's
fixative
and
retain
the
ease
of
collecting
tissues
in
formalin
 
a
single,
multipurpose
fixative
(
Foley
2001).
For
the
most
precise
staging
of
the
seminferous
epithelium
in
the
testis,
the
germ
cell
acrosome
should
be
stained
using
periodic­
acid
Schiff's
method
(
Clermont
1972).
Formalin
fixation
can
be
used
to
examine
the
seminferous
epithelium
if
it
is
then
embedded
in
plastic
and
stained
with
touluidine
blue
(
Russel
and
Frank
1978;
Ulvick
et
al.
1982).
Hematoxylin
and
eosin
or
touluidine
blue
are
common
stains
applied
to
gonadal
tissue.
Each
provides
a
different
view
of
tissue
structure.
Use
of
plastic
embedding
provides
greater
support
of
the
tissue
and
thinner
sections.
Samples
for
histopathological
examinations
should
be
collected
from
the
same
area
of
each
organ
and
serially
sectioned
along
the
long
axis
of
the
gonad.
Samples
should
be
examined
"
blind,"
without
knowledge
of
the
treatment.

Because
of
their
large
volume
of
yolk,
mature
oocytes
and
ova
have
been
difficult
to
preserve
for
microscopic
examination.
Immersion
fixation
of
follicular
oocytes
in
paraformaldehyde
and
glutaraldehyde
fixative
followed
by
a
second
immersion
in
fresh
fixative
to
assure
penetration
and
adequate
preservation
provides
the
best
fixation
of
the
ovarian
follicles
for
light
microscopy.
For
electron
microscopy,
tissues
are
trimmed
further
and
placed
in
osmium
tetroxide
before
sectioning
(
Bakst
1993).

Preparation
of
specimens
from
the
oviduct
or
excurrent
duct
for
light
microscopy
are
routine.
However,
the
secretory
function
of
the
oviduct
appears
to
develop
sooner
than
the
organs
gross
differentiation
(
Reddy
et
al.
1992);
therefore,
use
of
histochemical
or
immunocytochemical
techniques
to
detect
oviductal
secretion
of
fibrous
albumen
proteins
could
be
used
to
measure
the
maturation
of
the
organ.
These
oviductal
secretions
can
be
problematic
when
one
is
examining
the
tissue
with
scanning
electron
microscopy,
because
they
can
obstruct
the
luminal
surface.
Similarly,
a
clot
of
sperm
can
obscure
the
luminal
surface
in
the
excurrent
ducts
of
the
testis.
Battelle
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April
23,
2003
Procedures
are
available
that
reduce
the
incidence
of
these
interferences
(
Bakst
and
Howarth
1975;
Kirby
et
al.
1990).

Lectin
probes
have
been
used
to
distinguish
the
stages
of
avian
spermatogenesis,
by
binding
to
a
terminal
saccharide
of
a
glycoconjugate
expressed
on
either
a
spermatogonia,
spermtocyte,
spermatid,
or
Serotoli
cell
(
Bakst
and
Cecil
1988).
However,
fixatives
can
mask
this
binding.
Unfortunately,
the
fixative
did
not
mask
lectin
binding,
but
provided
adequate
tissue
preservation
contains
merucuric
chloride
(
sublimate
formalin).
Substitution
of
less
hazardous
zinc
chloride
could
be
possible,
but
has
not
been
evaluated.

There
are
fewer
professional
pathologists
with
experience
in
evaluating
avian
tissues
than
of
those
with
experience
in
mammalian
histology.
The
level
of
quanitification
and
codified
description
of
reproductive
organs
for
GLP
evaluations
that
has
developed
over
the
years
for
mammalian
toxicity
testing
is
lacking
in
avian
applications.
In
particular,
familiarity
with
the
stages
and
cycle
of
the
seminiferous
epithelium
of
the
testis
that
is
essential
to
injury
assessment
has
not
been
applied
to
avian
toxicity
studies.
However,
the
stages
of
the
germ
cells
in
the
Japanese
quail
and
poultry
are
well
described
and
used
to
evaluate
fertility
and
the
impact
of
endocrine­
moderating
chemicals
on
spermatogenesis.
Histological
examination
by
professional
pathologist
in
the
USA
costs
from
$
8
to
$
16
per
tissue,
when
conducted
under
GLP
criteria
for
mammalian
studies.

7.1.4
Histopathology
and
Organ
Weights
of
Nonreproductive
Tissues
The
thyroid,
adrenal
gland,
and
brain
are
potentially
affected
by
a
number
of
EDCs.
They
can
be
analyzed
by
classical
histological
techniques
for
evidence
of
abnormal
structure
that
generally
results
in
dysfunction.
However,
histopathological
examination
of
tissues
from
birds
exposed
to
potential
reproductive
toxins
in
avian
reproduction
toxicity
tests
has
been
limited
(
Mineau
et
al.
1994).
New
immunological
methods
can
provide
more
objective
and
precise
information
of
changes
in
morphology
and
function
in
tissues.
A
review
of
mammalian
reproduction
studies
indicates
that
the
adrenal
gland
is
the
most
susceptible
of
the
endocrine
glands
to
chemical­
induced
lesions
(
Ribelin
1984).
About
40%
of
the
studies
reporting
histological
effects
in
endocrine
tissue
were
compounds
that
damaged
adrenal
glands.
The
thyroid
was
affected
by15%
of
the
compounds.
As
noted
above,
methods
of
tissue
handling,
fixation,
staining
and
lesion
scoring
should
be
clearly
established
to
help
standardize
evaluations.

7.1.4.1
Brain.
In
recent
years,
several
research
groups
have
been
investigating
the
effect
of
steroids
on
the
innervation
of
the
septopreoptic
region
of
the
hypothalmus
of
birds.
The
medial
preoptic
nucleus
and
lateral
septum
are
sexually
dimorphic
and
highly
involved
in
the
activation
of
adult
sexual
behavior
of
Japanese
quail
(
Halldin
et
al.
1999;
Balthazart
and
Surelemont
1990
a,
1990
b;
Panzica
et
al.
1996).
Hormonal
manipulation
of
juveniles
and
adults
results
in
profound
morphological
changes
in
these
areas
of
the
brain,
including
changes
in
the
density
of
vasotocin
fibers
and
the
number
and
size
of
aromatase­
containing
neurons
(
Panzica
et
al.
1994).
These
changes
occur
in
response
to
photostimulation
and
aging,
but
also
as
a
result
of
exogenous
steroid
exposure
(
Panzica
et
al.
1996).
Although
examination
of
the
septopreoptic
tissue
can
provide
information
on
the
underlying
mechanisms
of
reproductive
depression,
measures
of
the
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behaviors
activated
by
this
area
are
highly
sensitive
to
changes
in
steroid
concentration
and
more
economical
to
perform.
Fixation
of
the
tissue
and
the
immunochemistry
on
serial
sections
of
the
septopreoptic
region
of
the
hypothalamus
are
relatively
lengthy
processes.
The
specific
antibody
for
the
immune
reaction
is
not
commercially
available,
and
quantitative
determination
of
the
density
of
the
immunoreactive
structures
requires
computerized
image
analysis.

7.1.4.2
Thyroid.
The
close
link
between
secretory
function
of
endocrine
tissue,
such
as
the
thyroid,
and
proliferative
activity
agrees
with
the
frequent
observation
in
toxicity
studies
that
a
common
response
to
excessive
stimulation
is
endocrine
hyperplasia
neoplasia
(
Pawlikowski
1982).
Generally,
therefore,
organ
weights
should
be
obtained
prior
to
histological
examination
of
these
organs.
However,
excising
and
trimming
firmly
bound
organs
such
as
the
thyroid
gland
for
organ
weight
determination
is
not
advisable
because
of
the
risk
of
disruption
of
histological
sections.
The
thyroid
is
a
small,
firmly
attached
organ
deeply
embedded
in
tissue
and
is
difficult
to
excise
and
trim.
Not
only
does
this
introduce
variation
in
organ
weight
measurements,
but
the
degree
of
handling
required
can
so
strongly
affect
the
quality
and
orientation
of
sections
as
to
offset
the
benefit
of
organ
weight
data.
Therefore,
thyroid
tissue
should
not
be
excised
for
organ
weight,
but
removed
with
minimal
handling
for
histological
preparation
(
Greaves
1999).
A
means
of
obtaining
thyroid
weights
is
to
excise
the
organ
within
the
surrounding
tissues
and
fix
it
in
preservative
prior
to
trimming.
Subsequently,
trimming
and
weighing
the
preserved
gland
will
minimize
damage
to
the
gland
and
allow
for
more
consistent
weights.
Thyroid
hypertrophy
can
then
be
quantified
histologically.
Enlarged
thyroids
may
reflect
either
a
hyper­
or
hypofunctioning
gland
(
Wentworth
and
Ringer
1986).
Initially,
examination
of
the
thyroid
should
be
completed
on
the
controls
and
high­
dosed
groups.
Histopathology
of
the
thyroid
can
be
conducted
on
other
groups
if
warranted
by
the
detection
of
lesions
in
the
thyroid
tissue
of
the
high
dosed
birds.
Immunocytochemical
techniques
have
superseded
trandioal
tinctorial
stanis
for
the
demonstration
of
poylpetide
hormones
(
Greaves
1999).

7.1.4.3
Adrenal
Gland.
As
noted
above,
the
adrenal
gland
appears
to
be
most
susceptible
of
the
endocrine
organs
to
chemically­
induced
structural
change.
Susceptibility
appears
to
be
due
to
the
accumulation
of
lipophilic
toxins
in
the
lipid
stores
of
the
cortical
cells
and
the
metabolic
conversion
of
chemicals
to
reactive
toxic
compounds
(
Capen
2001).
In
addition,
such
changes
appear
to
be
frequently
associated
with
alterations
in
reproductive
organ
function
and
structure
(
Maronpot
1987).
Even
so,
the
range
of
histological
responses
in
the
adrenal
gland
is
small
(
Tucker
1998).
The
most
commonly
induced
morphological
change
is
hypertrophy
with
increased
vacuolar
degeneration
(
Ribelin
1984).
Cortical
(
interrenal)
hypertrophy
resulting
from
impaired
steroidogenesis
and
the
accumulation
of
precursors,
such
as
neutral
fats,
in
the
cortical
cells
is
found
in
animals
treated
with
either
endogenous
or
exogenous
steroids.
The
hypertrophy
can
be
so
great
that
it
impairs
cellular
function.
Administration
of
adrenocorticotropic
hormone
(
ACTH)
will
also
cause
hypertrophy
of
the
cortex
from
increased
size
of
the
cells.
In
contrast,
exogenous
steroid
treatment
causing
depressed
ACTH
levels
results
in
atrophy
of
the
cortex
in
mice.
Cortical
hyperplasia
may
also
result
from
hormonal
disruption,
and
propylthiouracil
has
been
shown
to
cause
proliferation
in
chromaffin
cells
medulla
of
mammals
(
Tucker
1998;
Ribelin
1984).
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Because
of
adhering
fat,
adrenal
glands
can
be
difficult
to
excise
cleanly
for
organ
weight
measurements.
To
avoid
variation
in
adrenal
weights,
a
standardized
necropsy
protocol
for
the
tissue
should
be
established.

7.1.5
Japanese
Quail
Male
Cloacal
Gland
Measures
The
cloacal
gland
is
a
secondary
sex
characteristic
of
male
Japanese
quail
that
develops
in
response
to
testosterone
and
has
been
used
to
measure
androgen
status
in
the
male
during
sexual
maturation
(
Ottinger
and
Brinkley
1979a,
1979b).
Typically,
the
length
and
width
of
the
gland
is
measured
with
calipers,
and
the
area
is
recorded
as
the
product
of
longest
length
and
greatest
width
of
the
gland.
Coefficients
of
variation
are
typically
low
(
4
to
10)
for
this
measure.
Measurement
of
just
the
length
of
the
cloacal
gland
has
also
been
used
successfully
to
demonstrate
the
effect
of
hormone
treatment
on
maleness
in
Japanese
quail
(
Hutchison
1978)
and
cloacal
gland
volume
in
cubic
centimeters
has
also
been
used
to
assess
reproductive
activity
(
CV
of
4%)
using
the
following
formula
4/
3Bab2
(
2)

where
a
is
half
of
the
length
of
the
long
axis
and
b
is
half
of
the
length
of
the
short
axis
(
Chaturvedi
et
al.
1993).

Treatment
with
xenoestrogens
in
ovo
has
been
shown
to
significantly
decrease
the
area
of
the
gland
in
the
sexually
mature
male
(
Halldin
et
al.
1999),
and
cloacal
gland
swelling
has
been
used
as
a
reliable
indicator
of
androgenic
activity
in
females
treated
with
testosterone
(
Phillips
et
al.
1997).
Treatment
of
males
with
estrogen
results
in
reduced
gland
area
and
deformities
in
the
cloacal
orifice
(
Phillips
et
al.
1997;
Halldin
et
al.
1999).
As
a
measure
that
can
be
taken
repeatedly
during
the
course
of
a
reproductive
test,
it
is
a
useful
endpoint
for
monitoring
maturation
and
androgen
status
in
P1
and
F1
parent
populations.
The
amount
of
foam
in
the
cloacal
glands
is
also
used
to
determine
male
reproductive
fitness.
The
cloacal
gland
is
palpated
and
the
proteinaceous
foam
measured
with
calipers
and
reported
in
units
of
square
millimeters
(
Sachs
1967;
Hutchison
1978).
However,
foam
measurements
can
be
highly
variable.
Date
of
actual
foam
formation
is
used
as
a
maturation
endpoint
for
the
male
Japanese
quail.
This
measure
is
applicable
to
the
P1
(
if
a
prematuration
exposure
is
used)
and
F1
chicks
as
they
approach
breeding.

7.2
Sexual
Differentiation
(
Including
Time
of
Onset
of
Egg­
laying)

As
described
above
in
Section
7.1.2,
in
ovo
exposure
to
estrogenic
compounds
will
modify
sex
organ
differentiation
in
birds,
feminizing
male
sex
organs
and
causing
Müllerian
duct
abnormalities
in
genetic
females.
Failure
of
male
gonadal
differentiation
in
the
presence
of
ecoestrogens
may
not
persist
from
embryonic
exposure
into
adulthood,
but
abnormalities
are
conserved
in
females
(
Rissman
et
al.
1984;
Adkins­
Regan
et
al.
1995).
Ovotestes
regress
after
hatch
and
disappear
by
sexual
maturation
(
Scheib
and
Reyss­
Brios
1979).
Reorganization
of
the
POM
in
the
hypothalmus
as
a
result
of
exposure
to
extrogenic
compounds
can
result
in
apparently
normal
males
that
lack
sexual
behavioral
differentiation
(
Berg
et
al.
1999).
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Sexual
differentiation
endpoints
include
an
array
of
morphological
and
behavioral
landmarks
that
are
easily
recorded
during
the
course
of
a
reproductive
toxicity
test.
The
sequence
of
behavioral
and
morphological
landmarks
of
sexual
differentiation
in
male
Japanese
quail
has
been
described
(
Ottinger
and
Brinkley
1979a,
1979b).
Their
onset
and
maximum
expression
are
highly
correlated
in
time
with
serum
testosterone
levels.
Table
7­
1
lists
these
landmarks
in
sequence
of
their
appearance
during
sexual
maturation.
Also
listed
are
estrogen­
dependent
landmarks
that
include
appearance
of
dimorphic
plumage
and
the
onset
of
egg­
laying
in
females.
Age
at
sexual
maturity
was
among
the
most
sensitive
measures
of
impaired
reproduction
in
the
female
birds
exposed
to
lead
and
was
detected
at
dietary
concentrations
that
caused
no
body
weight
loss
or
overt
signs
of
toxicity
(
Edens
et
al.
1976).
Onset
of
egg­
laying
was
delayed
by
up
to
2
weeks
in
lead­
treated
birds.
In
turn,
peak
egg
production
was
also
delayed
and
in
most
of
the
treatment
groups.
Similar
delays
in
onset
of
egg­
laying
have
been
observed
in
female
quail
in
a
restricted
feeding
program
(
Zelenka
et
al.
1984).
Timing
of
the
transition
from
somatic
growth
to
gonadal
growth
(
sexual
maturity)
is
influenced
by
accumulation
of
energy
reserves
in
the
juvenile
stage.
Therefore,
chemicals
such
as
OP
pesticides
that
alter
growth
rate
will
influence
the
onset
of
sexual
maturity,
especially
in
the
female
(
Zelenka
et
al.
1984).

7.3
Secondary
Sex
Characteristics
Development
of
secondary
sexual
characteristics
in
males
and
females
are
predominantly
determined
by
differences
in
steroid
secretion
of
the
gonads
of
each
sex.
Perturbations
in
the
development
of
these
characteristics
can
affect
the
reproductive
performance
of
the
animal
(
Bortone
et
al.
1989;
Bortone
and
Davis
1994).
Because
secondary
sex
characteristics
in
quail
are
hormonally
controlled,
they
may
be
useful
endpoints
in
endocrine
disruption
detection.
Major
secondary
sex
characteristics
of
Japanese
quail
include
body
size,
distinctive
plumage
dimorphism,
and
the
development
of
a
foam­
producing
cloacal
gland
and
sternotracheal
(
syringeal)
muscles
in
males
(
Adkins
1975;
Balthazart
et
al.
1983;
Schumacher
and
Balthazart
1984).
Formation
of
medualary
bone
in
females
generally
begins
10
days
before
egg­
laying
under
the
influence
of
estrogen
and
testosterone,
and
could
be
considered
a
secondary
sexual
characteristic
of
female
quail.
It
is
readily
induced
in
males
by
exposure
to
estrogens
(
Johnson
2000).
Classic
radiographic
techniques
or
X­
ray
bone
densiometry
can
be
used
to
detect
changes
in
medulary
bone
formation
(
Schreiweis
et
al.
2001).
Initial
equipment
cost
range
from
about
$
14,000
to
$
35,000
for
digital
formats.
Some
hand­
held
systems
are
available
at
lower
cost
that
have
had
use
in
in
vivo
measurements
of
humerus
and
ulna
radiographic
density
evalautions
for
poultry
growth
traits;
however,
these
systems
use
radioisotopes
with
relatively
short
half­
lives
that
must
be
replaced
as
often
as
every
6
months
at
$
1300
per
replacement,
and
image
quality
diminishes
as
the
isotope
decays.

In
New
World
quail
such
as
the
bobwhite,
plumage
dichromatism
is
thyroid­
estrogen
based:
that
is,
when
the
bird
is
adequately
saturated
with
thyroid
hormones
and
estrogen,
it
induces
the
development
of
female­
like
plumage.
Lack
of
estrogen
results
in
male
phenotype
(
Hagelin
and
Ligon
2001).
Low
thyroid
hormone
concentrations
result
in
asexual,
juvenile­
type
feathers.
In
the
past,
it
was
generally
thought
that
male
plumage
was
under
androgen
control
and
was
the
primary
criterion
for
female
birds'
mating
decisions.
However,
it
is
now
recognized
that
traits
such
as
testosterone­
mediated
breeding
displays
and
body
size
reflect
male
condition
and
are
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used
by
females
of
some
species
to
select
mates
(
Hagelin
and
Ligon
2001).
It
is
possible
for
very
high
testosterone
exposure
to
result
in
female
plumage
in
males
due
to
the
aromatization
of
testosterone
to
estrogen
that
would
superimpose
the
estrogen­
dependent
female­
type
plumage
on
the
male.
Also,
posthatch
treatment
of
male
Japanese
quail
with
estradiol
monobenzoate
has
been
shown
to
result
in
plumage
that
resembled
that
of
female
Japanese
quail
(
Hutchison
1978).
Changes
in
plumage
characteristics
has
been
reported
as
number
of
individuals
per
group
displaying
spotted
(
female
phenotype)
or
rufous
colored
(
male
phenotype)
breast
feathers.
The
width
of
the
brown
spots
covering
the
breast
region
has
also
been
used
to
quantify
degree
of
feminization
in
males
(
Hutchison
1978).

The
cloacal
gland
(
Section
7.1.5)
is
a
secondary
sex
characteristics
that
is
used
to
determine
maturation
of
male
Japanese
quail.
Bobwhite
males
lack
this
gland.
Maturation
is
determined
by
size
and
morphological
features
of
the
gland
and
by
it
secretory
activity
(
Wada
et
al.
1992).
The
gland
begins
to
enlarge
as
the
bird
approaches
sexual
maturation
and
takes
on
a
red­
colored
appearance.
A
proteinaceous
foam
is
also
produced
in
response
to
circulating
androgen
and
secretion
of
foam,
but
not
fluid,
is
a
sign
of
maturation.
Foam
quantity
has
also
been
measured
as
an
additional
correlated
variable
to
plasma
testosterone,
but
the
measure
is
subject
to
collection
variability.
These
endpoints
have
also
been
used
to
provide
qualitative
information
that
could
be
used
to
support
evidence
of
endocrine
disruption
(
Wada
et
al
1992;
Halldin
et
al.
1999).
Sternotracheal
muscle
weight
(
combined
left
and
right
muscles)
has
been
used
occasionally
to
evaluate
the
effect
of
estrogenic
treatment
in
male
Japanese
quail
(
Hutchison
1978).

Most
of
the
measures
for
these
secondary
sexual
characteristics
are
relatively
simple,
though
somewhat
subjective,
and
can
aid
in
interpretation
of
reproductive
results.

7.4
Sex
Ratio
Direct
exposure
of
chicks
and
adults
or
material
transfer
of
an
endocrine­
active
compound
and/
or
its
transformation
products
to
the
egg
can
produce
and
inappropriate
estrogen
signal
that
can
cause
permanent
sex
reversal
of
behavior
in
male
birds
and
transient
feminization
of
gonadal
tissue,
if
exposure
is
discontinued
(
Scheib
and
Reyss­
Brios
1979;
Berg
et
al.
1999).
Female
quail
exposed
to
antiestrogens
in
in
ovo
display
masculine
behaviors
as
adults
(
Schlinger
and
Arnold
1995;
Ottinger
and
Abdelnabi
1997).
Sex
ratios
skewed
toward
a
high
number
of
females
have
been
found
in
populations
of
birds
exposed
to
estrogenic
contaminants
in
the
Great
Lakes
(
Feyk
and
Giesy
1998).
Although
the
ovotestis
is
usually
discernable
from
a
normal
testis,
at
high
doses
of
estrogenic
contaminants,
it
can
be
difficult
to
distinguish
an
ovary
from
an
ovotestis,
and
alternate
method
of
sex
determination
will
need
to
be
used.
Sex
ratio
should
be
determined
for
a
representative
group
of
the
F1
and
F2
14­
day
survivors.
To
assure
that
the
chemical
has
had
opportunity
to
transfer
to
egg,
hatchlings
from
one
of
the
later
hatches,
for
example,
at
Week
4,
should
be
genetically
sexed
at
hatch.
When
the
chicks
reach
14
days
of
age,
they
should
be
necropsied,
the
presence
of
abnormal
gonad
recorded,
and
the
relative
amount
of
ovarian
tissue
determined
in
ovo
testes
for
the
males
only.
Gonadal
weights
obtained,
presence
of
oviduct
on
right
side,
and
the
steroid
status
should
be
determined
for
all
chicks.
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Available
laboratory
techniques
for
determining
sex
include
chromosomal
(
karyotyping)
and
DNA
probe
 
that
is,
the
Western
Dot
Blot
and
polymerase
chain
reaction
(
PCR)
methods.
Of
these
methods,
DNA­
sexing
is
preferred
for
monomorphic
or
immature
birds.
Karyotyping
involves
growing
the
sampled
tissue,
such
as
feather
pulp,
in
culture
for
7
to
9
days
and
microscopically
identifying
the
Z
and
W
chromosomes
in
a
stained
preparation
based
on
morphological
features.
Commercial
laboratories
charge
between
$
35
and
$
100
per
sample.
However,
karyotyping
is
not
considered
to
be
as
reliable
as
the
new
DNA
probe
methods.
DNA
probes
are
labeled
DNA
fragments
cloned
from
chromosomes.
DNA
probes
have
recently
been
developed
that
can
accurately
distinguish
the
DNA
in
the
sex
chromosomes
of
male
and
female
birds.
In
Western
Dot
Blot
assays,
each
DNA
sample
is
placed
onto
a
nylon
membrane
using
a
dot
blot
apparatus.
The
membrane
is
then
hybridized
with
a
chemoluminescent,
sex­
specific
DNA
probe
and
exposed
to
an
X­
ray
film
to
illuminate
the
differences
between
male
and
female
samples.
This
method
is
very
reliable
when
sufficient
amounts
of
high­
molecular­
weight
genomic
DNA
can
be
collected.

PCR
methods
use
a
single
set
of
primers
to
simultaneously
amplify
homologous
segments
of
the
CHD­
W
and
related
Z­
linked
gene,
CHD­
Z
(
Griffiths
and
Kom
1997).
Because
the
two
CHD
products
are
of
the
same
size,
a
restriction
enzyme
is
employed
to
cut
a
fragment
of
the
CHD­
Z
product
before
gel
electrophoresis
(
Ellegren
1996;
Griffiths
et
al.
1996).
This
assay
has
been
recently
modified
(
Griffiths
et
al.
1998)
to
use
two
PCR
primers
that
anneal
to
conserved
regions
of
the
gene
and
amplify
across
an
intron
that
differs
in
length
between
in
the
CHD­
W
and
CHD­
Z
genes
(
Khan
et
al.
1998).
Thus,
the
PCR
products
are
of
different
sizes,
and
a
restriction
enzyme
step
is
not
required
to
differentiate
between
the
sexes.
Both
dot
blot
and
PCR
methods
are
extremely
accurate
and
can
make
use
of
blood
or
feather
samples.
Because
only
a
drop
of
blood
is
needed,
claw­
clipping
will
provide
a
sufficient
sample
if
the
blood
spots
are
simply
collected
on
filter
paper.
One
feather,
freshly
plucked
in
a
manner
that
obtains
cells
from
the
calamus,
is
an
adequate
sample
(
Taberlet
and
Bouvet
1991).
Blood
and
feather
samples
for
DNA­
sexing
require
no
special
storage
and
can
be
archived
for
years.
Experience
is
vital
to
perform
these
assays.

Costs
at
commercial
laboratories
for
sexing
birds
using
DNA
probes
range
from
about
$
17
to
$
20,
and
$
22
to
$
25
per
sample
for
blood
and
feather
samples,
respectively.
Equipment
cost
to
outfit
a
laboratory
for
dot
blot
assays
can
be
as
low
as
$
2000
to
$
3000.
For
PCR
methods,
initial
equipment
cost
can
be
$
5000­
$
10,000
for
manual
methods
and
as
much
as
$
75,000­$
100,000
for
highly
automated,
high
through­
put
fluorescent
TaqMan
analysis.
If
manual
methods
are
Battelle
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compared,
400
samples
could
be
completed
in
one
day
by
dot
blot,
but
it
would
require
3
days
to
complete
the
same
number
by
PCR.

7.5
Biochemical
Measures
There
are
many
hormonal
actions
of
compounds
that
are
categorized
as
EDCs.
Endocrine
disruption
includes
estrogenic,
antiestrogenic,
androgenic,
and
antiandrogenic
effects,
growth
factor
modulation,
cytokine
modulation,
modulation
of
hormone
metabolism,
and
many
other
activities
(
Jimenez
1997).
The
primary
function
of
estrogenic
substances
is
control
of
ovulation,
and
hence,
reproduction.
Secondary
functions
of
estrogenic
substances
include
gender
determination,
development
of
secondary
sex
characteristics,
regulation
of
mating
and
breeding
behaviors,
and
regulation
of
calcium
and
water
homeostasis.
The
major
differences
between
the
effects
of
estrogen
in
mammals
and
egg­
layers
are
the
production
of
the
egg
yolk
protein
VTG
in
the
liver,
and
eggshell
formation
in
oviparous
species.
In
birds,
the
primary
sites
of
estrogen
production
are
the
gonads,
but
the
brain
also
contains
significant
aromatase
activity,
suggesting
that
the
brain
is
another
major
source
of
estrogen.

Estrogen
production
is
regulated
through
a
negative
feedback
loop
by
the
pituitary
peptide
hormones,
GnRH,
leutinizing
hormone
(
LH),
and
FSH,
a
system
that
has
been
studied
in
the
Japanese
quail
primarily
within
the
context
of
reproductive
aging
(
reviewed
by
Ottinger
et
al.
1997).
From
the
current
understanding
of
reproductive
biology,
almost
any
hormone
involved
in
the
reproductive
cycle
is
a
potential
biochemical
marker
for
endocrine
disruption.
The
transport
of
steroid
hormones
through
the
body,
however,
depends
on
the
presence
of
various
serum
transporter
proteins,
which
may
differ
across
vertebrate
classes.
Steroid
hormones
readily
pass
through
cell
membranes
and
interact
with
receptors
either
in
the
cytosol
or
in
the
nucleus.
Steroid
hormones
are
metabolized
primarily
by
the
liver,
where
enzymes
make
them
biologically
inactive
and
water­
soluble.
The
water­
soluble
conjugates
are
released
into
the
blood
and
excreted
in
the
urine
or
bile.
Consequently,
measures
of
liver
enzyme
activity
or
steroid
byproducts
in
excrement
are
also
potential
biochemical
measures
of
endocrine
disruption.
GnRh
is
currently
being
investigated
as
a
potential
endpoint
for
detecting
endocrine
disruption
in
avian
reproduction
toxicity
tests
by
Mary
Ann
Ottinger
and
colleagues.
To
date,
GnRH
system
appears
to
be
robust,
and
therefore,
the
GnRH
endpoint
does
not
appear
to
be
a
sensitive
indicator
of
endocrine
effects.

Endocrine
disruption
is
only
one
mechanism
by
which
a
chemical
can
interfere
with
reproduction
and
development.
Thus,
there
is
continued
debate
about
whether
endocrine
disruption
should
be
narrowly
defined
to
pertain
only
to
subcellular/
cellular
effects
associated
with
the
nuclear
hormone
receptor
signaling
pathway,
or
to
be
more
inclusive
of
all
aspects
of
production,
release,
transport,
metabolism,
binding
action,
and
elimination.
Others
argue
that
endocrine
activity
of
xenobiotic
compounds
is
meaningful
only
if
changes
occur
to
the
whole
organism,
and
therefore
testing
should
include
methods
for
measuring
effects
at
all
levels
of
biological
organization.
In
practice,
the
test
methods
to
assess
biochemical
endpoints
vary
considerably,
and
include
production
of
gene
products,
cell
proliferation
assays,
tissue
responses,
VTG
induction,
hormone
assays,
egg
production
and
fertility
studies,
and
development
of
secondary
sex
characteristics
(
Jimenez
1997;
Fairbrother
2000).
Specific
test
methods
that
have
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April
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2003
been
performed
on
birds
include
subcellular
measures
of
receptor
binding,
gene
activation
and
protein;
DNA
interactions;
tissue
endpoints
of
hormone
concentration
or
enzymatic
activity;
and
whole­
organisms
studies
with
a
focus
on
blood
plasma
concentrations
of
analyte
(
Fairbrother
2000).

7.5.1
Vitellogenin
and
Other
Biomarkers
of
Hepatic
Metabolic
Changes
All
oviparous
species
produce
VTG,
a
protein
absent
in
mammals.
Both
male
and
female
livers
may
produce
VTG,
given
sufficient
estrogenic
stimulation.
VTG
production
is
stimulated
primarily
by
17$­
estradiol;
estrone
is
only
5%
to
10%
as
effective.
VTG
is
released
from
the
liver
into
the
bloodstream,
where
it
binds
with
free
calcium
ions,
resulting
in
elevated
total
plasma
calcium,
as
more
ions
are
released
from
bone
storage
sites.
Induction
of
vitellogenesis
in
Japanese
quail
leads
to
increases
in
the
plasma
concentrations
of
organically­
bound
phosphorus,
bound
calcium,
and
total
protein
(
Robinson
and
Gibbins
1984).
In
female
birds,
oocytes
take
up
the
VTG
and
hydrolyze
it
to
produce
the
yolk
proteins,
phosvitin,
and
lipovitellin.
The
synthesis
of
1,25­
hydroxycholecaciferal
in
the
kidney
is
also
affected
indirectly
by
estrogen,
where
increased
levels
of
1,25­
hydroxycholecaciferal
increase
absorption
of
calcium
through
the
gastrointestinal
tract.
Exposure
to
environmental
estrogens
such
as
OC
pesticides
and
phytoestrogens
(
Robinson
and
Gibbins
1984)
induces
this
egg­
forming
system
and
is
easily
assessed
in
birds
by
measuring
their
VTG
plasma
levels.
However,
VTG
is
present
in
the
plasma
of
female
birds
before
ovulation.
Therefore,
it
cannot
be
used
as
a
biochemical
marker
of
endocrine
disruption
in
reproductively
active
females,
because
the
natural
production
of
VTG
will
obscure
the
contribution
of
EDCs.

The
VTG
structure
varies
among
species
and
vertebrate
classes.
Wang
and
Williams
(
1980),
for
example,
identified
two
distinct
VTGs
from
white
leghorn
roosters
based
on
amino
acid,
phosphorus
composition,
peptide
maps,
immunological
reactivity,
and
relationship
to
the
yolk
lipovitellins.
The
authors
concluded
from
these
studies
that
the
two
proteins
are
distinct
gene
products
that
serve
as
precursors
to
different
lipovitellin
polypeptides.
Similar
findings
were
reported
for
the
Japanese
quail
(
Gibbins
and
Templeton
1982).
These
results
have
implications
for
the
development
and/
or
use
of
nucleic
acid
markers
of
endocrine
disruption,
such
as
messenger
ribonucleic
acid
(
mRNA),
in
that
a
mechanistic
understanding
of
VTG
production
will
be
required
to
select
the
most
appropriate
mRNA
target
for
study
and
detection.
Heppell
et
al.
(
1995)
attempted
to
develop
a
universal
VTG
assay
by
developing
mono­
and
polyclonal
antibodies
against
a
conserved
region
of
fish
VTG
sequences.
The
monoclonal
antibodies
did
react
with
chicken
plasma
as
measured
by
enzyme
linked
immunosorbent
(
ELISA)
and
Western
Blot
assays,
but
not
with
the
same
sensitivity
or
specificity
as
it
did
with
the
different
fish
species.
More
importantly,
however,
there
was
no
conclusive
evidence
that
the
monoclonal
antibodies
specifically
react
to
VTG
in
the
plasma,
especially
in
the
chicken,
and
no
data
were
shown
relative
to
the
reaction
of
the
so­
called
universal
polyclonal
antibody
with
bird
plasma.
Thus,
VTG
assays
continue
to
be
species­
specific
and
difficult
to
compare
across
species
or
vertebrate
classes.

Current
methods
for
detecting
VTG
levels
in
oviparous
animals
principally
rely
upon
tissue
endpoints
and
immunological
techniques
such
as
ELISA
and
radioimmunoassay
(
RIA).
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Induction
of
vitellogenesis
has
been
measured
indirectly
in
quail
by
determining
plasma
levels
of
protein­
bound
phosphorus,
total
calcium,
and
total
protein
by
conventional
methods
(
Robinson
and
Gibbins
1984).
Sodium
dodecyl
sulfate­
polyacrylamide
electrophoresis
(
SDS­
PAGE)
and
rocket
immunoelectrophoresis
have
also
been
described
for
quail
plasma
(
Robinson
and
Gibbins
1984).
VTG
was
successfully
detected
and
quantified
in
plasma
from
male
Japanese
quail
injected
with
estrogen
analogs
or
zearlenone
by
the
rocket
electrophoretic
technique,
but
the
method
was
unable
to
detect
VTG
in
male
birds
injected
with
the
weakly
estrogenic
compounds
o,
p1­
DDT,
chlordecone,
or
methoxycholor.
ELISA
and
RIA
systems
are
based
on
the
highly
specific
reversible
reaction
between
an
antigen
 
VTG,
in
this
case
 
and
a
specific
antibody.
Of
the
two
methods,
RIA
can
be
more
sensitive
because
it
does
not
require
dilution
of
samples
to
reduce
the
interferences
encountered
in
ELISA
techniques.
However,
ELISA
methods
do
not
require
the
use
of
unstable
reagents
or
radioisotopes
and
are
easier
to
set
up.
Both
ELISA
and
RIA
assays
are
readily
adapted
to
production
as
commercial
kits
or
to
routine
use
in
commercial
laboratories,
once
specific
antibodies
have
been
developed.
Cost
per
sample
for
VTG
analysis
in
fish
by
these
methods
range
between
$
15
to
$
25.

As
noted
above,
there
is
no
universal
VTG
antibody
available.
Until
very
recently,
the
necessary
specific
and
highly
reactive
antibody
for
quail
VTG
had
not
been
produced,
and
no
specific
adaptation
of
either
method
for
avian
VTG
is
described
in
the
current
literature.
However,
Masaru
Wada
(
College
of
Arts
and
Sciences,
Tokyo
Medial
and
Dental
University)
has
recently
developed
a
reportedly
highly
sensitive
quail
VTG
ELISA
assay
kit
in
collaboration
with
Transgenic,
Ltd.,
in
Kumamoto
Prefecture,
Japan.
The
assay
was
developed
using
a
rabbit
anti­
Japanese
quail
lipovitellin
antibody
and
is
being
evaluated
for
cross­
reactivity
with
other
species.
This
method
will
be
presented
at
the
European
Union
SETAC
2002
Invited
Symposium
on
Avian
Endocrine
Disruption.
Wada
has
also
developed
an
assay
for
circulating
very
lowdensity
lipoprotein,
another
estrogen­
sensitive
yolk
lipoprotein
precursor
in
quail.
Very
lowdensity
lipoprotein
is
separated
from
the
plasma
matrix
in
this
method
by
using
an
automated
lipoprotein
assay
system.
It
is
then
purified
using
high­
pressure
liquid
chromatography,
and
is
detected
enzymatically.
No
data
on
the
sensitivity
and
validation
procedures
for
these
assays
are
available
at
this
time.

Tissue
endpoints,
such
as
VTG
induction
or
enzyme
activity
measures,
continue
to
be
the
preferred
biochemical
markers
of
endocrine
disruption.
As
noted
above,
however,
there
is
a
lack
of
standard
protocols
and
most
assays
require
species­
specific
antibodies
due
to
structural
differences
in
the
VTG
peptides
in
different
vertebrate
classes.
The
VTG
test
alone
may
also
necessarily
miss
other
potential
estrogenic
effects,
and
the
relative
sensitivity
of
the
VTG
process
compared
with
other
estrogen­
induced
peptide
and
protein
responses
has
not
yet
been
established.
Until
such
time,
it
should
not
be
assumed
that
the
VTG
induction
test
would
be
a
sensitive
predictor
of
estrogenicity
of
xenobiotics
in
all
oviparous
species
(
Fairbrother
2000).
Major
limitations
with
in
vivo
assays
are
that
they
use
highly
complex
responses
that
can
be
modulated
through
mechanisms
that
do
not
directly
involve
steroid
receptors,
and
therefore
are
not
necessarily
selective
for
substances
that
act
through
these
receptors.
Nonetheless,
in
vivo
studies
are
essential
for
examining
endocrine
activity,
because
they
account
for
pharmacodynamic
and
pharmocokinetic
interactions.
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No
in
vitro
assay
for
avian
VTG
has
been
developed,
as
exists
for
fish.
However,
avian
hepatocytes
have
been
cultured
for
use
in
chemical
toxicity
assays
(
Hugla
et
al.
1996;
Kennedy
et
al.
1996)
indicating
that
there
is
potential
for
the
development
of
an
in
vitro
screening
assay
for
VTG
in
birds.

Not
only
is
the
liver
the
major
site
of
VTG
synthesis
in
birds,
but
it
is
also
the
site
of
many
other
enzymatic
processes
that
directly
or
indirectly
contribute
to
reproductive
fitness.
For
example,
birds
that
consume
petroleum­
contaminated
foods
develop
an
increased
ability
to
metabolize
the
circulating
contaminants
through
the
action
of
a
substrate­
inducible
mixed
function
oxidase
system.
Although
the
primary
function
of
the
system
is
to
rid
the
organism
of
the
contaminants,
it
may
also
accelerate
the
turnover
of
some
endogenous
substrates,
such
as
steroid
hormones.
Indeed,
studies
conducted
with
halogenated
aromatic
hydrocarbons
(
HAHs)
have
shown
a
link
between
HAH­
exposure,
liver
enzyme
function
and
health
effects,
such
as
developmental
toxicity,
hepatotoxicity,
endocrine
disruption,
immunotoxicity,
and
death.
Although
the
exact
role
of
cytochrome
P450
in
HAH
toxicity
is
not
clear,
both
the
toxic
and
biochemical
responses,
such
as
cytochrome
P450­
induction,
appear
to
be
mediated
by
a
shared
mechanism
that
involves
changes
in
gene
expression
initiated
by
binding
of
HAH
to
the
aryl
hydrocarbon
receptor.
Cytochrome
P450­
induction
in
cultured
cells
therefore
serves
as
a
sensitive
marker
of
the
activation
of
aryl
hydrocarbon
receptor­
dependent
pathways,
and
measures
of
liver
enzyme
activity
could
be
useful
indicators
of
endocrine
disruption.

Liver
enzyme
activities
in
birds
have
been
studied
most
thoroughly
with
respect
to
HAH
and
polychlorinated
biphenyl
(
PCB)
exposures.
The
principal
biomarker
continues
to
be
cytochrome
P450
1A­
induction,
assessed
by
measuring
7­
ethoxyresorufin­
O­
deethylase
(
EROD)
activity
using
7­
ethoxy
resorufin
as
substrate
and
a
fluorescent
plate
reader.
Cytochrome
P450
1A
concentration
is
typically
quantified
via
immunological
techniques,
such
as
RIA,
ELISA,
or
Western
Blot.
Kennedy
et
al.
(
1996),
for
example,
determined
concentration­
dependent
effects
of
HAHs
in
primary
hepatocyte
cultures
prepared
from
the
embryos
of
four
breeds
of
chicken,
pheasants,
turkeys,
three
breeds
of
duck,
and
herring
gulls.
Results
from
this
study,
and
comparison
with
prior
in
vivo
or
in
ovo
studies,
suggest
that
it
could
be
possible
to
predict
the
sensitivity
of
a
species
to
in
ovo
lethality
by
HAHs
from
the
relative
sensitivity
of
hepatocyte
cultures
to
EROD­
induction.
A
practical
implication
of
this
research
is
that
hepatocyte
cultures
could
be
used
to
estimate
the
sensitivity
of
rare
or
endangered
wild
bird
species.

Brunstrom
and
Halldin
(
1998)
also
assessed
EROD­
induction
in
chicken
embryo
livers
to
examine
whether
there
are
similar
interspecific
differences
in
the
EROD­
inducing
potencies
of
aryl
hydrocarbon
receptor
agonists
compared
with
embryo
toxicities.
The
EROD
assay
was
also
extended
to
hen,
turkey,
domestic
duck,
Japanese
quail,
eider
duck,
and
common
tern
eggs.
The
authors
found
good
agreement
between
EROD­
inducing
potency
and
embryo
toxicity,
measured
as
lethality
or
malformations,
which
supports
the
notion
that
species
differences
in
ERODinduction
reflect
differences
in
an
aryl
hydrocarbon­
receptor­
mediated
enhancement
of
Cytochrome
P450
1A
levels.
The
authors
note,
however,
that
differences
in
chemical
injection
method,
carrier
solution,
and
embryo
age
are
known
to
affect
the
extent
of
EROD­
induction
or
activity.
Further,
the
relative
potencies
of
HAHs
or
PCBs
can
vary
considerably
among
different
Battelle
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April
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2003
avian
species;
the
chicken
appears
to
be
the
most
sensitive
of
the
birds.
Freeman
and
McNabb
(
1991)
pointed
out
that
many
enzyme
activity
assays
have
never
been
validated
to
specifically
measure
initial
velocity
conditions,
such
that
many
published
estimates
of
enzyme
activity
could
actually
underestimate
the
true
enzymatic
activity
of
the
cells
or
tissue.
Assay
standardization
and
validation
are
therefore
prerequisite
to
the
use
of
enzyme
activity
as
a
biomarker
for
endocrine
disruption.

Other
enzyme
activities
measured
in
hepatic
tissues
or
cell
cultures
include
naphthalene­
metabolizing
activity
in
mallard
ducks,
using
a
radio­
labeled
(
I125)
substrate
to
measure
specific
activity
as
nanomoles
naphthalene/
minute/
milligram
microsomal
protein
(
Gorsline
and
Holmes
1981).
A
5'­
deiodinase
activity
has
been
used
as
a
biomarker
in
the
Japanese
quail;
it
is
an
assay
that
is
based
on
initial
velocity
conditions
(
Freeman
and
McNabb
1991).
There,
initial
velocity
conditions
were
defined
as
those
where
the
enzyme
activity
increases
proportionally
with
increasing
enzyme
concentration
and
increases
linearly
and
proportionally
with
increasing
incubation
time.
In
this
study,
the
authors
measured
the
release
of
I125
from
rT3
in
liver
homogenate
and
several
other
developing
tissues
from
12
h
to
2
days
of
age.
Of
particular
import
to
other
enzyme
activity
measures
was
a
demonstration
that
different
sets
of
assay
conditions
could
be
selected
for
making
quantitative
measurements
under
initial
velocity
conditions,
and
that
a
validated
assay
can
then
be
used
to
compare
the
effects
of
different
endocrine
disruptor
treatments.

The
estrogen­
synthesizing
enzyme
aromatase,
a
member
of
the
cytochrome
P450
family,
has
been
colocalized
with
estrogen
receptors
in
the
brain
of
Japanese
quail
(
Dellovade
et
al.
1995)
by
immunocytochemical
techniques
and
has
been
assayed
in
other
contexts
(
Balthazart
et
al.
1996;
George
and
Wilson
1982;
Schlinger
and
Arnold
1992),
but
not
specifically
as
a
biochemical
measure
of
endocrine
disruption.
In
birds,
the
enzyme
plays
an
important
role
in
determining
sexual
characteristics
and
reproductive
behavior
(
Elbrechet
and
Smith
1992;
Schlinger
and
Arnold
1992).
Ottinger
and
coworkers
at
the
University
of
Maryland
are
currently
conducting
a
preliminary
application
of
this
technique
in
a
one­
generation
reproductive
test
using
northern
bobwhite
and
Japanese
quail
challenged
with
methoxychlor,
a
weak
estrogenic
compound.
If
it
proves
to
be
a
sensitive
indicator
of
endocrine
disruption
in
the
various
life
stages
of
the
birds,
modifications
to
the
histoimmunochemical
technique
to
convert
it
to
an
ELISA
would
be
useful
to
provide
a
more
routine
procedure
for
endpoint
detection.

Although
nucleic
acid
assays
are
typically
conducted
within
the
framework
of
deducing
metabolic
pathways
and
mechanisms,
measuring
mRNA­
or
DNA­
protein
interactions
can
also
be
used
to
assess
hepatic
metabolic
changes.
For
example,
Gordon
et
al.
(
1988)
studied
the
effects
of
estrogen
on
the
stability
of
mRNAs
that
code
for
the
yolk
precursor
proteins
apolipoprotein
and
VTG
II
in
white
leghorn
cockerels.
Total
RNA
was
isolated
from
liver
tissue
and
used
for
Northern
Dot
Blot
analyses.
Solution­
hybridization
assays
were
also
performed
to
measure
mRNA
decay
constants.
Gupta
and
Kanungo
(
1996)
used
total
RNA
isolated
from
the
liver
for
Northern
Blots
and
gel
mobility
shift
to
study
the
transcriptional
regulation
of
the
VTG
operon.
Edinger
et
al.
(
1997)
used
nucleic
acid
analysis
to
address
the
concept
of
hepatic
memory
in
chickens,
a
concept
that
stems
from
observations
that
after
the
initial
activation
of
the
Battelle
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89
April
23,
2003
egg
yolk
protein
genes
by
estrogen,
subsequent
responses
to
estrogen
occur
more
rapidly.
In
this
paper,
the
enzyme
deoxyribonuclease
(
DNAse)­
I
footprinting
was
performed
to
identify
DNA:
protein
complexes
in
the
estrogen
response
element.
Run­
on
transcription
assays
were
also
performed
to
determine
the
extent
of
or
delay
in
accumulation
of
apolipoprotein
mRNA
during
transcription.
Shimada
et
al.
(
1996)
used
Northern
Blot
and
slot
blots
to
study
the
induction
of
cytochrome
P450
mRNA.
Although
the
statistical
power
of
nucleic
acid
techniques
such
as
these
to
interrogate
metabolic
changes
in
mRNA
production
is
widely
known
in
the
literature,
they
have
yet
to
be
applied
as
endpoint
measures
of
endocrine
disruption.

7.5.2.
Plasma
and
Fecal/
Urate
Hormone
Concentrations;
Estrogen,
Testosterone,
GnRH,
T3/
T4,
TSH,
Corticosterone
The
hepatic
enzyme
measurements
described
above
require
test
animals
to
be
sacrificed,
a
practical
consideration
that
is
not
conducive
to
profiling
changes
in
endocrine
status
in
test
animals.
Thus,
assays
have
been
developed
to
measure
many
of
the
hormones
and
proteins
in
blood
plasma
and
urine.
With
the
advent
of
RIA
systems
in
the
1960s
and
1970s,
circulating
hormones
could
be
easily
and
accurately
quantified.
For
example,
in
a
study
with
quail,
the
minimum
detectable
dose
of
estrogen
was
3.8
pg
/
tube,
the
concentration
at
50%
binding
was
188
pg/
mL,
and
the
interassay
CV
was
7.6%
(
Soh
and
Koga
1994).
Androgen
assay
by
single
antibody
RIA
kit
has
been
validated
for
use
in
Japanese
quail
plasma
by
Ottinger
and
Mahlke
(
1984).
Intra­
assay
CV
was
5%.
Corticosterone
concentrations
were
measured
by
a
double
antibody
RIA
by
the
same
authors.
Originally
developed
to
measure
the
circulating
titers
of
sex
steroid
hormones
in
mammals,
RIA
assays
were
readily
adapted
for
use
in
birds
because
of
the
similarity
of
steroid
hormones
among
phyla
(
Wingfield
and
Farner
1975).
Serum
thyroid
hormones
have
also
been
quantified
in
Japanese
quail
treated
with
endocrine­
active
compounds,
such
as
DDT,
PCBs,
and
thiouracil,
by
RIA
using
commercial
mammalian
kits
(
e.
g.,
Grässle
and
Biessmann
1982).
RIA
methods
for
gonadotropins
are
sufficiently
accurate
to
detect
changes
of
20%
in
mean
blood
hormone
levels
with
group
sizes
of
20
or
more
(
Thorell
and
Larson
1978).
RIA
continues
to
be
the
main
technique
for
hormonal
analysis
in
plasma
and
other
sample
sources
(
Ottinger
et
al.
2001;
Jones
and
Satterlee
1996;
Watson
et
al.
1990;
Jones
et
al.
1992;
Adkins­
Regan
et
al.
1995;
Schlinger
and
Arnold
1992;
Wingfield
and
Farner
1975;
Nisbet
et
al.
1999;
Millam
et
al.
1998;
Abdelnabi
et
al.
2000;
Marai
et
al.
2000;
Bluhm
et
al.
1984)
due
in
part
to
the
ease
with
which
specific
antibodies
can
be
developed,
and
the
sensitivity
of
radioactive
assays.
However,
fluorescent
immunoassays
such
as
ELISA
are
becoming
more
prevalent,
thus
eliminating
the
hazards
of
radiolabeled
substrates
(
Tell
and
Lasley
1991;
Millam
et
al.
1998;
Alston­
Mills
et
al.
1989;
Otani
et
al.
1993).
Despite
the
number
of
studies
on
circulating
hormones
in
birds,
there
are
relatively
few
studies
concerning
the
relationship
between
circulating
hormone
levels
and
endocrine
disruption.
Gorsline
and
Holmes
(
1982)
investigated
the
mechanism
whereby
petroleum
contaminants
alter
adrenocortical
function
by
studying
the
distribution,
metabolic
clearance
rate,
and
estimated
in
vivo
secretory
rate
of
corticosterone
in
mallard
ducks.
Results
showed
that
lowering
of
the
plasma
corticosterone
concentration
in
petroleum
exposed
birds
is
associated
with
a
decline
in
corticosterone
secretory
rate.
Biessmann
(
1982)
studied
the
effects
and
mode
of
action
of
PCBs
on
gonads
and
sex
hormone
balance
in
juvenile
quail
during
sexual
maturation
by
measuring
plasma
concentrations
of
17$­
estradiol,
testosterone,
5­"­
dihydrotestosterone,
and
progesterone.
17$­
estradiol
and
Battelle
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April
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2003
calcium
were
unaffected
by
PCB.
Cavanaugh
and
Holmes
(
1982)
studied
plasma
estrogen
and
progesterone
concentrations
to
determine
whether
impaired
reproductive
measures
in
mallard
ducks
exposed
to
petroleum
are
due
to
an
endocrine
dysfunction
in
the
ovary.
None
of
their
present
evidence
suggested
that
circulating
petroleum
hydrocarbons
can
stimulate
estrogenic
change
by
binding
to
estrogen
receptors.
Some
hydrocarbons
seem
to
impair
reproductive
cyclicity
in
a
manner
suggesting
impairment
of
gonadal
steroid
hormone
synthesis.
The
authors
suggest
this
second
type
of
effect
was
probably
responsible
for
the
disturbances
observed
during
the
study.
A
subsequent
study
(
Cavanaugh
and
Holmes
1987)
suggested
a
high
probability
that
petroleum
products
accumulate
preferentially
in
steroidogenic
cells
in
the
developing
ovaries,
such
that
petroleum
compounds
can
interfere
directly
with
gonadotropin­
dependent
steroid
hormone
synthesis.

Unfortunately,
frightening
or
painful
stimuli
can
cause
instantaneous
changes
in
endocrine
systems,
particularly
the
hypothalamic­
pituitary­
adrenal
axis.
Disturbances
such
as
movement
of
the
cage
or
handling
of
the
animal
can
alter
some
circulating
steroid
concentrations.
Therefore,
blood
sampling
methods
that
subject
the
animal
to
minimal
stress
will
result
in
more
accurate
concentrations
of
hormone
concentrations
in
the
circulation.
Application
of
fecal/
urate
sampling
to
reproduction
toxicity
testing
in
birds
is
a
means
of
sampling
for
steroids
with
minimal
disturbance
to
the
birds.

Measures
of
the
steroid
or
steroid
metabolite
content
of
feces
have
been
used
in
conservation
biology
for
over
a
decade.
These
measures
have
been
shown
to
reliably
detect
hormone
status
and
adrenal
activity
in
a
wide
array
of
mammalian
species
(
Wasser
et
al.
2000)
and
avian
species
(
Tell
and
Lasley
1991;
Wasser
et
al.
1997).
It
is
currently
being
adapted
for
use
in
monitoring
endocrine
changes
in
birds
(
Brewer
et
al.
2002a,
2002b;
J.
Clark,
A.
Faribrother,
L.
Brewer
and
R.
Bennett,
personal
communication,
2002,
Effects
of
exogenous
estrogen
on
mate
selection
by
female
house
finches
[
Carpodacus
mexicanus],
submitted
paper).
The
method
involves
collecting
fecal
samples,
or
fecal/
urate
samples
in
the
case
of
birds,
extracting
the
steroid
or
metabolites,
and
analyzing
the
extract
by
RIA.
Extraction
methods,
originally
involving
labor­
intensive
sequences
of
extraction
and
chromatography
steps,
have
been
replaced
by
a
rapid
method
validated
by
Wasser
et
al.
(
1991).
Current
fecal
extraction
methods
are
based
on
modifications
by
Brown
et
al.
(
1994)
and
Wasser
et
al.
(
1994).
The
estrogen
and
testosterone
content
of
the
extracts
are
analyzed
by
double­
antibody
I125
RIA
using
commercially
available
kits.
Recovery
of
17$­
estradiol
from
spiked
fecal
samples
has
been
reported
at
99.2%
±
9.1%.
Assay
sensitivity
for
estrogen
was
1.25
pg/
tube,
and
the
interassay
CV
was
reported
to
be
6.7%.
In
the
testosterone
assays,
recovery
of
testosterone
was
also
99.2%
±
9.1%,
the
assay
sensitivity
was
0.1
ng/
mL
and
the
interassay
CV
was
11.1%
to
14.2%
(
Wasser
et
al.
1991).
Similar
recovery
and
CV
are
obtained
for
progesterone
analyzed
by
single­
antibody
I125
RIA
that
cross­
reacts
with
progesterone
metabolites
(
Brown
et
al.
1994;
Wasser
et
al.
1994).
The
fecal
steroid
values
are
comparable
to
serum
steroid
secretory
profiles
measured
in
a
number
of
wild
species
(
Wasser
et
al.
1991,
1995,
2000).
Development
of
an
assay
to
measure
glucocorticoid
stress
hormone
in
fecal
material
has
only
recently
been
developed.
Assays
for
these
hormones
were
difficult
to
develop
because
they
undergo
extensive
and
rapid
metabolism
prior
to
excretion
that
is
both
species­
specific
and
subject
to
further
transformation
by
gut
flora
(
Erikson
1971;
Palme
et
al.
1997;
Bahr
et
al.
2000).
Using
a
commercial
I125
corticosterone
RIA
assay
(
ICN
2
Field
preservation
techniques
have
been
developed.

Battelle
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91
April
23,
2003
Pharmaceuticals,
Costa
Mesa,
California),
Wasser
et
al.
(
2000)
directly
compared
serum
and
fecal
concentrations
of
corticosterone
in
the
spotted
owl
(
Strix
occidentalis
caurina)
and
showed
that
the
metabolites
measured
in
the
feces
accurately
reflected
the
adrenal
stress
response
induced
by
a
natural
stress
(
removal
to
a
new
location)
and
challenge
by
ACTH.
The
interassay
CV
was
5.5%
for
a
high
sample
(
27%
bound)
and
3.0%
for
a
low
sample
(
62%
bound).
Intra­
assay
CV
was
5.18%
for
30
pairs.

Because
fecal/
urate
measures
of
steroid
hormones
are
noninvasive,
they
can
provide
both
more
cost­
effective
methods
for
determining
hormone
status
in
test
birds
and
much­
needed
endpoints
that
can
be
correlated
to
field
investigations.
2
Sampling
blood
is
an
invasive
procedure
that
involves
capture
and
withdrawal
of
blood;
because
it
stresses
the
birds,
it
should
be
avoided
during
egg­
laying.
This
limits
sampling
for
steroid
hormones
to
prematuration
periods
and
the
termination
of
the
study.
Sampling
of
blood,
as
noted
above,
requires
a
large
expenditure
of
labor
and
can
be
difficult
to
accomplish
within
an
appropriate
period
of
a
circadian
cycle.
With
fecal
sampling,
samples
can
be
collected
over
the
entire
exposure
period
with
minimal
effort.
Typically,
validation
involves
direct
comparison
between
blood
and
fecal
levels,
and
the
hormones
of
interest
over
one
or
more
reproductive
cycles
for
reproductive
steroids,
and
ACTH
challenge
studies
for
glucocorticoids.
A
study
correlating
fecal/
urate
steroid
concentrations
with
house
finch
nesting
behavior
was
recently
completed,
and
a
second
study
comparing
excreted
steroid
concentrations
with
plasma
levels
during
the
reproductive
cycle
of
Japanese
quail
is
currently
in
progress
under
funding
from
the
American
Chemistry
Council
(
L.
Brewer,
personal
communication,
2002).
An
indirect
validation
for
life
cycle
studies
is
to
track
fecal
hormone
concentrations
over
time
in
the
species
of
concern,
comparing
the
monitored
values
with
expected
biologically
relevant
profiles.
An
evaluation
of
the
effect
on
assay
results
of
potential
interferences
such
as
phytoestrogens
that
may
be
present
in
diet
and
in
excrement
is
needed.
Cost
per
sample
from
noncommercial
laboratories
(
e.
g.,
university
laboratories)
in
the
U.
S.
range
from
$
10
to
$
15
up
to
about
$
25.
Fecal
steroid
analysis
was
extensively
researched
at
San
Diego
Zoo
as
a
noninvasive
means
of
sexing
monomorphic
avian
species,
but
as
of
yet
is
not
currently
commercially
available.

The
use
of
feces
for
noninvasive
monitoring
of
thyroid
hormones
has
not
been
reported;
however,
there
is
a
high
probability
that
similar
assays
can
be
adapted
to
these
hormones
because
they
are
primarily
excreted
through
the
bile.
There
is
growing
interest
among
conservation
biologist
to
develop
a
thyroid
assay
from
feces
owing
to
the
importance
of
thyroid
hormones
as
an
index
of
starvation
(
S.
Wasser,
personal
communication,
2002).

Regardless
of
the
assay
method
or
sample
matrix,
thyroid
measures
should
be
viewed
with
caution.
Plasma
T3
levels
are
of
doubtful
value
for
diagnosing
hypothyroidism
because
of
a
common
syndrome
that
evolves
in
animals
experiencing
various
nonthyroidal
illnesses,
stress,
or
starvation.
This
syndrome,
called
Sick
Euthyroid
Syndrome,
or
Low­
T3
Syndrome,
could
develop
to
protect
the
body
from
catabolic
processes
that
accompany
these
conditions
(
Davison
et
al.
1985).
Although
T4
would
then
appear
to
be
the
better
choice
for
evaluating
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hypothyroidism
in
birds,
even
plasma
T4
concentrations
can
be
influenced
by
handling,
bleeding
(
Williamson
and
Davison
1985a),
food
intake
(
Williamson
and
Davison
1985b),
and
increased
corticosterone
concentration
(
Davison
et
al.
1985).
Because
of
the
short
half­
life
of
T3
and
T4
in
avian
blood,
plasma
fluctuations
of
T3
and
T4
render
it
difficult
to
document
hypo­
or
hyperthyroidism
from
a
single
sample
(
Lumeij
1994;
Oglesbee
et
al.
1997),
such
as
would
be
taken
at
necropsy.
Here
the
ability
to
monitor
thyroid
hormones
over
a
period
of
time
via
sampling
the
feces
could
enhance
the
interpretive
power
of
thyroid
assays.

Whereas
these
prior
studies
demonstrate
that
circulating
hormones
and
peptides
are
readily
measured
in
birds,
the
obvious
lack
of
mechanistic
understanding
currently
precludes
their
effective
use
as
biochemical
markers
of
endocrine
disruption.
Current
methods
for
assessing
wildlife
health
effects
are
generally
targeted
at
detecting
effects
rather
than
mechanisms,
and
may
not
adequately
evaluate
effects
on
the
endocrine
system.
This
is
particularly
true
for
exposures
that
occur
during
critical
developmental
periods
(
Jimenez
1997).
In
this
regard,
previously
highlighted
methods
and
approaches
for
assessing
endocrine
disruption
must
be
considered
within
the
mechanistic
black
box
that
defines
risk
assessment.
For
estrogen
or
an
estrogenic
substance
to
exert
its
effects,
for
example,
it
must
bind
to
an
estrogen
receptor
with
sufficient
affinity
and
specificity
to
elicit
the
downstream
production
of
species­
appropriate
proteins.
Thus,
similarities
among
species
hormones
or
estrogen
receptor
structures
could
be
important
in
determining
whether
or
not
a
chemical
elicits
an
estrogenic
or
antiestrogenic
response.
Further,
receptor
binding
assays
are
limited
because
they
only
measure
the
ability
of
a
chemical
to
bind
to
the
estrogen
receptor
and
do
not
include
any
measure
of
its
ability
to
pass
through
the
cell
membranes
and
contact
the
nuclear
receptors,
nor
do
they
reveal
whether
the
receptor
binding
initiates
mRNA
transcription.
Receptor
binding
assays
also
suffer
from
limitations
imposed
by
the
artificial
situation
whereby
substances
are
tested
for
their
ability
to
compete
with
small
amounts
of
radiolabeled
tracer
estrogen
for
binding
sites.
This
is
different
from
the
in
vivo
situation,
where
substances
compete
with
greater
amounts
of
natural
hormones
that
generally
have
a
much
higher
affinity
for
the
estrogen
receptor
(
Fairbrother
2000).

8.0
RESPONSE
TO
ESTROGEN
AGONISTS
AND
ANTAGONISTS
The
potential
for
environmental
chemicals
to
mimic
the
effects
of
estrogen
in
birds
was
first
reviewed
by
Rattner
et
al.
(
1984).
The
potential
for
such
interactions
to
result
in
feminization
of
males
(
both
in
wildlife
and
in
humans)
focused
attention
on
the
endocrine
disrupting
mode
of
action
of
reproductive
toxicants
(
Colborn
et
al.
1996.).
As
a
result,
a
large
amount
of
work
has
been
done
in
the
past
decade
to
develop
sensitive
assays
for
detection
of
estrogenic
effects
of
xenobiotics,
mostly
focusing
on
mammalian
systems,
but
more
recently
assessing
applications
to
Battelle
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April
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2003
fish
and
birds
(
Ankley
et
al.
1998;
DiGuilio
and
Tillett
1999),
and
invertebrates
(
DeFur
1999).
Initial
concerns
focused
on
estrogen
mimics,
primarily
due
to
binding
to
the
estrogen
receptor
and
stimulating
down­
stream
responses;
however,
scant
attention
has
been
paid
to
detection
of
antiestrogenic
effects
of
chemicals,
such
as
receptor
blockers.

8.1
Sexually
Mature
Life
Stages
All
vertebrate
classes
share
some
similarities
of
response
to
estrogenic
stimulation,
whereas
certain
aspects
of
avian
physiology
respond
differently
(
Fairbrother
2000).
Common
responses
are
development
and
expression
of
secondary
sex
characteristics,
reproductive
behaviors,
control
of
follicular
growth
and
maturation,
and
calcium
regulation.
Adult
oviparous
species,
such
as
birds,
herpetofauna,
and
many
fishes,
also
require
estrogen
for
VTG
synthesis,
oviduct
development
and
maturation,
and
shell
gland
function.
Estrogen
may
regulate
seasonal
reproductive
cycles
through
stimulation
of
the
pineal
gland
to
produce
melatonin.

8.1.1
Sensitivity
to
17$­
estradiol
or
Synthetic
Estrogen
Exposure
Estrogen
increases
production
of
VTG
by
increasing
the
number
of
copies
of
VTG
mRNA
in
hepatocytes.
VTG
production
can
be
under
the
control
of
one
gene,
for
example
in
the
chicken,
or
of
more
than
one
gene,
as
in
quail.
Alternatively,
different
forms
of
vitellogenic
peptides
may
result
from
posttranslational
processes.
Differential
effects
of
estrogen
on
species
with
the
various
production
modes
have
not
yet
been
determined.
VTG
production
is
stimulated
primarily
by
17$­
estradiol
(
see
Section
7.5.1);
potency
of
synthetic
estrogens,
such
as
diethylstilbesterol,
on
VTG
production
varies,
but
generally
is
less
than
that
of
estradiol.
Estrogen
also
plays
a
role
in
passerine
male
singing
behavior
and
on
copulatory
behaviors
of
both
sexes
(
reviewed
by
Fairbrother
2000).
Distinct
estrogen­
receptive
neurons
have
been
located
in
the
brain,
using
[
H3]
17$­
estradiol.

In
general,
neuroanatomimical
distribution
in
the
brain
of
the
steroid­
receptive
cells
is
similar
across
species.
Enstrom
et
al.
(
1997)
conducted
a
study
of
mate
choice
among
dark­
eyed
juncos
(
Junco
hyemalis),
in
which
estradiol
levels
were
manipulated.
Treatment
with
estradiol
enhanced
female
sexual
behavior
and
promoted
precopulatory
displays,
although
estradioltreated
females
had
significantly
smaller
ovaries
than
control
females.

Turner
and
Eliel
(
1978)
studied
the
ability
of
DDT
and
its
metabolites
(
o,
p'­
DDT,
p,
p'­
DDT,
o,
p'­
DDE,
and
o,
p'­
DDD)
to
compete
with
H3­
estradiol
for
binding
to
the
estrogen
receptor
on
Japanese
quail
oviduct
cells.
Only
o,
p'­
DDT
competed
significantly
for
binding
sites,
but
a
large
molar
excess
(
20,000
X)
was
required.
This
suggests
that
o,
p'­
DDT
has
a
very
weak
affinity
for
the
estrogen
receptor
and
likely
will
not
elicit
a
true
hormone­
specific
response
in
these
cells.

Another
chlorinated
insecticide,
chlordecone,
also
known
as
kepone,
has
been
shown
to
have
estrogenic
activity
at
doses
lower
than
that
which
causes
systemic
toxicity
(
McFarland
and
Lacy
1969).
The
greatest
effect
occurs
when
the
pituitary
is
intact,
because
chlordecone
stimulates
the
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April
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hypothalamic­
pituitary
axis
to
release
FSH.
It
also
is
possible
that
the
pituitary
trophic
hormones
increased
the
responsiveness
of
the
oviduct
to
direct
action
by
the
chemical.
Chlordecone
also
may
block
the
release
of
LH,
and
prolong
the
release
of
FSH
and
ovarian
estrogen
secretion.
It
binds
to
nuclear
estrogen
receptors
in
the
magnum
and
shell
gland
regions
of
the
oviduct,
and
mimics
estrogen
effects
in
these
locations,
although
its
potency
is
only
about
1/
1000
of
that
of
17$­
estradiol
(
Rattner
et
al.
1984).
In
males,
depressed
cloacal
gland
activity
following
chlordecone
exposure
could
be
due
to
inadequate
LH
release
and
associated
depressed
testosterone
secretion.

8.1.2
Antiestrogens
There
are
no
reports
in
the
literature
of
effects
of
avian
exposures
to
antiestrogenic
chemicals.
Pharmaceuticals
such
as
raloxofene,
tamoxifen,
and
danazol
have
been
developed
for
use
in
women.
Raoloxofene
and
tomoxofen
are
selective
antagonists
blocking
the
estrogen
receptors
on
the
uterus
and
breast
tissue,
but
are
agonists
in
other
tissues,
such
as
those
in
bone.
Danazol
also
binds
to
and
blocks
numerous
steroid
receptors,
including
those
for
estrogen
and
testosterone,
but
its
primary
use
is
through
the
inhibition
of
production
of
FSH
and
LH
by
the
pituitary.
Tamoxifen
has
been
used
as
an
antagonist
in
estrogen
research
in
poultry
and
exotic
bird
medicine
(
Lupu
2000),
in
which
it
is
effective
in
controlling
egg­
laying.
Recent
investigations
in
sex­
specific
neural
development
have
shown
that
tamoxifen
is
effective
in
inhibiting
song
circuit
development
in
the
male
zebra
finch
(
Holloway
and
Clayton
2001).

8.1.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
Study
of
the
potential
for
excess
estrogen
to
affect
behavior
and
reproduction
of
adult
birds
has
been
conducted
primarily
though
the
administration
of
the
endogenous
hormone
17$­
estradiol.
Although
this
regime
definitely
mimics
the
natural
action
of
estrogen,
it
has
been
criticized
by
some
because
its
efficacy
could
be
affected
by
endogenous
negative
feedback
cycles.
Further,
it
does
not
provide
a
means
of
determining
the
consequences
of
effects
on
synthetic
or
degradative
or
metabolic
pathways,
such
as
cytochrome
P450
systems,
for
estrogen.
Use
of
synthetic
estrogens
such
as
ethinyletradiol
(
EE2),
which
is
a
widely
used
contraceptive,
or
DES
also
provides
a
reasonable
model
in
the
avian
system,
but
is
susceptible
to
the
same
shortcomings
as
use
of
17$­
estradiol.
There
is
a
lack
of
suitable
chemicals
to
serve
as
known
antiestrogenic
controls
for
bird
studies.
However,
measurement
of
hormonally­
driven
behaviors
such
as
singing,
copulatory
receptivity,
and
nest
building,
and
concentrations
of
circulating
or
excreted
hormones
such
as
estradiol
and
estrone
will
provided
the
necessary
information
for
determination
of
general
mode
of
action
of
reproductively
active
chemicals.

8.2
Juvenile
Life
Stages
8.2.1
Sensitivity
to
17$­
estradiol
or
Synthetic
Estrogen
Exposure
Sexual
dimorphism
in
terms
of
behavior
and
neuroendocrinology
has
been
well
studied
in
the
Japanese
quail.
Female
quail
are
demasculinized
by
endogenous
estrogens
(
Hutchison
1978),
Battelle
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April
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2003
and
embryonic
treatment
with
estrogen
during
the
critical
period
of
brain
development
at
Days
4­
12
of
embryogenesis
results
in
an
irreversibly
depressed
response
of
copulatory
behaviors
in
the
adult
male
to
the
activating
effects
of
testosterone
(
Halldin
et
al.
1999).
Likewise,
estrogen
suppresses
development
of
the
singing
center
in
brains
of
embryonic
passerines.

Berg
et
al.
(
1999)
clearly
showed
that
the
synthetic
estrogens
EE2
and
DES
exhibit
significant
estrogenic
activity
during
embryonic
maturation
of
Japanese
quail,
resulting
in
feminization
of
male
embryos
and
malformation
of
the
Müellerian
ducts
in
females.
EE2
was
3
to
10
times
more
potent
than
DES,
as
indicated
by
a
dose­
dependent
increase
in
the
frequency
of
male
embryos
exhibiting
an
ovotestis.
Halldin
et
al.
(
1999)
then
studied
the
effects
of
embryonic
exposure
to
these
same
synthetic
estrogens
on
sexual
behavior
in
adult
Japanese
quail.
Sexual
behaviors
were
significantly
depressed
following
in
ovo
treatment
by
either
estrogen.
Testis
weight
asymmetry
was
significantly
increased
by
EE2
but
not
by
DES.
The
cloacal
gland
area
was
significantly
reduced
by
treatment
with
DES.
However,
plasma
testosterone
concentrations
did
not
differ
significantly
from
the
control
values
and
were
not
correlated
with
frequency
of
sexual
behaviors.
This
suggests
that
the
in
ovo
exposure
resulted
in
altered
sensitivity
of
the
brain
to
the
activating
effects
of
testosterone,
rather
than
affecting
the
ability
of
the
testes
to
produce
the
hormone.
Taken
together,
these
two
studies
indicate
that
EE2
is
more
potent
than
DES
during
embryonic
exposures.

Schumaker
et
al.
(
1989)
identified
Day
9
of
incubation
to
be
the
time
when
embryonic
development
of
Japanese
quail
is
most
sensitive
to
estrogenic
effects.
Estradiol
benzoate
(
EB)
treatment
demasculinized
sexual
behavior
and
cloacal
gland
growth
of
males.
However,
some
dependent
variables,
such
as
plasma
levels
of
luteinizing
hormone
and
crowing,
were
still
affected
by
EB
treatment
on
Day
14.
A
5
:
g
dose
of
EB
totally
suppressed
the
capacity
of
the
adult
male
to
show
copulatory
behavior
in
response
to
testosterone,
if
it
was
present
in
the
egg
on
Day
9
of
incubation.

Treatment
of
embryonic
Japanese
quail
and
chickens
by
egg
injection
on
Day
3
of
incubation
with
the
plastic
monomere,
bisphenol
A,
resulted
in
malformed
Müllerian
ducts
at
2
days
prior
to
hatch
in
female
quail
and
ovotestes
feminization
in
male
chickens
(
Berg
et
al.
2001).
Effective
doses
of
bisphenol
A
were
embryolethal
in
chickens,
but
not
in
quail.
DES
used
as
a
positive
control
showed
the
same
species­
specific
effects
on
Müllerian
duct
and
ovotestes
formation.
DES
appears
to
be
five
times
more
potent
than
bisphenol
A
in
the
quail,
but
only
three
times
more
potent
in
the
chicken.
Thus,
target
organs,
gender
effects,
and
sensitivity
to
xenobiotics
appear
to
be
species­
specific.
The
flame­
retardant,
tetrabromobisphenol
A,
had
no
sublethal
effects
on
the
developing
embryo
of
either
species.
Both
of
these
compounds
are
readily
excreted
in
the
bile,
and
the
authors
acknowledged
that
it
is
possible
that
female
birds
do
not
deposit
sufficiently
high
levels
in
the
egg
to
result
in
the
same
effects
observed
in
the
laboratory
egg
injection
studies.

Sexual
differentiation
is
not
a
unitary
process
in
quail:
various
responses
differentiate
at
embryonic
ages
between
Days
9
and
12,
and
require
different
levels
of
estrogen.
The
process
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also
is
progressive,
and
some
aspects
of
behavior
and
morphology
are
not
complete
until
the
posthatching
period.
Yoshimura
et
al.
(
2000)
documented
changes
in
gonadal
growth
and
histology,
age
of
sexual
maturation,
and
ovarian
weights
of
Japanese
quail
exposed
to
DES
during
the
posthatch
period
,
Day
0
to
Day
53.
Age
of
sexual
maturation
was
delayed
in
males
as
a
result
of
slower
testicular
development.
Semen
production
and
copulation
activity
appeared
to
be
unaffected,
as
measured
by
egg
fertility
rates.
In
females,
ovarian
development
also
was
delayed
during
early
posthatch
lifestages,
although
the
age
of
sexual
maturation
was
no
different
from
controls.
A
greater
number
of
ovarian
interstitial
cells
were
found
in
DES­
treated
birds
than
in
controls.
The
authors
suggest
that
these
effects
may
be
due
to
binding
of
DES
to
the
estrogen
receptor,
stimulating
downstream
activities.

Feminization
of
male
gull
embryos
occurred
following
injections
of
DDT
at
concentrations
similar
to
those
found
in
gull
eggs
from
southern
California
(
cited
in
Halldin
et
al.
1999),
suggesting
that
environmental
contaminants
may
have
similar
effects
on
embryonic
development
as
the
estrogens
reviewed
above.
However,
relative
potency
of
the
various
chemicals
is
not
known.

8.2.2
Antiestrogens
Very
little
is
known
about
antiestrogenic
properties
of
xenobiotics
administered
to
birds.
McMurry
and
Dickerson
(
2001)
and
others
showed
that
2,3,7,8­
tetrachlorodibenzo­
p­
dioxin
(
TCDD)
is
both
antiestrogenic
and
antiandrogenic
in
northern
bobwhite
quail.
TCDD
is
approximately
as
potent
as
EE2
and
decreases
eggshell
gland
weight
in
a
dose­
response
fashion
when
injected
into
eggs
prior
to
the
start
of
incubation.
Indole­
3­
carbinol
is
a
naturally
occurring,
phytoantiestrogen,
although
it
causes
minimal
effects
 
decreased
weight
gain
 
in
developing
bobwhite
embryos
(
McMurry
and
Dickerson
2001).
The
result
of
the
inhibition
of
aromatase
in
5­
day­
old
chicken
embryos
by
5­(
p­
cyanophenyl)­
5,6,7,8­
tetrahydroimidaxo
[
1,5­"]
pyridine
hydrochloride
was
that
all
hatchlings
developed
as
phenotypic
males
(
Elbrecht
and
Smith
1992).
Sex­
reversed
females
developed
bilateral
testes
that
were
capable
of
complete
spermatogenesis
and
had
the
physical
appearance
and
behavior
of
normal
males.
Similar
effects
occurred
after
exposure
to
ergosterol
biosynthesis­
inhibiting
fungicides
that
also
inhibit
aromatase
P450
enzymes
(
Dawson
2000).

8.2.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
In
birds,
the
developing
embryo
is
the
most
sensitive
life­
stage
to
the
activity
of
estrogens
or
their
antagonists.
Changes
that
occur
during
either
in
ovo
or
posthatch
exposure
will
persist
throughout
the
life
of
the
bird
and
influence
adult
reproduction,
even
if
exposure
occurs
only
in
the
early
life
stages.
Because
birds
develop
in
hard­
shelled
eggs,
they
are
most
likely
to
be
exposed
to
such
substances
throughout
embryogenesis,
including
the
most
sensitive
stage
of
development,
which
is
Day
9
in
the
quail.
Some
xenobiotics
 
OC
pesticides,
for
example
 
require
metabolic
degradation
into
secondary
metabolites
prior
to
exerting
estrogenic
effects.
It
is
not
clear
whether
such
degradation
can
occur
in
the
egg,
or
whether
the
secondary
products
must
be
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deposited
by
the
hen
during
egg
formation.
Regardless,
once
present
in
the
egg,
these
products
cannot
be
excreted
as
readily
as
they
are
in
mammals,
even
though
clearance
of
metabolic
wastes
by
the
bile
does
occur.
Therefore,
they
may
continue
to
pose
a
risk
throughout
the
incubation
period.
Hatchlings
can
continue
to
be
exposed
for
several
days
immediately
posthatch,
because
they
rely
on
the
resorption
of
the
yolk
sac
as
an
energy
source
during
that
time.
It
is
clear
that
sexual
development
continues
during
the
posthatch
period
and
can
be
significantly
influenced
by
increased
levels
of
estrogen
during
that
time.
Both
the
natural
and
synthetic
estrogens
are
reasonable
models
for
use
in
demonstrating
estrogenic
effects
during
these
life­
stages.
Further
studies
of
estrogenic
xenobiotics
that
require
metabolic
activation
will
determine
whether
or
not
this
occurs
in
the
embryo,
and
if
so,
its
relationship
to
the
sensitive
developmental
period.

9.0
RESPONSE
TO
ANDROGEN
AGONISTS
AND
ANTAGONISTS
Birds
appear
to
be
affected
by
androgenic
or
antiandrogenic
substances
to
a
much
lesser
extent
than
they
are
affected
by
estrogens
and
antiestrogens.
Because
males
are
the
homozygous
sex
in
birds,
sexual
dimorphism
results
from
a
demasculinization
of
female
embryos
under
the
influence
of
estrogen;
lack
of
estrogen
results
in
production
of
phenotypic
males
(
see
Section
3.2).
In
contrast,
excess
or
insufficient
testosterone
is
unlikely
to
affect
female
birds,
unless
levels
are
so
low
that
there
is
no
substrate
for
the
action
of
aromatase,
and
therefore
no
production
of
estrogens.
Adult
male
and
female
birds
have
equivalent
levels
of
circulating
plasma
testosterone.
It
has
been
shown
that
17$­
estradiol
administered
to
the
adult
male
quail
induces
receptivity,
but
females
treated
with
testosterone
fail
to
show
male
sexual
behaviors
(
reviewed
by
Balthazart
et
al.
1983).
Consequently,
less
research
has
been
focused
on
understanding
potential
effects
on
testosterone­
active
xenobiotics
in
birds
than
on
estrogen­
related
substances.

9.1
Sexually
Mature
Life
Stages
9.1.1
Sensitivity
to
Androgenic
Steroid
Exposure
Testosterone
exerts
its
influence
through
binding
to
cell
receptors,
entering
the
target
cell,
and
then
undergoing
conversion
into
a
number
of
metabolites.
These
metabolites
bind
to
nuclear
receptors
and
stimulate
DNA
to
produce
appropriate
physiological
responses.
For
example,
the
5"­
reduced
metabolites
(
5"­
dihydrotestosterone
[
5"
DHT]
and
5"­
androstane­
3",
17$­
estradiol
[
5",
3"­
diol])
activate
strutting,
crowing,
and
cloacal
gland
development.
The
production
of
the
5"­
reduced
metabolites
is
much
higher
in
male
cloacal
glands
than
in
females
(
Balthazart
et
al.
1983).
However,
in
the
brain
of
birds,
a
specific
enzyme,
5$­
reductase,
reduces
testosterone
to
5$­
dihyrotestosterone
(
5$­
DHT)
which
then
is
converted
to
5$­
androstane­
3",
17
­
estradiol(
5B,
3"­
diol).
These
$­
metabolites
are
inactive;
it
has
been
shown
that
injections
of
5$­
DHT
do
not
activate
sexual
behavior
or
development
of
secondary
sex
characteristics.
The
pituitary
gland
and
syringeal
muscles
used
in
singing
of
females
produced
more
of
the
5$­
reduced
metabolites
than
was
generated
in
these
locations
by
males
(
Balthazart
et
al.
1983).
Because
circulating
plasma
levels
of
testosterone
do
not
differ
significantly
between
males
and
females
in
photostimulated
Japanese
quail,
it
is
likely
that
sexual
dimorphism
is
a
result
of
the
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differences
in
metabolic
activation/
deactivation
reactions
rather
than
in
the
capacity
of
ovarian
or
testicular
tissues
to
produce
testosterone
(
Balthazart
et
al.
1983).

Adkins­
Regan
and
Ottinger
(
1988)
demonstrated
a
pronounced
diurnal
rhythm
in
rate
of
clearance
of
injection­
induced
elevations
in
plasma
testosterone
levels
in
Japanese
quail,
with
faster
clearance
rates
occurring
during
the
day
than
at
night.
In
addition,
many
birds
follow
a
cranial
cycle
as
well.
They
are
seasonal
breeders,
responding
to
increasing
length
of
photostimulation
by
enlargement
of
the
gonads
and
onset
of
copulatory
behaviors.
Some
birds
molt
just
prior
to
onset
of
reproductive
capacity,
and
most
begin
their
molt
shortly
after
completing
the
breeding
and/
or
brood­
rearing
phase
of
the
reproductive
cycle.
At
the
onset
of
short
day
lengths,
gonads
are
receptive
to
the
inhibitory
effects
of
reduced
amount
of
light.
Eventually,
the
gonads
become
insensitive
to
the
gonadosuppressive
effect
of
the
short
photoperiod,
and
reproductive
activity
increases.
The
onset
and
length
of
the
period
of
sensitivity
of
the
gonads
to
photosuppression
varies
by
species.
Treatment
of
day­
old
Japanese
quail
chicks
with
high
levels
of
testosterone
proprionate
resulted
in
the
development
of
testes
that
were
always
sensitive
to
the
effects
of
short
day
length,
and
therefore
never
were
stimulated
to
return
to
breeding
condition.
The
same
testosterone
treatment
in
female
chicks
resulted
in
suppressed
ovarian
development.

Stoehr
and
Hill
(
2001)
addressed
the
question
of
interference
of
hormones
on
molting
and
plumage
coloration.
They
found
that
male
birds
exposed
to
exogenous
testosterone
close
to
their
molt
were
likely
to
molt
into
plumage
that
was
duller
than
that
which
they
previously
displayed.
Males
that
had
been
treated
with
testosterone
earlier
in
the
year
did
not
show
as
marked
an
effect.

9.1.2
Antiandrogens
Inhibitors
of
the
enzyme
that
reduces
testosterone
to
its
active
5"­
metabolites
have
been
used
as
testosterone
antagonists
for
the
treatment
of
prostate
cancer.
However,
it
is
not
clear
whether
inhibitors
of
5$­
reductase,
such
as
finasteride,
will
have
any
physiological
effect
on
birds.
The
level
of
circulating
testosterone
will
increase,
at
least
transiently,
which
may
have
an
effect
on
the
amount
of
testosterone
that
is
changed
to
the
5"­
metabolites.
In
humans,
finasteride
is
marketed
under
the
name
Proscar
by
Merck
&
Co.,
Inc.,
for
the
treatment
of
enlarged
prostates;
it
shrinks
the
size
of
the
organ.
Treatment
with
the
5$­
reduced
metabolites
does
not
result
in
any
antiandrogenic
effects.

Cyproterone
acetate
(
CyA)
is
an
antiandrogenic
substance
that
has
been
shown
to
suppress
nestsoliciting
behavior
and
ovarian
development
of
ring
doves
(
Columba
palumbus),
copulatory
behavior
and
external
morphology
of
Japanese
quail,
and
courtship
behavior
of
zebra
finches
(
Taeniopygia
guttata)
(
Suresh
and
Chaturvedi
1987).
The
interaction
of
CyA
with
lengthening
photoperiod,
which
is
a
reproductive
stimulator,
in
the
red
headed
bunting
(
Emberiza
bruniceps)
was
studied
by
Suresh
and
Chaturvedi
(
1987).
Under
normal
day
lengths,
CyA
inhibited
testicular
growth
and
reduced
body
weight.
It
partially
arrested
the
activity
of
the
developing
gonad
during
the
photostimulatory
period
of
lengthening
days,
although
these
effects
were
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completely
and
quickly
reversed
at
the
cessation
of
treatment.
Effects
of
CyA
were
partially
offset
by
simultaneous
treatment
with
testosterone
proprionate.

Another
pesticide,
DDT,
and
its
degradative
p,
p'­
metabolites,
are
weakly
antiandrogenic,
and
TCDD
is
both
antiandrogenic
and
antiestrogenic
in
northern
bobwhite
quail,
depending
upon
dose
and
tissue
(
McMurray
and
Dickerson
2001).
Neither
of
these
OC
compounds
influences
the
levels
of
circulating
hormone.
Their
mode
of
action
is
similar
to
that
of
vinclozolin,
acting
as
androgen
receptor
antagonists.

9.1.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
Administration
of
excess
testosterone
or
its
active
metabolites
appears
to
have
little
effect
in
the
adult
bird.
The
one
notable
exception
is
the
lack
of
response
of
birds
to
increasing
day
length
for
the
spring
reproductive
season,
for
example.
Nevertheless,
several
chemicals
have
been
investigated
to
determine
whether
a
xenobiotic
substance
will
introduce
such
a
response.
Antiandrogenic
effects
appear
to
be
somewhat
more
pronounced,
although
none
was
of
the
same
magnitude
as
those
induced
by
estrogenic
substances.
Most
of
the
notable
changes
in
birds
exposed
to
antiandrogenic
substances
occurred
in
reproductive
behaviors.
This
lack
of
responsiveness
in
the
adult
bird
to
testosterone
manipulations
is
not
surprising,
because
sufficient
hormonal
differentiation
occurs
in
ovo
and
during
the
first
two
posthatch
weeks
to
maintain
gender­
appropriate
behaviors
(
Hutchinson
1978).
Altered
male
behaviors
in
birds
result
from
the
lack
of
testosterone,
not
elevated
levels
of
androgens.

9.2
Juvenile
Life
Stages
9.2.1
Sensitivity
to
Androgenic
Steroid
Exposure
The
inactive
testosterone
metabolite,
5$­
dihydrotestosterone,
had
no
demasculinizing
effects
if
administered
on
Day
9
of
embryonic
development
(
Schumacher
et
al.
1989),
indicating
that
it
has
no
estrogenic
activity.
Similarly,
no
behavioral
effects
occurred
in
the
adult
bird
as
a
result
of
embryonic
exposure
to
5$­
DHT.
Thus,
this
hormone
appears
to
be
inactive
in
quail.
Furthermore,
it
is
not
capable
of
being
acted
upon
by
aromatase
to
form
estrogens.
Embryonic
quail
have
high
5$­
reductase
activity
in
the
hypothalamus,
suggesting
that
they
transform
testosterone
into
this
inactive
metabolite
so
it
cannot
be
aromatized
into
estrogen
during
embryonic
development.
Thus,
although
5$­
DHT
is
categorized
as
a
testosterone,
it
has
no
activity,
and
therefore
is
neither
an
agonist
nor
antagonist.
TCDD
increases
testosterone
hydroxylation
in
herons,
with
the
position
on
the
ring
that
is
hydroxylated
dependent
upon
age
and
sex
(
Dawson
2000).
However,
treatment
with
TCDD
sufficient
to
cause
P450
induction
had
no
effect
on
circulating
testosterone
or
estradiol
concentrations,
suggesting
that
changes
in
hydroxylase
activity
were
compensated
for
by
endogenous
feedback
mechanisms.

Plants
produce
androgenic
substances
as
well
as
phytoestrogens.
The
ketone
fraction
of
both
wheat
germ
oil
and
gibberellic
acid
(
a
plant
hormone)
is
androgenic,
as
evidenced
by
a
positive
response,
increased
weight,
in
the
chick
comb
bioassay
in
male
chickens
treated
by
injection
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April
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from
1
to
35
days
of
age
(
Gawienowski
et
al.
1997).
Similar
treatment
with
these
two
substances
had
no
effect
on
female
chickens,
suggesting
that
their
potency
is
relatively
weak.

Hutchison
(
1978)
showed
that
exposure
of
6­
to
12­
h­
old
Japanese
quail
hatchlings
to
elevated
testosterone
through
implants
of
testosterone
proprionate
had
no
effect
on
the
differentiation
of
sexual
behavior
in
intact
males
or
females.
In
gonadectomized
birds,
57%
of
the
testosterone­
treated
females
displayed
male
sexual
behaviors,
male
vocalizations,
and
cloacal
gland
development.
Gonadectomized
birds
that
had
initially
been
treated
with
estradiol
benzoate
were
later
treated
with
testosterone
proprionate,
and
vice
versa.
Evidence
suggests
that
a
critical
period
during
embryonic
development
occurs
when
estrogen
eliminates
the
development
of
male
behaviors,
which
normally
occurs
only
in
females.
However,
estrogen
must
continue
to
be
present
in
the
posthatch
period
for
such
behaviors
to
become
established
in
females
as
adults.
Estrogen
given
in
adulthood
to
males
castrated
at
hatching
induces
both
male
and
female
behaviors,
whereas
estrogen
given
embryonically
demasculinizes
males.

9.2.2
Antiandrogens
Juvenile
Japanese
quail
exposed
to
aflatoxins
prior
to
and
during
sexual
maturation
developed
testes
approximately
half
the
size
of
testes
from
untreated
birds.
They
had
delayed
testicular
development
and
reduced
production
of
testosterone
(
Doer
and
Ottinger
1980).
Vinclozolin
((
R.
S.)­
3­
(
3,5­
dichlorophenyl)­
5­
methyl­
5­
vinyl­
1,3­
oxazolidine­
2,4­
dione),
a
dicarboximide
fungicide
applied
on
crops
and
ornamental
plants,
has
known
antiandrogenic
properties
in
mammals.
Similar
studies
were
conducted
with
Japanese
quail
(
McGary
et
al.
2001).
Embryos
exposed
on
Day
4
of
incubation
metabolized
vinclozolin
to
its
metabolically
active
form,
which
significantly
altered
GnRH
levels
in
male
hatchlings,
but
not
in
females.
The
onset
of
male
reproductive
behaviors
seemed
to
be
delayed,
with
the
number
of
mounts
and
cloacal
contacts
significantly
lower
at
the
beginning
of
the
reproduction
period.
There
were
no
measurable
effects
on
GnRH
levels
in
adults,
plasma
steroid
levels
in
either
hatchlings
or
adults,
proctodeal
foam
gland
growth
during
maturation,
or
relative
testicular
weight
at
7
weeks
of
age.
The
authors
speculated
that
observed
effects
were
due
to
vinclozolin
acting
as
an
androgen
receptor
antagonist
in
the
hypophyseal
region
of
the
brain.

9.2.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
There
has
been
little
interest
among
researchers
to
study
the
potential
consequences
of
exposing
avian
embryos
or
hatchlings
to
elevated
testosterone
levels.
Hutchison
(
1978)
and
others
clearly
established
that
estrogen
is
the
active
hormone
during
embryogenesis,
with
testosterone
levels
remaining
similar
in
both
sexes.
However,
because
testosterone
is
responsible
for
inducing
development
of
singing
centers
in
the
brain,
male
copulatory
behaviors,
and
the
cloacal
and
foam
glands,
chemicals
that
might
inhibit
the
action
of
testosterone
should
be
of
more
concern.
Unfortunately,
there
are
few
studies
of
the
effects
of
antiandrogens
on
the
developing
embryo
or
hatchling.
McGary
et
al.
(
2001)
produced
the
most
definitive
study
conducted
to
date
(
see
Section
9.2.2).
Further
work
needs
to
be
done
to
investigate
whether
or
not
reduced
levels
of
the
5"­
metabolites
are
more
likely
to
cause
adverse
effects
through
blockage
of
the
reductase
Battelle
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April
23,
2003
enzymes
than
is
reduction
of
the
parent
compound.
However,
there
likely
will
continue
to
be
less
interest
in
understanding
the
potential
for
xenobiotics
to
influence
the
androgen
system
in
birds
than
there
is
for
the
estrogen­
dependent
responses,
due
to
the
requirement
of
estrogen
for
demasculinization
of
genotypic
females.
This
is
significantly
different
from
effects
in
mammals,
where
masculinization
of
the
female
fetus
is
of
much
greater
concern,
due
to
the
requirement
for
elevated
testosterone
for
genotypic
males
to
develop
normally;
females
will
be
masculinized
under
these
conditions
regardless
of
endogenous
estrogen
production.
Thus,
results
from
mammalian
studies
designed
to
determine
endocrine­
disrupting
effects
are
likely
not
directly
applicable
to
the
avian
model.

10.0
RESPONSE
TO
THYROID
AGONISTS
AND
ANTAGONISTS
Relatively
little
work
has
been
done
to
assess
the
effects
of
xenobiotics
on
thyroid
activity
in
birds;
only
the
potential
for
thyroid
inhibition
has
been
studied.
However,
hyperthyroidism
can
result
after
the
cessation
of
treatment
by
thyroid
inhibitors
(
Peebles
and
Marks
1991).
Thyroid
function
can
be
affected
as
a
result
of
interference
with
pituitary
production
of
TSH,
iodination
and
coupling
of
tyrosine
residues
to
form
T4,
or
conversion
of
T4
into
T3
through
enzymatic
activation
in
the
liver.
Metabolism
and
excretion
of
T4
and
T3
also
could
be
affected,
and
it
is
possible
that
chemicals
bind
to
T3
cellular
receptors,
resulting
in
either
agonistic
or
antagonistic
effects.

10.1
Sensitivity
to
Thyroid
Stimulation
Various
crude
oils
were
shown
to
affect
thyroid
function,
as
measured
by
reduced
body
weights,
thyroid
hypertrophy,
and
increased
plasma
thyroxine
levels
in
several
species
of
seabirds:
herring
gulls,
black
guillemots
(
Cepphus
grylle)
and
adult
Leach's
petrels
(
Oceanodroma
leucorhoa)
(
Peakall
et
al.
1981).
Several
other
hormones,
such
as
circulating
corticosterone,
and
corticotrophic
hormone,
also
were
elevated.
The
authors
noted
that
the
effects
of
thyroxine
on
regulating
basal
metabolism
of
birds
are
equivocal.
Growth
in
several
bird
species
following
thyroxine
administration
ranged
from
none
to
moderate,
with
some
studies
indicating
significant
increases
in
body
weight
at
low
administered
doses,
and
reduced
body
weight
at
high
levels.
Peakall
et
al.
(
1981)
suggested
that
the
observed
effect
of
oil
on
circulating
thyroid
hormones
most
likely
is
a
compensatory
response
to
changes
in
osmoregulation
induced
by
alterations
in
the
intestinal
mucosa
due
to
oil
ingestion,
which
affects
both
osmotic
balance
and
nutrient
uptake.
This
indicates
that
crude
oil
would
not
be
classified
as
an
EDC,
because
there
was
no
direct
effect
of
the
substance
on
the
thyroid
or
steroid
reproductive
hormone
systems.

There
were
no
other
reports
found
in
the
literature
documenting
effects
of
thyroid
stimulation
in
birds,
either
through
direct
exposure
to
thyroid
hormone
treatment
or
through
exogenous
thyroid
agonists.
Battelle
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April
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2003
10.2
Inhibition
of
Thyroid
Function
It
has
been
suggested
that
maternal
thyroid
activity
influences
functional
eggshell
properties
in
birds,
playing
a
significant
role
in
egg
hatchability
(
Peebles
and
Marks
1991).
Exposure
of
high
growth
rate
Japanese
quail
to
low
amounts
(
0.2%)
of
dietary
propylthiouracil,
a
known
thyroid
inhibitor,
had
no
effect
on
embryonic
growth,
but
resulted
in
significantly
reduced
body
water
due
to
changes
in
eggshell
permeability
(
Peebles
and
Marks
1991).
Although
molt
and
plumage
characteristics
are
thought
to
be
primarily
controlled
by
testosterone
(
Stoehr
and
Hill
2001),
thyroid
hormone
may
play
a
role
as
well.
Propylthiouracil
treatment
of
American
kestrels
(
Falco
sparverius)
resulted
in
changes
in
the
width
of
subterminal
bands
on
tail
feathers,
feather
reflectance,
and
duration
of
the
molt
(
Quinn
et
al.
2002).
However,
the
potential
for
xenobiotics
to
result
in
similar
responses
is
unclear.
PCBs
appear
to
have
a
bimodal
response
on
the
thyroid
system:
low
levels
are
stimulatory,
and
higher
concentrations
result
in
inhibition
(
Quinn
et
al.
2002).
Nevertheless,
when
kestrels
were
fed
Aroclor
1242
at
concentrations
that
significantly
decreased
circulating
thyroid
hormone
levels,
no
measurable
change
in
plumage
characteristics
resulted
(
Quinn
et
al.
2002).
High
dietary
concentrations
of
DDT,
toxaphene,
and
PCBs
all
resulted
in
enlarged
thyroid
glands
and
reduced
body
weights
of
adult
bobwhite
quail
after
4
months
of
continuous
exposure
(
Hurst
et
al.
1974).
Low
doses
of
PCBs
reduced
the
size
of
the
thyroid,
whereas
similar
doses
of
DDT
and
toxaphene
had
no
effect.
The
authors
suggested
that
DDT
and
toxaphene
could
decrease
the
effective
level
of
T3,
thus
enhancing
TSH
secretion,
causing
the
thyroid
to
grow
in
size.
DDT
could
affect
T3
through
changes
in
liver
metabolic
enzymes
or
through
direct
action
on
pituitary
secretion
of
TSH.
The
authors
provided
no
explanation
for
the
bimodal
response
to
PCB
exposure.
Grässle
and
Biessmann
(
1982)
also
studied
the
effects
of
DDT
and
PCBs
on
quail
thyroid
systems.
They
showed
that
Japanese
quail
exposed
to
either
the
OC,
DDT,
or
the
PCB,
Aroclor
1254,
for
up
to
120
days
had
significantly
decreased
T4
levels
and
altered
thyroid
histology
suggestive
of
glandular
inactivity,
but
little
to
no
change
in
circulating
T3
levels.
The
authors
suggested
that
PCBs
have
a
different
mode
of
action
than
does
propylthiouracil,
possibly
acting
on
the
hypothalamus­
pituitary­
thyroid
axis
rather
than
at
the
glandular
level.
Furthermore,
there
was
no
relationship
between
T4
or
T3
levels
and
eggshell
breaking­
strength
for
either
OC
or
propylthiouracil
exposed
birds,
supporting
the
conclusion
that
thyroid
hormone
activity
does
not
play
a
significant
role
in
reducing
eggshell
quality.

Dawson
(
2000)
reviewed
the
effects
of
various
OCs
on
growth
and
other
thyroid
functions
of
young
birds.
Such
effects
were
first
investigated
in
the
late
1960s
and
early
1970s
in
pigeons
exposed
to
DDT
and
dieldrin,
then
in
gulls
(
Larus
sp.)
and
pigeon
guillemots
(
Cepphus
columba)
exposed
to
PCBs.
Changes
in
mass
and
histology
of
the
thyroid
were
measured,
but
were
not
sufficiently
severe
to
result
in
gross
physiological
effects.
Mallards
(
Anas
platyrhynchos)
exposed
to
PCBs
for
5
weeks
in
a
repeated
dose
exposure
showed
a
slight
increase
in
T3
at
doses
1000
times
that
used
in
rodent
studies
(
Fowles
et
al.
1997).
Three­
week
old
herring
gulls
(
Larus
argentatus)
exposed
to
tetrachlorobenzene
showed
no
changes
in
T4,
but
significantly
decreased
levels
of
T3.
Conversely,
in
ovo
exposure
of
chickens
to
tetrachlorobenzene
showed
no
change
in
T3
and
an
increase
in
T4.
In
ovo
exposure
of
chickens,
pigeons,
and
great
blue
herons
(
Ardea
herodias)
to
TCDD
resulted
in
no
effects
on
T3
or
T4
levels,
even
when
EROD
and
P450
enzymes
were
substantially
induced.
Field
studies
of
Battelle
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103
April
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2003
thyroid­
related
effects
due
to
exposure
of
birds
to
PAHs
were
also
inconclusive,
because
eggs
were
contaminated
with
both
PCBs
and
polychlorinated
dibenzofurans.
Nevertheless,
no
significant
changes
in
circulating
levels
of
T3
or
T4
were
documented.

10.3
Relevance,
Sensitivity,
Use
History,
Uncertainty
There
has
been
relatively
little
interest
in
studying
the
potential
effects
of
xenobiotic
chemicals
on
the
avian
thyroid
system.
Early
studies
were
conducted
to
determine
whether
the
thyroid
played
a
significant
role
in
eggshell
quality,
but
as
it
became
clear
that
other
hormones
or
cellular
mediators
,
such
as
prostaglandins,
were
more
directly
related
to
eggshell
synthesis,
interest
waned.
Effects
of
thyroid
function
on
metabolic
rate,
such
as
weight
gain
in
embryos
or
juveniles,
was
studied
briefly
in
the
poultry
science
field,
but
again
did
not
appear
to
play
as
significant
a
role
as
did
other
dietary
parameters.
More
recently,
the
role
of
thyroid
hormone
in
plumage
development
was
investigated,
but
also
shown
to
be
equivocal.
It
may
be
that
the
thyroid
system
is
relatively
robust
and
not
particularly
sensitive
to
xenobiotic
effects.
Generally,
there
is
an
excess
of
circulating
T4,
and
approximately
30%
to
40%
of
normal
T4
levels
are
sufficient
to
produce
an
adequate
amount
of
T3.
Therefore,
unless
a
chemical
acts
directly
on
conversion
of
T4
to
T3,
or
binds
to
T3
receptors,
sufficiently
high
doses
would
be
required
that
systemic
toxicity
is
likely
to
result
prior
to
the
onset
of
signs
of
thyroid
dysfunction.
To
date,
only
propylthiouracil,
which
is
a
known
thyroid
inhibitor
that
is
used
pharmacologically
to
control
Graves
disease
in
humans,
is
known
to
inhibit
the
conversion
of
T4
to
T3.
Propylthiouracil,
along
with
several
other
pharmacological
agents,
also
inhibits
the
action
of
thyroid
peroxidase,
thereby
reducing
the
amount
of
T4
produced.

Measurement
of
thyroid­
specific
endpoints
is
relatively
simple,
because
T4
and
T3
hormone
structures
are
well
conserved
across
species,
and
therefore
circulating
levels
are
easily
measured
with
commercially
available
ELISA
kits.
Thyroid
weights
also
can
be
measured
at
necropsy,
and
body
weights
 
especially
weight
gain
in
juveniles
 
are
routinely
measured
in
all
avian
studies.
Therefore,
identification
of
thyroidogenic
modes
of
action
of
xenobiotics
can
readily
be
detected
during
the
avian
two­
generation
test.
It
is
likely,
however,
that
thyroid­
related
fitness
endpoints
will
be
much
less
sensitive
than
other
hormone­
related
effects
and
can
occur
at
exposure
concentrations
that
also
result
in
generalized
systemic
toxicity.

11.0
CANDIDATE
PROTOCOLS
Candidate
protocols
for
the
development
of
an
avian
two­
generation
toxicity
test
guidelines
include
established
life
cycle
("
one­
generation")
reproduction
tests
(
ASTM,
OECD,
and
EPA)
and
a
proposed
short­
term
life
cycle
test
using
proven
breeders
(
OECD
2000).
Two
principal
designs
under
consideration
for
an
avian
two­
generation
reproduction
test
are
a
proven
breeder
design
and
a
pre­
egg­
laying
exposure
design,
based
on
a
draft
guideline
proposed
by
OECD
(
1999)
and
suggested
modifications
of
the
EPA
OPPTS
850.2300
one­
generation
avian
Battelle
Draft
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April
23,
2003
reproduction
test
guideline
(
EPA
1996)
proposed
by
EDSTAC
(
EPA
1998)
for
the
development
of
a
two­
generation
guideline
with
endocrine
endpoints
from
which
the
OECD
(
1999)
proposed
guideline,
in
part,
evolved.
Each
of
these
existing
and
proposed
guidelines
is
reviewed
and
their
strengths
and
weaknesses
discussed
and
contrasted.
Table
11­
1
compares
these
guidelines
by
test
species,
husbandry,
test
procedures,
test
design,
endpoints,
and
reportable
results.

Detailed
reviews
of
the
one­
generation
avian
reproductive
test
guidelines
and
their
strengths
and
weakness
were
presented
by
Bennett
and
Ganio
(
1991)
and
OECD
(
1996).
The
advantages
and
disadvantages
of
design
and
endpoint
components
of
the
draft
OECD
guideline
for
a
two­
generation
avian
reproduction
test
were
reviewed
in
detail
by
Bennett
et
al.
(
2001).
One­
generation
reproduction
tests
are
conducted
with
bobwhite
and
mallard
ducks.
ASTM
also
permits
other
nonpasserine
species,
and
OECD
allows
the
use
of
the
Japanese
quail.
The
proposed
two­
generation
guidelines
limit
the
tests
to
the
two
quail
species.
Battelle
Draft
105
April
23,
2003
Table
11­
1.
Comparison
of
Avian
Reproductive
Toxicity
Tests
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

TEST
ANIMALS
Species
JQ,
BW
Bobwhite
(
BW(
b)),
mallard
duck
(
MD(
c)),
other
nonpasserines
Japanese
quail
(
JQ(
d)),
BW,

MD
BW,
MD
JQ
Age
Approaching
first
breeding
season;
Successful
fertilization
should
have
taken
place
before
start
of
pretreatment
BW:
$
6
months
old
at
onset
of
lay
Birds
of
similar
age;
age
in
first
season
must
be
±
10%

of
mean
age
of
group;
if
proven
breeders
used,

must
be
same
age
in
years
BW:
20­
24
weeks
±
1
week
JQ:
proven
breeders
±
½
week
MD:
9­
12
months
±
2
week
$
7
months
old;
birds
approaching
first
breeding
season;
same
age
±
1
month
Approaching
first
breeding
season
(
4
wk
old)

Criteria
for
use
Birds
should
appear
healthy
and
free
of
abnormalities
or
injury
that
could
affect
test
results
Birds
should
not
receive
medication
beginning
1
week
prior
to
start
of
acclimation
Successful
fertilization
demonstrated
prior
to
test
Parental
mortality
during
last
2
weeks
of
acclimation
should
not
exceed
3%

All
birds
should
be
from
same
hatch
Birds
from
one
source
and
strain.
Birds
in
poor
physical
condition,

deformed,
or
with
plumage
that
differs
from
that
of
wild
birds
must
not
be
used
Birds
from
the
same
population
of
well­
known
parentage
Free
of
disease
and
injury
Population
should
not
be
used
if
>
3%
of
either
sex
die
or
become
debilitated
during
acclimation
period
Birds
from
same
source
and
strain
with
known
breeding
histories,
lighting
regimes,
disease
record,

drug
or
medication
administered
Not
selected
for
resistance
to
toxic
substances
Not
used
in
a
previous
test
Phenotypically
indistinguishable
from
wild
stock
Birds
should
appear
healthy
and
free
of
abnormalities
or
injury
that
could
affect
test
results
Birds
should
not
receive
medication
beginning
1
week
prior
to
start
of
acclimation
Onset
of
egg
laying
should
have
taken
place
before
start
of
treatment
Parental
mortality
during
last
2
weeks
of
acclimation
should
not
exceed
3%

All
birds
should
be
from
same
hatch
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
106
April
23,
2003
One­
Generation
Test
Guidelines
HOUSING
Location
Adults
Young
indoors
in
pairs
(
1M:
1F)

To
minimize
injury,
etc.,
from
aggression,
males
and
females
may
be
housed
separately;
pairs
placed
together
for
$
1
H/
day
for
5
days/
week
to
maintain
fertility
In
groups
preferably
by
treatment
group
Indoors;
outdoors
for
some
species
In
pairs
(
1M:
1F)
or
in
groups
containing
no
more
than
1M
Not
specified
Indoors
preferred;
outdoors
permissible
In
pairs
(
1M:
1F)
or
in
groups:

BW/
JQ:
1M/
2F
MD:
1M:
3F
In
groups
by
pen
of
origin
Together
if
birds
individually
marked
indoors
BW:
in
pairs
(
1M:
1F)
or
in
groups
(
1M:
2F)

MD:
in
pairs
(
1M:
1F)
or
in
groups
(
1M:
3F)

In
groups
by
pen
of
origin
Together
if
birds
individually
marked
indoors
in
pairs
(
1M:
1F)
by
lineage,

males
from
one
line
paired
with
females
from
another
line
To
minimize
injury,
etc.,
from
aggression,
males
and
females
may
be
housed
separately;
and
house
together
long
enough
to
maintain
fertility
In
groups
preferably
by
treatment
group
F1
cohort
for
breeding
paired
(
1M:
F)
at
4
wk
old
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
107
April
23,
2003
Pens
Size
(
cm2/
bird)

Construction
BW:
625
JQ:
1
week
old:
50
2
week
old:
75
3­
4
week
old:
100
>
4
week
old:
625
Stainless
or
galvanized
steel
or
other
inert
materials.
Wire
pens
with
slanting
floors
and
egg­
catchers
recommended
for
adults.
Food
troughs
should
be
covered
with
wire
grid
to
minimize
food
spillage
Adequate
size
From
literature:

BW:
>
760
JQ:
>
200
MD:
>
5000
Stainless
steel,
galvanized
steel,
and
material
coated
with
perfluourocarbon
plastics
preferred.
Any
nontoxic
material
not
capable
of
excessive
sorption
of
test
substance,

not
dissolved
by
water
or
loosened
by
pecking
are
acceptable
BW:
1250
JQ:
750
MD:
5000
Not
specified
BW:
>
5000
MD:
>
10,000
Galvanized
or
stainless
steel
sheeting
for
common
walls
and
ceilings,
and
wire
mesh
for
floors
and
external
walls.
Material
coated
with
perfluorocarbon
plastics
also
acceptable
JQ
1
week
old:
50
2
week
old:
75
3­
4
week
old:
100
>
4
week
old:
625
Stainless
or
galvanized
steel
or
other
inert
materials.
Wire
pens
with
slanting
floors
and
egg­
catchers
recommended
for
adults.
Measures
should
be
taken
to
reduce
food
spillage
(
e.
g.,
cover
food
troughs
with
wire
grid)

ENVIRONMENT
Temperature
(
°
C)

Adult
Egg
Storage
Hatching
Brooding
16­
27
13­
16
37­
37.5
35­
38
first
week
30­
35
second
week
23­
27
third
week
21
12­
16
39
Upper
brooder
temperature
is
species­
specific;
provide
gradient
within
brooder
down
to
21
22
±
5
BW/
JQ:
15­
16
MD:
14­
16
37.5
BW/
JQ:
35­
38
first
week,

decrease
by
4­
5/
week
MD:
32­
35
first
week,

decrease
by
3­
4/
week
21
16
37.5
35
to
22
temperature
gradient
within
brooder
measured
at
2.5­
4
cm
above
floor
16­
27
13­
16
37­
37.5
35­
38
first
week
30­
35
second
week
23­
27
third
week
16­
27
fourth
week
on
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
108
April
23,
2003
Percentage
RH
(%)

Adult
Egg
storage
Incubation
Hatching
Young
40­
80
55­
75
50­
70
70­
75
40­
80
45­
70,
higher
for
MD
at
high
elevations
65
70
45­
70,
higher
for
MD
50­
75
BW:
50­
65
JQ:
50
 
70
MD:
60­
65
BW/
JQ:
70­
75
MD:
75­
85
BW/
JQ:
50­
75
MD:
60­
75
BW/
JQ:
55­
75
MD:
60­
85
55
55­
80
70
45­
70,
higher
for
MD
40­
80
55­
75
50­
70
70­
75
40­
80
Lighting
Intensity
(
Lux)

Adult
(
h
of
light)

Young
(
h
of
light)
Sunlight
spectrum
$
10
16­
17
JQ:
16­
17
Sunlight
spectrum
65
8
prior
to
photostimulation
(
8
weeks)

17
thereafter
14
7­
8
for
8
weeks
16­
18
thereafter
Transition
dawn/
dusk
65
7­
8
for
6­
8
weeks
16­
17
thereafter
or
increased
by
15
min/
day
from
17
14;
15­
30
min
transition
at
dawn/
dusk
Daylight
visual
spectrum
automatically
controlled
$
10
at
level
of
feeder
16­
17
JQ:
16­
17
Ventilation
1
to
15
changes/
h
recommended
As
in
CCAC
(
1984,
1993)
Good
ventilation
Good
ventilation
(
rate:
4
changes/
h
in
winter;
15/
h
in
summer
suggested)
8
to
15
changes/
h
recommended
Egg
turning
Storage:
optional
Incubation:
yes
Hatching:
no
Storage:
optional
Storage:
optional
Incubation:
yes
Hatching:
no
Storage:
yes
daily
Incubation:
yes
Storage:
optional
Incubation:
yes
Hatching:
no
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
109
April
23,
2003
Feed
Ad
libitum
Must
meet
nutrient
requirements
of
the
species
Caloric
and
water
content
of
diet
must
be
reported
Extra
calcium
added
to
adult
diet
if
same
diet
used
for
chicks
and
adults
Ad
libitum
Any
unmedicated
commercial
diet
that
meets
minimum
nutritional
standards
Description
of
basal
diet:

analyze
for
contaminants
Ad
libitum
Avoid
use
of
chemicals
or
medication
Contaminant­
free
as
possible
(
no
pesticides,

heavy
metals)
Ad
libitum
Must
meet
nutrient
requirements
of
JQ
Caloric
and
water
content
of
diet
must
be
reported\

Sufficient
space
for
feeding
must
be
provided
during
first
week
after
hatching
so
weak
birds
have
access
Extra
calcium
added
to
adult
diet
if
same
diet
used
for
chicks
and
adults
Water
Ad
libitum
Sufficient
space
for
drinking
must
be
provided
during
first
week
after
hatching
so
weak
birds
have
access
Ad
libitum
Ad
libitum
Ad
libitum
Water
bottles
or
automatic
water
devices
recommended
Bacitracin
or
one
of
its
forms
can
be
added
to
drinking
water
of
young
birds
if
necessary
Ad
libitum
Sufficient
space
for
drinking
must
be
provided
during
first
week
after
hatching
so
weak
birds
have
access
TEST
PROCEDURES
Quarantine
at
least
2
weeks
Acclimation
$
2
weeks
BW:
photostimulation
6
weeks
prior
to
acclimation
period
$
1
week
Incompatible
birds
rearranged
or
replaced
$
2
weeks
$
2
weeks,
can
coincide
with
quarantine
$
2
weeks
If
necessary
photostimulate
birds
during
acclimation
(
onset
of
laying
can
occur
during
acclimation)
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
110
April
23,
2003
Test
Substance
Mix
to
obtain
homogenous
distribution
in
diet.
Use
of
premix
is
advisable.

Frequency
of
diet
preparation
chosen
so
degradation
and
volatilization
of
test
substances
$
80%
of
initial
concentration.
Frequency
of
diet
renewal
#
1X/
day
and
$
1X/
week.
Can
keep
frozen
until
use;
Stability,

homogeneity
tests
required
Physical,
chemical,

biological
properties
Analyses
to
determine
stability
in
diet,
frequency
of
diet
preparation,

homogeneity
in
diet
Chemical
identification
data;

water
solubility;
vapor
pressure;
structural
formula;

purity;
chemical
stability
in
water,
light,
and
diet;

octanol/
water
partition
coefficient;
biodegradability
Cannot
use
guideline
for
highly
volatile
or
unstable
substances
Must
have
characteristics
that
allow
uniform
mixing
in
diet
Chemical
name,
source,

composition:
major
ingredients
and
percentage
of
each
Impurity,
known
physical
and
chemical
properties
(
solubility,
volatility,

degradation
rate)

Use
technical
grade
Mix
to
obtain
homogenous
distribution
in
diet.
Use
of
premix
is
advisable.

Frequency
of
diet
preparation
chosen
so
degradation
and
volatilization
of
test
substances
$
80%
of
initial
concentration.
Frequency
of
diet
renewal
#
1X/
day
and
$
1X/
week.
Can
keep
frozen
until
use;
Stability
analyses
under
conditions
on
test
prior
to
start
or
during
rangefinding
,
verify
during
main
test.
Check
stability
at
end
of
1st
feeding
period
and
at
end
of
last
Homogeneity
analysis
prior
to
test
of
at
1st
mix
for
study
Administration
Route
Carrier/
diluent
Maximum
carrier
in
feed
(%)
In
the
diet
A
vehicle
of
negligible
toxicity,

such
as
food
or
corn
oil,
water
Acetone
can
be
used
if
allowed
to
completely
evaporate
prior
to
feeding
<
2
A
constant
amount
should
be
added
to
each
test
group
and
control
diet
to
keep
caloric
value
and
moisture
content
equal
between
dosage
groups
In
the
diet
Solvent
or
other
material
Determine
concentration,

stability,
homogeneity
of
test
substance
in
diet
Stability,
volatility
determine
frequency
of
diet
preparation
and
storage
method
<
2
In
the
diet
Water,
corn
oil
Should
not
interfere
with
toxicity
of
test
substance
<
2
In
the
diet
Distilled
water
preferred.
If
not
water­
soluble,
could
dissolve
in
reagent
grade
evaporative
diluent
such
as
acetone,
methylene
chloride.
Solvent
should
be
completely
evaporated
prior
to
feeding
Other
carriers:
corn
oil,

propylene
glycol,
gum
arabic
(
arcacia)

<
2
Equivalent
amount
of
diluent
should
be
added
to
control
diet
In
the
feed
or
water
A
vehicle
of
negligible
toxicity,
such
as
food
or
corn
oil,
water
Acetone
can
be
used
if
allowed
to
completely
evaporate
prior
to
feeding
<
2
A
constant
amount
should
be
added
to
each
test
group
and
control
diet
to
keep
caloric
value
and
moisture
content
equal
between
dosage
groups
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
111
April
23,
2003
Number
of
dietary
concentrations/
choice
of
concentration
One
or
multiple
If
multiple,
then
at
least
three
plus
control.
Highest
concentration
should
be
level
expected
to
reveal
significant
effects
on
adult
health
or
reproductive
parameters.

Highest
dose
should
not
cause
mortality
or
other
severe
signs
of
parental
toxicity.
If
no
significant
reproduction
effects
expected
at
lower
test
concentration,
then
highest
should
be
the
expected
concentration
of
the
chemical
in
the
environment.

If
this
concentration
is
at
lower
than
1000
mg/
kg
diet,
no
need
to
test
above
1000
mg/
kg.

Lowest
concentration
should
not
impact
adult
health
or
reproductive
parameters
Intermediate
concentration
should
be
geometrically
spaced
between
the
highest
and
lowest
doses.
2
methods
to
establish
treatment
levels:
control
+
3
levels;
geometrically
spaced
control
+
1,
or
more
expected
or
known
environmental
concentration
If
nonstatistical,
$
1
concentration
must
produce
effect
or
contain
0.1%
of
test
material,
or
be
100X
the
highest
field
or
measured
concentration
At
least
3
+
control
Results
of
a
dietary
LC50
test
(
TG
205)

Highest
concentration
should
approximate
½
of
LC
10.

Lower
concentrations
should
be
geometrically
spaced
at
fractions
of
highest
dose
(
e.
g.
1/
6
and
1/
36
of
the
highest
dose)
3
+
control
Higher
2
treatment
concentrations
will
be
multiples
such
as
5
of
the
lowest
treatment
level.
The
highest
treatment
levels
usually
will
be
below
lethal
levels.
Highest
nonlethal
level
is
estimated
from
a
dietary
LC50.
Concentrations
should
include
an
actual
or
expected
field
residue
exposure
level.
Not
specified
Choice
on
basis
of
toxicological
data
from
range
finding
test,
prior
avian
tests,

and/
or
tests
with
mammalsHighest
concentration
should
be
level
expected
to
reveal
significant
effects
on
adult
health
or
reproductive
parameters.

Highest
dose
should
not
cause
mortality
or
other
severe
signs
of
parental
toxicity.
If
no
significant
reproduction
effects
expected
at
lower
test
concentration,
then
highest
should
be
the
expected
concentration
of
the
chemical
in
the
environment
with
addition
of
5X
safety
factor.

Lowest
concentration
should
not
impact
adult
health
or
reproductive
parametersIntermediate
concentration
should
be
geometrically
spaced
between
the
highest
and
lowest
doses.

Minimum
number
of
pens
per
concentration
Sufficient
pairs,
such
as
20,
to
ensure
16
breeding
pairs
in
the
control
groups
during
egglaying
Penmates
can
be
replaced
during
pretreatment
If
statistic
approach
used,

enough
to
give
detection
level
of
25%
at
"=
5,

power=
0.8
Nonstatistical
approach,

use
$
16
12
pairs
or
BW/
JQ:
12
groups
MD:
8
groups
BW:
12
pairs
or
groups,
20
pairs
in
control
group
MD:
12
pairs
or
8
groups
Sufficient
pairs,
such
as
20,

to
ensure
16
breeding
pairs
in
the
control
groups
to
end
of
treatement
period.

Penmates
can
be
replaced
during
pretreatment
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
112
April
23,
2003
Treatment
period
prior
to
egg­
laying
(
weeks)
no
treatment
prior
to
egglaying
$
10
for
persistent
compounds
<
10
for
nonpersistent;

sometime
during
egg­
laying
10­
12
8­
12
no
treatment
prior
to
egglaying
P1:
6
weeks
after
laying
established
F1
breeders
from
hatch
through
6
wk
post­
fertility
F1
chicks
from
hatch
to
termination
at
14
days
F2
not
treated
Post
egg­
laying
treatment
period
(
weeks)
6
(
8)
MD/
BW:
may
be
unnecessary
to
collect
more
eggs
than
would
be
laid
in
the
wild
with
2
clutches
Terminate
when
control
pens
produce
25
eggs,
or
6
weeks
after
50%
of
control
hens
have
laid
1
egg
8­
10
8­
12
At
least
6
Egg
collection
At
least
once
daily
for
6
weeks
Daily
until
control
pens
produce
25
eggs,
or
6
weeks
after
50%
of
control
hens
have
laid
1
egg
Daily
for
last
10
weeks
of
treatment
Daily
for
last
8­
10
weeks
At
least
once
daily
for
6
weeks
Eggs
hatched
Entire
pretreatment
and
treatment
period
Entire
egg­
collection
period
Entire
egg­
collection
period
Entire
egg­
collection
period
Entire
pretreatment
and
treatment
period
Candling
Day
0
for
cracks
Fertility/
early
embryo
viability:

BW:
Day
11
JQ:
Day
8
Embryo
survival:

BW:
Day
20­
21
JQ:
Day
15­
16
Day
0
for
cracks
At
1
and
2
weeks
of
age
for
fertility
Prior
to
incubation
to
detect
cracks
Day
0
for
cracks
Fertility/
early
embryo
viability:

BW:
Day
11
MD:
Day
14
Embryo
survival:

BW:
Day
18
MD:
Day
21
Day
0
for
cracks
Fertility/
early
embryo
viabilityDay
8
Embryo
survival:
Day
15­
16
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
113
April
23,
2003
Storage
period
Maximum
1
week
Weekly
Weekly
or
biweekly
BW:
Weekly
or
biweekly
MD:
biweekly
Maximum
1
week
Incubation
period/
hatch
BW:

Day
20­
21/
Day
24­
25
JQ:
Day
15­
16;
Day
17­
18
BW:
Day
21/
Day
23­
24
JQ:
Day
16/
Day16­
17
MD:
Day
23/
Day
25­
27
BW:
Day
21/
Day
24
:
Day
15­
16/
Day
17­
18
Rearing
period
(
days)
14
14
14
14
14
Brood
from
6th
wk
used
to
establish
F1
breeding
pairs
ENDPOINTS
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
114
April
23,
2003
Recommended
additional
endocrine
endpoints
Gross
morphology
and
histology
Size/
weight
of
gonads,
brain,

thyroid,
adrenals
Histology
of
thyroid,
brain,

adrenals,
gonads
Testicular
spermatid
counts
and
morphology
Gross
abnormality
of
genital
tract
Cloacal
gland
area
Plasma
and
fecal/
urate
hormones
Steroid
hormones,
estradiol,

testosterone,
corticosterone,

VTG
(
males),
thyroid
hormones,
TSH
Brain
chemistry:
GnRH,

catecholamine,
aromatase,

foam
gland
test
Fresh
egg
weight
Growth
curve
for
young
Behavior
of
young
and
adults
Effects
on
multiple
generations
Genetic
sex
ratio
at
hatchinge
For
12
genetic
males
and
12
genetic
females/
group
at
14
days
of
age:
presence
at
necropsy
of
structures
on
right
side,
histologically
determined
relative
amount
of
cortex
and
medulla,
and
development
of
oocytes,

serum
sex
steroids
Both
sexes
at
14
days
old:

organ
weights
including
brain,
body
weights,
wing
and
bone
length,
thyroid
weight,
skeletal
x­
ray;
if
differences
appear,
then
thyroid
histopathology
should
be
performed
on
all
groups,
otherwise,
on
highdose
and
control
groups
only
All
surviving
chicks
at
14
days
of
age:
subjected
to
visual
cliff
test,
challenged
with
cold
stress
test,
and
nest
attentiveness
test
Reproductive
capability
of
offspring
Gross
morphology
and
histology
Size/
weight
oftestes
Sex
of
F1
chicks
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
115
April
23,
2003
RESULTS
Validity
of
test
(
quality
criteria)
Test
substance
concentration
in
diet
is
satisfactorily
maintained
and
reported
Losses
of
<
20%
initial
concentrations
acceptable.

Higher
loss
rates
must
be
investigated,
explained
$
16breeding
pairs
of
control
birds
that
have
produced
eggs
must
survive
to
end
of
treatment
period
All
control­
group
mortalities
should
be
explained
In
controls,
hatching
success
for
incubated
eggs
laid
during
fifth
and
sixth
week
of
exposure
should
be
$
50%

Rate
of
viability
should
remain
$
80%

$
10
breeding
pairs
of
controls
should
survive
until
end
of
test
BW
chick/
MD
duckling
productivity
in
control
groups
does
not
average
12
or
10,
respectively,
14­

day­
old
survivors
per
pen
over
a
10­
week
period
Average
eggshell
thickness
in
control
groups
is
BW:
<
0.19
mm
MD:
<
0.34
mm
>
10%
of
adult
control
birds
die
Test
substance
concentration
in
diet
is
satisfactorily
maintained
and
reported
Losses
of
<
20%
initial
concentrations
acceptable.

Higher
loss
rates
must
be
investigated,
explained
$
16breeding
pairs
of
control
birds
that
have
produced
eggs
must
survive
to
end
of
treatment
period
All
control­
group
mortalities
should
be
explained
Parental
mortality
during
last
two
weeks
of
acclimation
should
exceed
3%

If
control
reproductive
parameters
do
not
meet
typical
values
(
provided
in
Annex
2),
procedure
and
husbandry
conditions
should
be
checked
for
problems
PARAMETER
OECD
Revised
Draft
Guideline,
April
2000
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite
Short­
Term
Life
Cycle
Test
(
Proven
Breeders)
ASTM
E1062­
86
Standard
Practice
for
Conducting
Reproductive
Studies
with
Avian
Species(
a)

Life
Cycle
Test
OECD
206
Avian
Reproduction
Test
Life
Cycle
Test
EPA
OPPTS
850.2300
Avian
Reproductive
Test
e
Life
Cycle
Test
OECD
First
Draft
Guideline,

December
1999
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
Two­
Generation
Test
(
P1
Proven
Breeders)

Battelle
Draft
116
April
23,
2003
One­
Generation
Test
Guidelines
Treatment
of
data
Numerical
data
should
be
presented
in
tabular
form,

separating
clearly
the
pretreatment
and
treatment
data
Treated
groups
compared
with
control
group
by
methods
in
MacLeod
(
1994)

Methods
that
compare
individual
performance
before
and
during
exposure
using
covariate
analysis
can
be
used
If
there
appears
to
be
delayed
toxic
responses
after
1­
2
gamete
cycles,
results
should
be
evaluated
in
1­
2
week
increments
to
avoid
reduction
in
the
power
of
the
test
to
detect
effects
when
responses
from
all
treatment
weeks
are
averaged
together
NOAEL
(
mg/
kg
body
weight/
day)
should
be
determined
for
all
health
and
reproductive
parameters
evaluated
Analyze
continuous
variables
(
body
weight,

eggshell
thickness)
by
ANOVA
or
general
linear
models
Separate
means
by
multiple
comparison
procedures
(
e.
g.,
Dunnett's
test)

Discrete
variables
such
as
count
of
eggs
laid,
cracked
eggs,
or
14­
day­
old
survivors
can
be
analyzed
by
contingency
tables
 
chisquare
analysis
Derived
variables
such
as
14­
day­
old
survivors
as
percentage
of
eggs
laid
can
be
compared
by
ANOVA
percentages
should
be
arcsin
transformed
and
weighted
ANOVA
performed
Test
groups
individually
compared
with
control
group
by
statistical
procedure
designed
in
study
plan
(
e.
g.,

ANOVA,
NOAEL)
Experimental
groups
compared
with
controls
by
ANOVA
Regression
analysis
is
highly
desirable
if
the
data
and
number
of
dose
levels
allow
Sample
units
are
individual
pens
Numerical
data
should
be
presented
in
tabular
form
All
adult
health
data
should
be
recorded
per
individual
bird
(
food
consumption
per
pair).
Since
P1
parental
pair
is
primary
statistical
unit
and
F1
parental
pair
is
secondary
unit,
all
reproductive
data
should
be
related
by
lineage.

Raw
data
should
be
reported
by
pen.
Measurement
of
endpoints
made
on
adult
birds
will
be
evaluated
by
comparing
values
obtained
from
birds
from
treated
groups
with
values
obtained
from
control
birds.
An
NOEC
expressed
in
mg/
kg
diet
and
mg/
kg
body
weight
per
day
should
be
determined
for
all
health
and
reproductive
parameters
evaluated
a)
Reapproved
in
1991;
discontinued
in
2000.

b)
BW
Bobwhite
(
Colinus
virginianus).

c)
MD
Mallard
duck
(
Anas
platyrhynchos).

d)
JQ
Japanese
quail
(
Coturnix
japonica).

e)
EDSTAC
(
EPA
1998)
recommended
extension
for
a
two­
generation
avian
reproduction
test
using
the
Japanese
quail
and
a
pre­
breeding
exposure
for
the
P1
generation.
The
additional
endpoints
recommended
by
EDSTAC
for
a
two­
generation
test
a
summarized
in
bold
under
the
"
Recommended
additional
endocrine
endpoint"
section
of
this
table
in
the
OPPTS
850.2300
column.
Battelle
Draft
117
April
23,
2003
11.1
Life
Cycle
("
One­
Generation")
Reproduction
Tests
The
three
one­
generation
avian
reproduction
toxicity
test
guidelines
considered
in
this
discussion
are
ASTM
E1062­
86,
OECD
206,
and
EPA
OPPTS
850.2300.
The
last
guideline,
OPPTS
850.2300
(
EPA
1996),
represents
a
harmonization
of
previous
test
guidelines
developed
by
the
Office
of
Prevention,
Pesticides
and
Toxic
Substances,
EPA.
ASTM
E1062­
86
was
established
in
1986,
reapproved
in
1991,
and
discontinued
in
2000.
No
replacement
protocol
has
been
proposed.
OECD
206
is
similar
to
the
OPPTS
850.2300
guideline.
A
proposed
short
term
life
cycle
(
proven
breeder)
guide
(
OECD
Revised
Draft
Guideline,
April
2000,
Proposal
for
a
New
Avian
Reproduction
Toxicity
Test
in
Japanese
Quail
or
Northern
Bobwhite)
is
also
discussed.

All
of
the
life
cycle
tests
were
designed
to
be
first­
line
screening
tests
for
identifying
potential
reproductive
effects
in
birds
exposed
to
environmental
chemicals.
All
use
a
similar
array
of
measurement
endpoints
to
evaluate
effects
of
chemicals
on
components
of
reproduction
that
reflect
the
reproductive
success
of
the
female
and
prerecruitment
survival
of
young.
The
endpoints
are
also
selected
to
aid
in
identifying
cause­
and­
effect
relationships.

11.1.1
OPPTS
850.2300
The
test
simulates
a
chronic
dietary
exposure
for
the
purpose
of
detecting
potential
long­
term
effects
of
persistent
chemicals
or
those
chemicals
that
are
repeatedly
or
continuously
applied
to
the
environment.
It
takes
into
account
the
measured
or
estimated
residues
in
the
environment
with
the
goal
of
determining
both
the
highest
and
the
lowest
dietary
concentration
that
produces
an
observable
adverse
effect
on
an
array
of
reproduction
measurements.

This
guideline
provides
an
excellent
simulation
of
chronic
exposure
for
chemicals
that
are
present
in
the
environment
for
long
periods
of
time,
either
because
of
their
refractory
nature
or
application
in
the
environment.
It
measures
the
potential
for
bioaccumulation
of
the
test
substance
in
tissues
and
its
deposition
into
eggs
and
detects
toxicity
and
reproductive
injury
from
long­
term
exposure.
The
harmonized
guideline
provides
statistical
information
and
allows
for
application
of
regression
analysis
if
the
number
of
treatment
levels
and
the
data
are
adequate.

However,
the
20­
week
continuous
dietary
exposure
period
is
not
realistic
for
most
contemporary
chemicals
and
pesticide
exposure
scenarios.
The
exposure
protocol
also
does
not
account
for
the
rapidity
with
which
dietary
exposure
of
chemicals
can
affect
reproductive
performance
in
birds
and
fails
to
trigger
a
test
for
substances
that
pose
a
risk
to
reproductive
success
from
short­
term
exposure.
In
addition,
the
length
of
laying
period
appears
to
reduce
statistical
power,
because
it
approaches
the
biological
limits
of
some
birds.
It
thereby
introduces
high
variability
within
groups,
because
some
birds
remain
in
production
while
others
no
longer
lay
eggs.

A
major
weakness
of
the
OPPTS
test
guideline
that
has
become
apparent
as
past
data
have
been
reviewed
is
that
the
statistical
power
of
the
test
is
low
for
some
endpoints,
making
it
difficult
to
detect
effects
that
could
be
biologically
significant.
Battelle
Draft
118
April
23,
2003
Also,
reliance
on
using
estimated
environmental
concentrations
to
determine
exposure
concentrations,
although
sound
from
a
realism
perspective,
is
problematic
when
significant
differences
are
detected
in
all
dose
groups
or
in
none.
If
no
effects
are
observed,
it
cannot
be
determined
whether
the
dietary
concentrations
are
truly
below
those
causing
reproductive
deficits
or
whether
effects
could
not
be
detected
because
of
inadequate
test
design.
There
is
also
no
information
on
how
close
the
tested
concentrations
are
to
those
that
cause
effects.
In
contrast,
if
effects
are
observed
at
all
dietary
concentrations,
no
information
is
obtained
on
the
impact
of
lower
concentrations
on
reproductive
performance.
Because
of
the
usually
limited
concentration
selection,
there
is
little
predictive
capability
in
protocol
design
that
would
aid
in
the
assessment
of
potential
risk
of
a
chemical
for
a
new
use
at
higher
or
lower
concentrations
than
those
originally
tested.

The
guideline
does
not
evaluate
exposures
to
life­
stages
other
than
the
adult
and
egg.
Therefore,
many
potential
effects
on
reproduction
may
not
be
detected.
With
the
exception
of
eggshell
thickness,
most
of
the
endpoints
measured
are
not
indicators
of
endocrine
disruption,
and
although
the
survivability
of
the
P1
offspring
is
measured,
their
reproductive
capacity
is
not.
As
a
one­
generation
test,
there
is
no
evaluation
of
chemicals
on
the
F2
progeny.

11.1.2
OECD
206
The
OECD
one­
generation
test
is
similar
to
OPPTS
850.2300
and
shares
many
of
the
same
strengths
and
weaknesses.
However,
it
differs
in
three
major
ways
from
the
OPPTS
protocol.
First,
the
Japanese
quail
is
an
acceptable
species
in
the
OECD
206
guideline.
Use
of
the
Japanese
quail
with
its
extended
breeding
period
could
reduce
the
variability
in
egg
production
and
related
parameters
that
is
encountered
in
the
OPPTS
protocol,
because
the
bobwhite
and
mallards
approach
the
end
of
the
reproductive
limits
after
10
weeks
of
egg­
laying.
Secondly,
the
OECD
protocol
recommends
that
Japanese
quail
be
proven
breeders
before
they
are
used
in
the
test.
The
test
begins
with
dietary
exposure
of
the
birds
under
short­
day
conditions
(
non­
egg­
laying)
for
8
weeks
prior
to
the
initiation
of
a
long­
day
photoperiod
to
bring
the
birds
into
breeding
condition.
Proven
breeders
are
birds
that
were
reproductively
successful
previously
and
that
start
the
test
approaching
their
second
breeding
season.
Using
firs­
year
breeders
in
a
test
in
which
the
treatment
is
initiated
prior
to
onset
of
breeding,
as
in
the
OPPTS
guideline,
does
not
allow
for
removing
infertile
or
incompatible
pairs,
because
the
infertility
could
be
a
result
of
treatment.
Use
of
the
proven
breeders
reduces
the
variability
and
increase
the
power
of
the
test.
Thirdly,
the
OECD
protocol
establishes
dietary
treatment
concentrations
based
on
a
range
finding
test
that
does
not
necessarily
incorporate
an
estimated
environmental
concentration
such
that
the
concentrations
tested
are
based
on
toxic
response
(
½
LC10).
The
test
is
therefore
less
likely
to
produce
the
situation
described
above
for
the
OPPTS
guideline,
where
the
lack
of
effects
in
all
treatment
groups
cannot
be
attributed
to
concentrations
truly
below
those
causing
reproductive
effects,
because
the
design
of
the
test
could
have
been
inadequate
to
detect
effects.
Battelle
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April
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2003
11.1.3
ASTME1062­
86
The
ASTM
guideline
was
designed
to
be
flexible
in
its
approach
to
screening
for
reproductive
effects.
It
could
be
used
to
duplicate
the
OPPTS
850.2300
test
design
or
to
create
a
design
that
attempts
to
overcome
some
of
the
weaknesses
of
the
both
the
OPPTS
and
OECD
206
guidelines.
Design
options
were
provided
in
varying
detail
for
testing
nonpersistent
chemicals,
limiting
collection
of
egg
production
to
within
the
biological
capacity
of
the
species,
assuring
that
the
power
of
the
test
was
adequate
to
detect
effects,
and
selecting
treatment
concentrations
linked
to
toxic
response
rather
than
field
concentration.

Unlike
the
other
one­
generation
studies,
the
ASTM
guideline
suggested
that
the
exposure
period
for
nonpersistent
test
substances
may
be
shortened
and
dietary
concentrations
decreased
to
simulate
declines
under
field
conditions.
However,
the
recommendations
were
vague,
and
no
discussion
or
criteria
for
selecting
one
of
the
optional
exposure
scenarios
were
provided.
Dietary
exposure
prior
to
the
onset
of
egg
production
could
be
less
than
10
weeks,
or
for
some
chemicals,
exposure
can
be
initiated
during
egg­
laying.
Although
the
potential
benefit
of
using
pre­
exposure
data
as
covariates
for
birds
is
increased
power;
the
disadvantage
is
the
inability
to
determine
whether
the
chemical
affected
the
onset
of
egg­
laying.

Whereas
the
OPPTS
850.2300and
OECD
206
guidelines
recommend
a
post
egg­
laying
exposure
and
an
egg­
collection
period
of
10
weeks,
the
ASTM
protocol
recommended
an
egg
collection
period
for
the
bobwhite
and
mallard
equivalent
to
laying
two
clutches,
or
25
eggs.
This
egg­
laying
period
is
within
the
biological
limits
of
these
species
and
could
reduce
one
source
of
variability
that
is
unrelated
to
chemical
treatment.
However,
the
variability
of
proportional
measurements
based
on
the
numbers
of
eggs
laid
could
increase
as
a
result
of
the
reduced
numbers
of
eggs
collected.
Also,
the
shortened
egg­
collection
period,
could
be
too
short
to
detect
effects
that
are
delayed
or
increase
in
severity
during
treatment.
For
example,
effects
such
as
those
on
early
germ
cells
in
the
Japanese
quail
would
not
be
manifested
until
about
3
weeks
after
exposure
is
initiated.
If
birds
were
exposed
prior
to
egg­
laying,
and
the
onset
of
egg­
laying
were
delayed,
the
resulting
delay
in
peak
egg
production
would
be
overlooked
by
the
shortened
egg­
collection
period,
and
an
overestimate
of
the
severity
of
impact
could
be
inferred.

The
ASTM
protocol
also
provides
a
statistical
means
for
estimating
the
number
of
replicates
needed
to
assure
sufficient
power
to
detect
selected
effects.
Although
useful
and
made
readily
calculable
by
means
of
an
appended
table,
the
information
on
the
coefficients
of
variation
for
various
endpoints,
in
particular
endocrine
endpoints,
in
the
species
or
strain
being
used
may
not
be
available
to
the
investigator.

The
remaining
strength
of
the
ASTM
guideline
is
the
use
of
toxicity­
based
information
to
establish
the
treatment
concentrations
so
as
to
avoid
the
shortcomings
of
using
an
expected
field
concentration
criteria
discussed
in
Section
11.1.1.
The
disadvantages
of
using
toxicity­
based
criteria
is
the
need
for
a
range
finding
test(
s)
to
establish
the
appropriate
dietary
concentrations
and
the
diminished
usefulness
of
tissue
and
egg
residues
if
the
test
concentrations
are
considerably
different
from
the
expected
environmental
concentrations.
Battelle
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120
April
23,
2003
Other
options
of
the
ASTM
protocol
included
the
use
of
natural
incubation
and
the
potential
to
use
behavioral
endpoints
and
examine
multigenerational
effects.
Little
information
is
provided
for
these
options.

11.1.4
OECD
Revised
Draft
Proposal
(
April
2000)
for
a
New
Test
Guideline,
Avian
Reproduction
Toxicity
Test
in
the
Japanese
Quail
or
Northern
Bobwhite
The
April
2000
revised
draft
proposal
for
a
new
test
guideline,
Avian
Reproduction
Toxicity
Test
in
the
Japanese
Quail
or
Northern
Bobwhite
(
ARTT
2000),
is
similar
to
the
optional
exposure
scenarios
of
the
ASTM
guideline
in
that
it
has
a
limited
exposure
period.
To
accommodate
bioaccumulating
substances
that
may
require
lengthy
exposure
periods
to
achieve
steady
state
concentrations
in
the
test
subjects,
both
the
OECD
206
and
the
OPPTS
850.2300
guidelines
prescribe
8
to
10­
week
exposures
prior
to
egg
laying
and
a
similar
exposure
period
after
egg
laying
has
begun.
Exposure
in
the
ARTT
2000
guideline
is
limited
to
6
weeks
after
egg
production
has
been
established,
limiting
the
applicability
of
the
guideline
to
nonbioaccumulating
chemicals.
However,
contemporary
chemicals
are
more
likely
to
be
nonpersistent
which
reduces
the
severity
of
this
limitation.

The
short
exposure
period
of
the
ARTT
2000
guideline
is
a
result
of
an
effort
to
increase
the
statistical
power
to
detect
treatment
effects.
By
using
pairs
of
birds
that
are
proven
breeders,
pre­
treatment
differences
in
birds
can
be
used
to
correct
post­
treatment
effect
measurements
and
reduce
the
variability
within
treatment
groups;
thereby
making
it
more
likely
that
treatment
differences
will
be
detected.
Also
contributing
to
reduced
variability
in
the
test
is
the
ability
to
remove
unfertile
and
incompatible
pairs
prior
to
start
of
treatment.
The
disadvantage
of
this
approach
is
the
inability
to
detect
effects
on
reproductive
maturation.

Although
the
OECD
206
guideline
also
provides
for
a
proven
breeder
exposure
scenario
for
the
Japanese
quail,
it
is
able
to
retain
the
advantage
of
increased
power
from
using
pre­
exposure
data
as
covariates
for
birds
while
at
the
same
time
providing
an
extended
exposure
period
of
up
20
weeks
by
using
birds
approaching
their
second
breeding
season
and
reproductive
data
from
the
previous
season.
Increased
cost,
space
utilization
etc.,
is,
of
course,
required
to
obtain
the
baseline
data.

Although
liver,
spleen,
and
testes
weights
and
descriptive
records
of
abnormal
behavior
are
required,
this
guideline
lacks,
as
do
the
other
one­
generation
tests,
reproductive
evaluation
of
the
P1
offspring
or
endpoints
that
effectively
detect
endocrine
disruption.

11.2
Two
Generation
Life
Cycle
Test
The
two
principal
designs
being
considered
for
an
avian
two­
generation
reproduction
test
are
a
proven
breeder
design,
where
reproduction
is
monitored
pre­
exposure,
and
a
pre­
egg­
laying
exposure
design,
where
effects
on
maturation
are
included.
The
(
EPA
1998)
recommendations
for
extending
the
EPA
OPPTS
850.2300
one­
generation
avian
reproduction
test
to
a
twogeneration
guideline
included
a
pre­
egg­
laying
exposure
regime
for
the
P1
generation.
A
proposed
two­
generation
guideline
(
OECD
1999)
was
developed
based
on
these
Battelle
Draft
121
April
23,
2003
recommendations,
but
suggested
a
proven­
breeder
design.
Bennett
et
al.
(
2001)
discussed
the
advantages
and
disadvantages
of
proven­
breeder
and
pre­
egg­
laying
exposure
protocols
in
detail.

11.2.1
Proven­
Breeder
Design
The
proven­
breeder
protocol
increases
statistical
power
by
1)
eliminating
pre­
egg­
laying
exposure
allowing
nonlayers
to
be
removed
prior
to
treatment
so
that
the
confounding
effects
of
nonbreeders
are
eliminated,
2)
introducing
a
pretreatment
period
during
peak
egg­
laying,
in
which
reproduction
data
are
collected
to
be
used
as
covariates,
and
3)
using
a
shorter
egg­
laying
period
(
6
weeks),
so
that
birds
are
laying
at
a
more
constant
rate
throughout
the
entire
exposure.
The
shorter
exposure
period
is
more
consistent
with
the
minimal
persistence
and
bioaccumulation
characteristics
of
contemporary
chemicals.
The
exposure
can
be
lengthened
somewhat
to
accommodate
longer­
lived
substances,
particularly
if
the
Japanese
quail
is
used.
Japanese
quail
maintain
a
longer
period
of
peak
egg­
laying
than
the
period
suggested
in
the
protocol
which
is
based
on
the
bobwhite.
A
major
improvement
in
this
design
over
that
of
the
one­
generation
protocols
is
the
increased
sensitivity
of
the
test
resulting
from
a
more
comprehensive
evaluation
of
the
P1
progeny
and
the
addition
of
endocrine
endpoints.

Although
increased
power
to
detect
effects
is
obtained
by
this
design,
it
comes
at
the
sacrifice
of
information
on
maturation
effects,
such
as
delayed
onset
of
egg­
laying.
However,
these
endpoints
can
be
measured
during
the
maturation
of
the
F1
generation.
Proven­
breeders
from
a
previous
breeding
cycle
could
also
be
used
to
reduce
variability
in
a
prebreeding
exposure
scenario.
The
retention
of
the
same
level
of
egg
production
and
fertility
over
multiple
breeding
cycles
in
aging
birds
may
be
unlikely.

The
statistical
concerns
that
motivated
the
proven­
breeder­
design
for
the
P1
generation
cannot
be
eliminated
in
the
F1
breeders,
because
they
receive
treatment
throughout
their
life
cycle.
Therefore,
because
mortality
is
likely,
the
test
is
vulnerable
to
problems
of
decreased
power
to
detect
effects
in
the
F1
reproduction
measurements.
Also,
if
delayed
effects
occurred,
the
6­
week
period
of
data
collection
could
be
insufficient
to
detect
the
changes.
Delayed
onset
of
egg­
laying
in
the
F1
would
also
result
in
a
delay
of
peak
egg
production.
Egg
production
may
not
otherwise
be
impaired,
but
could
appear
to
be
so
under
the
short
exposure
period
of
this
design.

Measurement
of
endocrine
status
is
significantly
enhanced
in
this
two­
generation
protocol.
Endpoints
include
measures
of
steroid
and
thyroid
hormones,
maturation
of
hormone­
mediated
secondary
sexual
characteristics,
organ
condition
,
and
spermatid
quality
and
number.
The
protocol
is
lacking
somewhat
in
assessing
endocrine
impacts
on
F1
and
F2
progeny.
However,
EDSTAC
(
EPA
1998)
focuses
on
endpoints
that
evaluate
effects
on
the
14­
day­
old
chicks.
Reproduction
endpoints
are
similar
to
those
of
the
one­
generation
studies.

A
major
weakness
of
both
two­
generation
protocols
is
the
reliance
on
detecting
effects
by
comparison
of
means.
There
is
potential
for
delayed
effects
in
both
the
P1and
F1
generations:
that
is,
delayed
manifestation
of
effects
on
spermatogonia
for
up
to
3
weeks
in
Japanese
quail
and
delayed
recruitment
of
yolk
to
maturing
oocytes
in
the
P1
generation,
and
delayed
onset
and
Battelle
Draft
122
April
23,
2003
peak
production
of
eggs
in
the
F1.
Therefore,
a
regression
approach
to
analyzing
the
data
would
better
define
the
level
and
pattern
of
toxicity.
Animal
numbers
can
be
reduced,
because
the
dependence
on
replication
is
diminished
in
regression
statistics.

11.2.2
Pre­
Egg­
laying
Exposure
Design
The
pre­
egg­
laying
protocol
is
similar
to
the
proven­
breeder
design,
except
the
birds
are
paired
and
exposed
to
the
dietary
treatment
prior
to
egg­
laying.
Exposure
prior
to
egg­
laying
could
impact
developing
systems
that
would
not
be
vulnerable
in
the
mature
reproductive
animal.
Such
exposures
could
cause
precocial
or
delayed
onset
of
egg­
laying
that
would
not
be
detectable
in
a
proven­
breeder
design.
Considerable
loss
of
power
has
been
demonstrated
when
nonproducing
pairs
cannot
be
removed
from
the
test
and
pretreatment
egg
production
cannot
be
used
as
covariates
to
reduce
sources
of
nontreatment
variation
(
Springer
and
Collins
1999).
It
is
possible
that
proven
breeders
approaching
their
second
breeding
season
could
be
used
so
that
exposure
could
begin
prior
to
the
onset
of
egg­
laying,
thus
obtaining
both
information
on
pre­
egg­
laying
effects
and
the
statistical
benefit
of
reducing
the
confounding
effects
of
nonbreeders.
However,
age­
effects
would
have
to
be
taken
into
account,
and
the
benefit
of
using
pretreatment
egg­
production
data
is
still
lost
by
this
option.

Because
much
of
the
information
obtained
from
a
two­
generation
reproduction
toxicity
test
will
be
related
to
the
P1
population,
it
is
important
that
the
reproductive
homogeneity
of
the
test
population
be
assured.
This
is
best
attained
by
not
only
accounting
for
equivalent
body
condition
(
body
size,
weight,
and
health)
in
the
test
groups,
but
also
equivalent
ability
to
produce
and
fertilize
eggs.
One
of
the
major
weaknesses
of
the
pre­
egg­
laying
design
is
that
it
cannot
assure
a
homogeneous
test
population
from
which
to
detect
changes
due
to
chemical
and
endocrine
challenge.

12.0
RECOMMENDED
PROTOCOL
AND
ADDITIONAL
DATA
NEEDS
12.1
Preferred
Test
Species
Of
the
few
bird
species
available
that
breed
successfully
under
laboratory
conditions,
the
Japanese
quail
is
the
preferred
test
species,
because
it
is
an
indeterminate
egg­
layer
that
matures
much
more
rapidly
than
other
species.
It
has
both
a
higher
rate
of
egg
production
and
a
longer
period
at
which
peak
production
is
maintained
than
the
northern
bobwhite.
In
addition,
the
reproductive
physiology,
endocrinology,
and
behavior
of
the
species
are
better
characterized
than
those
of
the
bobwhite.
Spermatogenesis
is
most
fully
described
in
the
Japanese
quail,
among
avian
species;
therefore,
the
minimum
length
of
exposure
required
for
germ
cell
effects
to
be
manifested
extragonadally
can
be
calculated.
The
cycle
of
the
seminiferous
epithelium
of
the
bobwhite
has
not
been
determined.
Also,
the
unique
cloacal
gland
of
the
male
can
be
used
as
a
measure
of
maturity
and
androgen
disruption,
as
can
the
sexually
dimorphic
plumage
of
the
species.
The
Japanese
quail
has
been
used
in
toxicity
tests
and
appears
to
be
of
comparable
sensitivity
to
environmental
chemicals
as
the
northern
bobwhite,
though
a
direct
comparison
of
their
relative
sensitivity
to
substances
that
disrupt
reproduction
and
endocrine
function
is
only
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currently
being
assessed.
Although
there
are
problems
such
as
strain
characteristics
and
high
sensitivity
to
inbreeding
associated
with
the
extensive
domestication
of
Japanese
quail,
the
advantages
of
using
this
species
outweigh
those
of
the
less
domesticated
bobwhite.

12.2
Exposure
Protocol
Currently,
there
are
insufficient
data
to
determine
the
combination
of
exposure
protocols
for
the
P1
and
F1
generations
that
is
the
most
robust
for
documenting
changes
in
ecologically
important
fitness
endpoints,
and
that
at
the
same
time
is
the
most
effective
in
determining
mechanism
of
action.
Therefore,
a
side­
by­
side
performance
evaluation
of
the
prebreeding
and
proven­
breeder
exposure
regimens,
each
one
combined
with
a
nontreated
and
a
worst­
case,
hatch­
throughegg
laying
F1
exposure
scenario
is
recommended
to
evaluate
the
sensitivity
and
cost­
benefit
of
these
protocols.
The
proven­
layer
exposure
has
advantages,
because
removing
infertile
pairs
provides
for
the
endpoints
to
be
assessed
from
an
established
and
homogeneous
fecundity
in
the
P1
generation.
In
particular,
the
primary
biological
endpoint
of
the
test,
the
number
of
F2
14­
day
old
survivors
per
P1
generation
pair
per
day,
can
be
assessed.
Likewise,
exposing
the
P1
generation
after
the
onset
of
egg­
laying
avoids
the
complication
of
delaying
maturation,
thereby
shifting
the
period
of
peak
egg
production
in
affected
birds,
in
some
cases
beyond
the
exposure
period.
If
reproduction
is
otherwise
not
affected,
this
asynchrony
in
production
could
result
in
misleading
conclusions
regarding
egg
production
and
associated
endpoints,
and
greatly
reduce
the
number
of
hatchlings
available
for
assessing
F1
survivorship
and
forming
the
F1
parental
generation.
There
are
also
considerable
statistical
benefits
in
the
proven­
breeder
scenario,
when
endpoints
are
evaluated
by
comparisons
of
means
to
the
control.

The
advantages
of
the
pre­
egg­
laying
exposure
design
are
the
ability
to
detect
affects
that
alter
maturation
(
e.
g.,
onset
of
egg­
laying,
foam
production)
and
to
have
long
enough
exposure
that
near
steady
state
concentrations
in
the
tissues
of
treated
birds
will
be
attained
during
the
exposure
period
and
adequate
assessment
of
impact
achieved
for
the
more
persistent,
bioaccumulating
substances.

Exposure
of
the
F1
from
hatch
through
egg­
laying
is
recommended
in
semblance
of
a
worst­
case
exposure,
because
it
allows
the
expression
of
effects
on
susceptible
growth
and
developmental
stages,
in
addition
to
effects
manifested
in
the
P1
generation.
For
substances
expected
to
cause
significant
juvenile
mortality,
a
subset
should
be
exposed
from
hatch
through
egg­
laying,
while
another
cohort
is
not
exposed.
The
time
of
onset
of
egg­
laying,
one
of
the
more
sensitive
measures
of
endocrine
disruption,
can
be
detected
under
this
exposure
regimen
and
could
minimize
the
need
to
detect
altered
maturation
using
a
prebreeding
exposure
scenario
in
the
P1
population.
However,
it
is
not
known
whether
the
previous
in
ovo
exposure
of
the
F1
chicks
would
confound
the
interpretation
of
the
response
compared
with
the
response
measured
in
P1
birds
that
lack
in
ovo
exposure.

Data
on
the
onset
of
sexual
maturation
in
the
P1
generation
could
be
obtained
from
the
current
one­
generation
reproduction
toxicity
tests
(
OPPTS
Guidelines
850.2300;
OECD
Guidelines
206)
at
little
added
cost.
The
one­
generation
tests
could
also
supply
yolk­
residue
data
that
would
provide
information
on
the
length
of
exposure
needed
for
maximum
transfer
of
the
test
substance
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to
the
egg.
Lastly,
dose­
response
data
from
the
one­
generation
tests
would
also
greatly
aid
in
selecting
appropriate
test
concentrations
for
the
multigeneration
test.

12.3
Appropriateness
of
Reproductive
Endpoints
In
general,
the
reproductive
fitness
endpoints
of
reproductive
output,
developmental
adequacy,
and
appropriate
behaviors,
for
example,
described
in
both
the
one­
generation
and
two­
generation
candidate
protocols,
provide
useful
data
on
the
reproductive
function
of
the
mated
pairs.
However,
because
the
male
has
opportunity
for
multiple
copulations,
egg
fertility
is
not
necessarily
a
sensitive
indicator
of
impact
on
male
fecundity.
Assessment
of
the
relative
fertility
of
the
male
at
the
onset
of
test
is
needed
in
a
proven­
layer
exposure
regimen
to
assure
that
the
groups
begin
on
test
with
equivalent
reproductive
capacity
in
males
as
well
as
females.
An
assay
such
as
the
sperm
mobility
test
or
sperm
penetration
of
the
perivitelline
layer
should
be
added
as
an
inexpensive
means
of
tracking
gender­
specific
changes
in
fertility.

Valuable
endpoints
for
evaluating
effects
on
reproduction
and
endocrine
system
function
in
adults
that
are
described
in
the
candidate
two­
generation
reproduction
tests
are
°
onset
of
sexual
maturation
(
first
egg,
foam
production)
°
cloacal
gland
area
°
male
copulatory
behavior
°
plasma
and
fecal/
urate
steroid
hormones
°
gross
morphology
and
histology
of
specific
organs,
such
as
liver,
spleen,
gonads,
brain,
thyroid,
and
adrenals.

Some
modification
of
these
endpoints
is
recommended
to
reduce
redundancy,
increase
the
cost­
effectiveness
of
the
test,
and
provide
higher­
quality
data.
Both
size
and
weight
measurements
of
the
organs
are
suggested
in
these
protocols;
however,
size
measurements
of
the
organs
provide
little
additional
data
at
increased
labor
cost
and
are
not
recommended.
Also,
considerable
timesaving
will
be
realized
during
necropsy
if
the
thyroid
and
adrenal
glands
are
excised
for
histological
examination
rather
than
for
organ
weight.
Removing
and
trimming
these
tissues
for
weight
measurements
is
time­
consuming
and
damaging
to
the
tissue.
Better
quality
data
are
obtained
from
histological
examination
of
these
tissues.
Histological
examinations
should
only
be
conducted
on
organs
from
birds
receiving
the
highest
dietary
concentration
of
the
test
substance
and
on
controls.
If
chemical­
induced
abnormalities
are
observed,
the
tissues
from
birds
in
the
lower
dose
groups
should
be
examined.

Other
endpoints
suggested
by
the
candidate
two­
generation
protocols
that
are
not
useful
at
this
time
are
spermatid
counts
and
morphology,
VTG,
and
several
brain
chemistry
measures.
Microscopic
enumeration
of
spermatids
and
morphological
abnormalities
of
sperm
are
timeconsuming
to
document
and
have
been
found
to
be
relatively
poor
indicators
of
reproductive
capability
in
male
birds.
A
major
effort
in
poultry
research
in
recent
years
has
been
the
development
of
more
reliable
assays
of
male
condition
based
on
measurements
of
sperm
function,
rather
than
semen
quality.
These
assays
are
mainly
based
on
measures
of
the
interaction
of
sperm
with
the
inner
or
outer
perivitelline
layer
of
the
egg.
They
do
not
require
Battelle
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April
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2003
special
expertise
or
physically
handling
the
bird
to
obtain
a
semen
sample
(
samples
can
be
obtained
from
the
same
eggs
as
those
used
for
egg
quality
tests)
and
are
inexpensive
to
perform.
Substitution
of
a
perivitelline
assay
for
the
spermatid
counts
is
recommended.
It
is
not
known
at
this
time
how
useful
VTG
assays
will
be
in
birds,
and
only
recently
has
an
assay
been
reported
that
uses
the
necessary
species­
specific
antibodies
for
quail.
If
it
is
as
sensitive
of
an
endpoint
as
it
has
been
shown
to
be
in
other
oviparous
animals,
it
could
be
a
valuable
inclusion
in
the
guideline
at
a
future
date.
Several
brain
chemistry
endpoints,
such
as
GnRH,
catecholamine,
aromatase,
and
vasotocin,
which
have
been
used
to
identify
and
investigate
hormonally
dimorphic
areas
of
the
avian
brain,
are
histochemical
techniques
requiring
considerable
expertise,
preparation,
and
computer
image
analysis.
It
should
be
noted
that
these
endpoints
are
currently
being
adapted
to
more
routine
laboratory
procedures
and
evaluated
for
their
potential
use
as
indicators
of
endocrine
disruption.
Few
data
are
available
in
the
literature
on
these
endpoints,
because
they
are
only
currently
under
testing.
Researchers
conducting
this
work
report
that
GnRH
appears
to
be
relatively
insensitive
as
an
endpoint
of
endocrine
disruption,
but
that
preliminary
data
from
dose­
response
tests
with
a
weak
estrogenic
compound
indicate
that
catecholamines
and
vasotocin
are
promising,
having
application
at
all
life­
stages
and
adaptability
to
routine
laboratory
procedures,
such
as
RIA.
These
procedures
are
obviously
terminal,
requiring
the
sacrifice
of
the
animal,
and
would
therefore
be
applied
to
brain
tissue
gathered
at
adult
necropsy
and
from
the
14­
day
chicks.

EDSTAC
(
EPA
1998)
endpoint
recommendations
mainly
focus
on
evaluation
of
the
14­
day
surviving
chicks.
Determining
the
genetic
sex
of
the
14­
day
old
survivors
at
hatch
provides
a
good
measure
of
in
ovo
disruption
of
endocrine
systems.
EDSTAC
(
EPA
1998)
then
recommends
selecting
a
subset
of
males
and
females
from
each
group
for
gross
and
histological
examination
of
gonadal
tissue.
The
gonadal
tissue
of
all
males
and
all
females
in
the
high­
dose
group
and
controls
should
be
weighed
and
its
external
appearance
described.
Relative
amount
of
cortex
and
medulla,
and
the
development
of
oocytes
should
be
histologically
determined
on
a
subset
of
males,
as
described
by
EDSTAC
(
EPA
1998).
Oviduct
weight
and
differentiation
should
also
be
determined.
However,
to
keep
costs
of
the
test
within
a
reasonable
range,
these
endpoints
should
be
measured
on
a
single
cohort
of
hatchlings,
preferably
from
the
latter
half
of
the
exposure
period,
to
assure
maximum
transfer
of
the
test
substance
to
the
yolk.
If
the
incidence
of
phenotypic
change
in
the
high­
dose
group
is
above
that
found
in
the
control
group,
the
remaining
treatment
groups
should
be
examined.
Companion
determination
of
circulating
steroid
concentrations
is
also
a
valuable
endpoint
suggested
by
EDSTAC
(
EPA
1998).
The
committee
also
suggested
the
determination
of
organ
weights,
particularly
the
thyroid
weight,
for
all
chicks.
As
discussed
above,
thyroid
weights
are
not
recommended,
because
of
the
difficulty
in
removing
this
tissue
without
severely
affecting
the
more
informative
histological
examination
of
the
tissue.
The
benefit
of
collecting
organ
weights
on
all
chicks
does
not
appear
to
warrant
the
cost
involved
and
is
not
recommended.
However,
the
wing
and
bone
length
measurements
would
provide
an
index
of
thyroid
function.
Although
skeletal
x­
ray
would
provide
an
additional
measure
of
thyroid
effects,
it
does
so
at
high
cost
and
also
is
not
recommended.
However,
x­
ray
could
also
be
used
to
detect
medullar
bone
formation
in
males
and
females
in
response
to
chemical
challenge.
A
battery
of
behavioral
tests
is
recommended
by
EDSTAC
(
EPA
1998)
for
the
14­
day­
old
chicks.
This
endpoint
has
merit,
though
the
composition
of
the
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April
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2003
test
battery
needs
further
investigation
to
refine
protocols,
validate
sensitivity,
and
assure
applicability
to
a
broad
range
of
chemicals.

12.4
Preferred
Methods
for
Quantification
of
Biochemical
Endpoints
The
sex
hormones,
corticosterone,
and
the
thyroid
hormones
can
be
determined
by
RIA
or
ELISA
methods.
Commercial
kits
are
available
and
are
adaptable
to
use
on
birds
and
samples
from
various
matrices.
RIA
methods
are
often
more
sensitive,
but
ELISA
techniques,
also
highly
sensitive,
lack
the
additional
cost
and
issues
involved
with
working
with
radioisotopes.
Because
many
of
the
hormones
respond
rapidly
to
stress,
handling,
and
bleeding
procedures,
the
preferred
sampling
method
is
from
fecal/
urate
samples,
rather
than
from
plasma.
Fecal
steroid
sampling
has
been
validated
in
a
wide
array
of
animals
including
birds,
and
evaluated
under
both
laboratory
and
field
conditions.
Sampling
feces
will
allow
periodic
sampling
of
hormone
status
rather
than
single­
point,
terminal
evaluations
that
can
be
misleading,
particularly
for
thyroid
hormones.
Steroid
hormones
can
also
be
sampled
during
egg­
laying,
by
this
manner,
without
the
danger
of
decreasing
egg
production
or
inducing
body
checks
in
eggs
from
handling
stress
of
the
hens.
Because
male
Japanese
quail
usually
must
be
removed
from
the
hen
several
times
during
a
week
to
prevent
injury
from
aggressive
behavior
by
either
mate,
monitoring
of
fecal
steroids
by
sex
can
be
accomplished
in
their
separate
home
cages.
Although
the
thyroid
hormones
have
not
been
measured
in
feces,
it
is
highly
likely
that
this
noninvasive
method
can
be
easily
applied
to
T3
and
T4.
Use
of
fecal/
urate
samples
for
hormone
monitoring
under
the
dosing
regime
of
the
two­
generation
laboratory
test
will
also
provide
a
direct
comparative
measure
for
field
collected
samples.

Determination
of
genetic
sex
by
DNA
methods
is
preferred
over
the
more
lengthy
and
costly
karyotyping
techniques.
Western
Dot
Blot
or
PCR
methods
are
equally
reliable;
the
dot
blot
technique
is
somewhat
more
rapid.

Several
promising
biochemical
methods
in
development
(
VTG,
catecholamine,
and
vasotocin)
are
being
adapted
to
RIA
or
ELISA
techniques
because
of
their
superior
sensitivity
and
ease
of
measurement.
Again,
additional
convenience
and
some
cost
savings
are
attained
when
ELISA
techniques
are
employed.
Battelle
Draft
127
April
23,
2003
12.5
Significant
Data
Gaps
Several
data
gaps
were
found
in
the
course
of
the
review.
Among
these
gaps
is
the
lack
of
clear
information
on
the
transfer
to
and
fate
of
xenobiotics
in
ovo.
Chemical
transfer
to
the
egg
has
been
variably
described
from
tissue
or
dietary
sources.
The
source
of
the
in
ovo
concentrations
of
test
substances
has
impact
on
decisions
regarding
exposure
duration
and
interpretation
of
endpoint
responses.
Further
study
of
estrogenic
xenobiotics
that
require
metabolic
activation
is
needed
to
determine
whether
or
not
this
occurs
in
the
embryo,
and
if
so,
its
relationship
to
the
sensitive
developmental
period.
Additional
work
is
also
needed
to
determine
whether
reduced
levels
of
the
5"­
metabolites,
through
blockage
of
the
reductase
enzymes,
are
more
likely
to
cause
adverse
effects
than
is
reduction
of
the
parent
compound.

Because
histopathological
evaluations
are
observer­
based,
the
procedures
for
their
use
must
be
standardized
to
produce
repeatable
results
that
can
be
verified
by
different
investigators.
Avian
histopathological
evaluations
in
the
context
of
toxicity
assessments
have
had
little
formalization
of
technique,
description,
or
morphometric
analysis
although
there
are
standard
practices
and
vocabulary
in
use
by
organizations
such
as
the
American
Association
of
Avian
Veterinarians
and
the
College
of
Veterinary
Pathology
Little
is
known
of
the
interactive
effects
of
endocrine­
active
substances.
In
particular,
most
test
diets
(
and
vegetable
oil
carriers)
contain
variable
amounts
of
the
natural
phytoestrogens
and
phytoandrogens.
How
these
chemicals
affect
the
bird's
response
to
the
test
substance
(
a
suspected
endocrine­
active
compound)
is
unknown.
Dietary
treatments
or
changes
to
eliminate
this
potential
interference
will
need
to
be
investigated.

Significant
genetic
differences
exist
between
strains
of
Japanese
quail
that
are
used
in
avian
reproductive
testing.
It
is
not
known
whether
these
differences
can
have
significant
effects
on
the
outcome
and
interpretation
of
the
test.
Further
investigations
into
traits
that
are
coselected
with
high
body
weight
or
high
fecundity
are
needed.
The
feasibility
and
criteria
for
establishing
and
maintaining
a
random­
bred
line
from
a
natural
source,
such
as
UBC,
or
a
standard
random­
bred
line
with
selected
qualities
for
toxicity
testing
needs
investigation.

If
ANOVA
methods
continue
to
be
applied
to
avian
reproduction
toxicity
tests,
a
statistical
approach
for
delayed
effects
must
be
investigated.
For
example,
in
a
6­
week
exposure
period,
only
during
the
final
3
weeks
of
this
period
will
the
full
effects
of
a
test
substance
on
fertility,
embryo
viability,
hatching
success,
and
sperm
quantity
and
quality
be
observed
because
of
the
about
21
days
needed
for
effects
on
spermatogonia
to
be
expressed
extragonadally.
No
effective
statistical
method
has
been
identified
for
analyzing
such
a
delayed
treatment
effect
so
far.
Springer
and
Collins
(
1999)
conducted
simulation
tests
using
an
adaptation
of
the
Roth
step­
down
trend
test
incorporating
covariates
for
the
last
3
weeks
(
Weeks
8
­
10)
of
a
standard
bobwhite
quail
reproduction
toxicity
test.
They
found
that
using
only
the
last
3
weeks
of
data
can
result
in
a
decline
in
the
power
of
the
test
to
detect
the
number
of
chicks
that
hatched
per
number
of
eggs
incubated,
and
the
number
of
hatchlings
that
survived
14
days.
Battelle
Draft
128
April
23,
2003
Though
seemingly
a
minor
data
gap,
the
lack
of
specific
information
on
husbandry
requirements
of
the
Japanese
quail
that
will
result
in
consistent
results
in
laboratory
toxicity
tests
is
important.
As
indicated
in
a
comparative
study
of
five
laboratories
using
the
same
source
of
birds
and
test
substances
(
Schlatterer
et
al.
1993),
a
number
of
parameters
differ
significantly
among
laboratories
(
e.
g.,
number
of
eggs
with
cracks,
food
consumption,
eggs
laid,
etc.),
potentially
resulting
in
different
conclusions
regarding
the
hazard
a
chemical
poses.
Species­
specific
information
needs
to
be
developed
for
dealing
with
fear,
social
stress,
injurious
pecking
,
and
other
behavioral
problems
of
Japanese
quail
in
laboratory
reproduction
tests.
These
behaviors
seriously
harm
the
birds'
welfare
and
productivity
(
Jones
and
Hocking
1999).
Much
can
be
learned
from
anecdotal
information
and
experience
of
flock
curators,
but
it
should
be
verified
for
the
various
growth
and
maturation
stages
of
the
quail
in
testing
situations.

13.0
IMPLEMENTATION
CONSIDERATIONS
Pre­
validation
considerations
that
are
important
to
the
development
of
the
avian
reproduction
test
include
the
following.
Because
there
is
not
enough
information
available
to
determine
the
combination
of
the
P1
and
F1
exposure
protocols
that
is
the
most
advantageous
for
evaluating
reproductive
effects
and
determining
mechanism
of
action,
a
direct
performance
comparison
of
the
proven­
breeder
and
prebreeding
exposure
regimens
combined
with
nontreatment
and
worstcase
hatch­
through­
egg­
laying
F1
exposure
scenarios
is
needed
for
the
selection
of
the
appropriate
exposure
regimen.

Considering
the
sensitivity
of
the
Japanese
quail
compared
with
that
of
the
northern
bobwhite,
the
effects
of
strain
selection
on
test
outcome
should
be
determined
prior
to
implementing
the
test
protocol
to
verify
the
sensitivity
of
the
test
species
and
to
minimize
nontreatment
variability
across
laboratories.
Likewise,
husbandry
practices
need
review
and
standardization
to
reduce
interlaboratory
variability
and
to
reduce
behavioral
problems
of
Japanese
quail
in
laboratory
reproduction
tests.

Avian
histopathological
evaluations
in
the
context
of
toxicity
assessments
have
had
little
formalization
of
technique,
description,
or
morphometric
analysis.
Because
histopathological
evaluations
are
observer­
based,
the
procedures
for
their
use
must
be
standardized
to
produce
repeatable
results
that
can
be
verified
by
different
investigators.

If
ANOVA
methods
continue
to
be
applied
to
avian
reproduction
toxicity
tests,
a
statistical
approach
for
delayed
effects
should
be
investigated.

T4/
T3
are
important
indicators
of
thyroid
function,
but
are
limited
in
value
when
obtained
from
plasma
samples
because
of
their
sensitivity
to
handling
and
bleeding,
and
because
plasma
fluctuation
of
these
hormones
render
it
difficult
to
document
hypo­
or
hyperthyroidism
from
a
single
sample.
These
hormones
are
excreted
through
the
bile.
Therefore,
development
of
T4/
T3
assays
in
fecal/
urate
samples
could
make
the
ability
to
monitor
thyroid
hormones
noninvasively
over
time
readily
available.
Battelle
Draft
129
April
23,
2003
PCR
methods
for
genetic
sex
determination
need
to
be
optimized
for
the
Japanese
quail.

The
interactive
effect
of
phytoestrogens
in
test
diets
needs
investigation,
and
concentration
limits
for
natural
phytoestrogens
in
feed
should
be
established
relative
to
the
potential
impact
of
these
compounds
on
test
outcome.

14.0
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